Training Manual

For professional staff

Seed Quality Evaluation

(Vegetable Crops) By

Muhammad Boota Sarwar

Published by

Facilitation Unit for Participatory Vegetable Seed And Nursery Production Program

Ministry of Food, Agriculture & Livestock, Islamabad

CONTENTS
Sr. Chapters No . 1 Introduction 2 Objective of Seed Quality Evaluation
Accuracy of the Results of Seed Analysis Recognized Standard Methods Reporting Results

Pag e No. 6 6
6 7 7

Seed Sampling
Definitions used in Seed Sampling Size of Seed Lot Preconditions for Seed Sampling Table-1: Maximum Seed Lot Weight of Vegetable Crops Table-2: Submitted Sample Weight of Vegetable Crops

8
8 9 9 10 11

Methods for Drawing Primary Seed Samples

A. Seeds in Bulk Table-3: Required Intensity of Primary Seed Samples for Seeds in Bulk B. Seeds in Sacks C. Seeds in Small Containers Table - 4: Required Intens i t y of Pr imary Seed Samples Seeds in Sacks or Mult ip l e Containers of 15 kg to 100 kg capaci ty D. Seeds in Processing or Conditioning Machines Table-5: Required Intensity of Primary Seed Samples for Seeds during Seed Processing E. GENERAL

12
12 12 12 13 13 for

13 14 14

Preparation of Working Sample in the Laboratory

Minimum Weight of Working Sample Obtaining the Working Sample Storage of Seed Samples Table-6: Working Sample Weight of Vegetable Crops

15
15 15 15 16 2

Mixing and Dividing Procedures

Mechanical Divider Method a. Centrifugal Divider b. Soil Divider Hand Mixing or Spoon Method

17
17 17 18 20

Purity Analysis
1. Objectives 2. Definitions: Seed Pure Seed Other Crop Seed Weed Seed Inert Matter 3. General Principles 4. Apparatus 5. Procedure for Separation of Components Table-7: Minimum number of decimal places required for weighment of components 6. Calculation and Expression of Results 7. Reporting of Results Table-8. Tolerances for comparing percentage of purity analysis components, to determine if two tests from same submitted sample are compatible

22
22 22 22 22 25 25 25 29 30 30 30 31 31 32

Determination of Other Seeds by Number

Objective Procedure Calculation and Expression of Results Reporting of Results

34
34 34 34 34

Verification of Species and Cultivars

Objective Preconditions for the Test General Principles Apparatus and Facilities Weights of Submitted Sample Table-9A: Minimum weight of Submitted Samples for Determination of Species / Cultivars Examination of seeds Examination of seedlings Examination of plants in glasshouse or growth chamber Examination of plants in field plots Calculating and expression of results Reporting results

35
35 35 35 36 36 36 37 37 38 38 39 39

10

Determination of Moisture Content

Definition

41
41 3

Sample Procedure: A: Constant temperature oven method Calculation of Results Tolerance B: Electric Moisture Meter

41 41 41 43 44 44

11 12

Determination of 1,000 Seed Weight

Definition Procedure

45
45 45

Purity Procedures for Coated Seeds

Note Definitions Obtaining the working sample Foreign seed determination

47
47 47 47 48

13

The Germination Test

Introduction Methods to be used Definitions Preparation of Seeds for Germination Tests Germination Conditions Germination Methods Table-10. Germination Methods for Vegetable Crop Species Special Germination Procedures Beta spp. Phaseolus vulgaris Vicia faba Coated seeds Tetrazolium testing Use of fungicides Treatments for Promoting Germination of Dormant Seeds Counts and Duration of Test Calculation and Reporting of Germination Results Retesting Evaluation of Tests Germination Tolerances Table-11. Maximum tolerated ranges in germination percentages of replicates for deciding when to retest Table-12. Maximum tolerated differences in germination percentages of tests for deciding which tests to average Seedling Descriptions: Family Azoaceae - New Zealand spinach Family Asteraceae - Lettuce Family Asteraceae Kinds other than lettuce Family Brassicaceae Cabbages, Radish, Turnip

49
49 49 50 51 52 57 58 61 61 61 61 62 62 63 63 66 67 69 70 72 74 77 79 80

Family Chenopodiaceae Beet, Spinach Family Cucurbi taceae Melons, Gourds, Squashes Family Fabaceae Beans, Cowpea Family Fabaceae Peas, Lathyrus Family Fabaceae enugreek F Family Liliaceae Asparagus Family Liliaceae Onion, Leek, Chives Family Malvaceae Okra Family Apiaceae & Solanaceae Carrot, Tomato, Chili, Eggplant etc

82 84 86 88 90 92 95 97 100 101 103 104

14

Biochemical tests for viability

Objectives Principle Reagent Procedure Calculation and Expression of Results Reporting Results

107
107 107 108 108 108 109

15

Seed Health Testing

Objective Definitions Principle Procedure Calculation of Results Reporting Results

110
110 110 111 111 114 114

16

Specific Instructions
Vegetable Crops of Family Solanaceae; Tomato, Chil i , Sweet pepper, Eggplant: Seed unit Submitted Sample Weight Working Sample Weight Purity Analysis Germination SEED HEALTH TESTING: The main seed-borne tomato pathogens with common names of the diseases they cause The main seed-borne eggplant pathogens with common names of the diseases they cause The main seed-borne pepper pathogens with common names of the diseases they cause Method for the Detection of Xanthomonas campestr i s pv. Vesicator i a on Pepper seed Method for the Detection of Tobamoviruses on Pepper seed (ISF)

120 122 122 122 123 124 125

Method for the Detect ionXanthomonas of campestr i s pv. vesicatoria on Pepper and Tomato seed (STA selective media assay) Method for the Detection of Xanthomonas campestris pv. vesicatoria on Pepper and Tomato seed (ISHI- vegetable selective media assay) Method for the Detect ionClav of ibacte r michiganensissubsp. michiganensis on Tomato seed Method for the Detection of Tobamoviruses on Tomato seed (Adapted from ISF) Method for the Detection of Pepino Mosaic Virus on Tomato seed (Adapted from ISF)

130 133

137 145 149

17

Specific Instructions
Vegetable Crops of Family Cucurbitaceae Melons, Gourds, Squashes Seed unit Submitted Sample Weight Working Sample Weight Purity Analysis Germination SEED HEALTH TESTING: The main seed-borne watermelon pathogens with common names of the diseases they cause The main seed-borne cucumber pathogens with common names of the diseases they cause The main seed-borne melon pathogens with common names of the diseases they cause The main seed-borne squash pathogens with common names of the diseases they cause

154
154 154 154 154 155 158 158 158 159 159

18

Specific Instructions
Vegetable Crops of Family Brassicaceae Cabbages, Cauliflower, Turnip, Radish Seed unit Submitted Sample Weight Working Sample Weight Purity Analysis Germination SEED HEALTH TESTING: The main seed-borne Brassica pathogens with

160
160 160 160 160 161 164 164

common names of the diseases they cause The main seed- borne radish pathogens with common names of the diseases they cause Detect ion of Phoma l i ngam on Brassica spp. Detect ion of Xanthomonas campestr i s pv. campestris on Brassica spp. Method for the Detection of Xanthomonas campestris pv. campestris in disinfested seed of Brassica spp.

164 165 171 188

19

Specific Instructions
Vegetable Crops of Family Fabaceae Peas, Beans 193 Seed unit 193 Submitted Sample Weight 193 Working Sample Weight 193 Purity Analysis 193 Germination 194 SEED HEALTH TESTING: 204 The main seed- borne peas pathogens with common 204 names of the diseases they cause 205 The main seed- borne Phaseo lu spathogens with common names of the diseases they cause The main seed- borne Broad beanpathogens with 207 common names of the diseases they cause Detect ion of Ascochyta pisi on Pisum sativum (Pea) [ISTA Validated Method] 208 Detection of Colletotrichum lindemuthianum on Phaseolus vulgaris (Bean) 216 Detection of Pea Early-Browning Virus and Pea Seed-borne Mosaic Virus on Pisum sativum 222 Detection of Pseudomonas savastanoi pv. phaseolicola on Phaseolus vulgaris Detection of Xanthomonas axonopodis pv. phaseoli 230 and Xanthomonas axonopodis pv. phaseoli var. fuscans on Phaseolus vulgaris 247

20

Specific Instructions
Vegetable Crops of Family Apiaceae Carrot Seed unit Submitted Sample Weight Working Sample Weight Purity Analysis Germination

265
265 265 265 265

SEED HEALTH TESTING: The main seed- borne carrot pathogens with common names of the diseases they cause The main seed- borne Parsn ip pathogens with common names of the diseases they cause The main seed- borne Pars l e y pathogens with common names of the diseases they cause The main seed- borne Ce le ry pathogens with common names of the diseases they cause Blot te r method for the detect ion Alteof rnar i a dauci on Daucus carota [ISTA] Detection of Xanthomonas hortorum pv. carotae on Daucus carota [ISTA] Alternaria dauci: ISTA Freeze Blotter test Alternaria dauci: Blotter/Pathogenicity test

268
268 268 269 269 270 277 312 313

266

21

Specific Instructions
Vegetable Crops of Family Chenopodiaceae Beetroot, Swiss chard, Spinach Seed unit Submitted Sample Weight Working Sample Weight Purity Analysis Germination SEED HEALTH TESTING: The main seed-borne Beetroot pathogens with common names of the diseases they cause The main seed-borne Spinach pathogens with common names of the diseases they cause

315
315 315 315 315 316 319 320

319

22

Specific Instructions
Vegetable Crops of Family Malvaceae Okra Seed unit Submitted Sample Weight Working Sample Weight Purity Analysis Germination SEED HEALTH TESTING: The main seed-borne Okra pathogens with common names of the diseases they cause

321
321 321 321 321 321 323

23

Specific Instructions
Vegetable Crops of Family Asteraceae Lettuce Seed unit

324

Submitted Sample Weight Working Sample Weight Purity Analysis Germination SEED HEALTH TESTING: The main seed- borne le t tuce pathogens with common names of the diseases they cause

324 324 324 324 327

24

Specific Instructions
Vegetable Crops of Family Asteraceae Lettuce Seed unit Submitted Sample Weight Working Sample Weight Purity Analysis Germination SEED HEALTH TESTING: The main seed-borne lettuce pathogens with common names of the diseases they cause

328
328 328 328 328 329 333

INTRODUCTION
Prime objective of seed quality evaluation is to make available high quality seeds and planting material having capacity to produce a plentiful crop of the required cultivar. Various evaluation and seed testing processes have been developed to assess the quality of seeds before sowing. The concept of seed quality may vary from the perspective of seed producer, Seed Company involved in seed marketing, the end user (farmer) and the government but anyhow the main objective of seed quality evaluation is to determine the value of seed for planting. Seed is a living biological product hence its behavior is not predictable with as sureness as is possible in case of nonliving inert products hence methods used for seed quality evaluation should be based on scientific knowledge of seed and the evaluation results should be reproducible universally within acceptable variance. Although broader definition of seed includes all parts of the plant which can be readily used as planting material but for the purpose of this manual the definition of seed is delimited to true seeds and seed units (fruits) marketed as seed. The text of the Training Manual that follows has been developed for professionals already acquainted with basics of seed testing procedures; to provide updated knowledge of the techniques used for evaluation of vegetable seeds of commerce.

10

It is acknowledged that Technical resources of ISTA, OECD and AOSA have been utilized to develop this Training Manual to present information on seed quality evaluation of vegetable crops ensuring credibility for seed transactions in international trade; with the hope that professional Seed Analysts working in public and private sector seed testing laboratories will benefit from the handbook.

PART-I

Basic Concepts of Seed Testing

11

1.OBJECTIVE OF SEED QUALITY EVALUATION

The objective of all methods and procedures used to analyze a representive sample of the seed lot is to get analytical information regarding the factors addressed in the specific seed standards and can include, depending on the crop and category of seed: 1. numbers per unit weight (per 25 g, per Kg, etc.) of other crop seeds, weed seeds and inert matter such as ergot/sclerotia; 2. percentages by weight of the components of the seed sample (pure seed, other crop kinds, weed seeds, inert matter, etc.); 3. moisture content in percentage; 4. percentages by number of germinable seeds, diseased seeds and seeds of other species.

Accuracy of the Results of Seed Analysis

12

Subject to the natural variations which occur in random sampling, the accuracy with which the results of seed analyses will represent a lot of seed depends on: a. The thoroughness of the blending of the lot of seed from which the sample is drawn; b. The care used in drawing the sample; c. The care with which a number of small samples drawn from several containers are mixed to form a composite sample representing a lot of seed or, when the lot is not homogeneous, the care taken in keeping unlike samples separate; d. The care with which the working sample is taken; e. The technical knowledge, skill and accuracy of the analyst; f. The condition of the equipment used.

Recognized Standard Methods: procedures set out in the:

The methods and

a. Procedures and Direct i ons for

b. c. d. e.

Seed Cert i f i c a t i on System in Pakis tan not i f i ed by Food and Agr icu l t u re Div is i on , Government of Pakis tan Rules for test i ng seed publ i shed by the US Associa t i on of Off i c i a l Seed Analyst (AOSA) Rules for Seed Test ing In ternat i ona l Rules for seed test publ i ng i shed by In ternat i ona l Seed Test ing Associa t i on ( ISTA) OECD Schemes for the Var ie ta l Cert i f i c a t i on or the Contro l of Seed Moving in In te rnat i ona l Trade Canadian Methods and Procedures for Test ing Seed

Reporting Results The Report of Analysis must contain the following information:
o

Ident i f i c a t i on number. This must be a unique laborato ry - ass igned number which i s ident i ca l
13

to

o o o o

o o

o o

the sample number and the identification number of any related worksheets; Lat in ame n of crop kind on which the reported analyses were conducted; Name of the laborato ry i ssu ing the Report ; A lo t ident i f i e r , such as seed lo t number, i f known Federa l Seed Cert i f i c a t i on and Regis t ra t i on Department (FSC&RD) Fie ld Inspect i on Report Number, i f known; Analyt i ca l resu l t s as requi red for speci f i c category of seed; Test method(s) used i f di f f e ren t f rom those prescr ibed in Methods and Procedures not i f i ed by Government of Pakis tan ; Signature of Accredi ted Analyst for the crop kind under analys i s ; Date test completed.

2. SEED SAMPLING
Seed quality evaluation requires inspection of seeds in a specific seed lot; whereas it is not practically possible to check each seed in the lot hence a representative sample of the seed lot is drawn and checked to assess quality of the whole seed lot. Statistical models developed on the basis of 99% probability of accuracy provide protocols for drawing a seed sample representing the seed lot in a reduced form. The seed sample is then subjected to various visual examinations and tests for assessing planting quality of the seed lot. Therefore accuracy of seed testing results directly depends on accuracy of protocol followed for drawing and dispatch of seed sample.

Definitions used in Seed Sampling:

14

1. True Seed or Botanica l Seed : Mature ovule is called true seed; without attachment of any other part of the flower or fruit. 2. Seed Units: Fruits, with or without other parts of the flower, commercially marketed as seed are called seed units as is the case of caryopsis, achenes, schizocarps etc. 3. Seed Lot: A seed lot is a specified quantity of seed, physically identifiable, in respect of which reproducible results can be drawn on the basis of sampling. 4. Primary Sample: A primary sample is a small portion taken from one point in the lot. 5. Composite Sample: The composite sample is formed by combining and mixing all the primary samples taken from the lot. 6. Submitted Sample: A submit ted sample is a sample that is to be submitted to the testing laboratory and may comprise either the whole of the composite sample or a sub-sample thereof. The submitted sample may be divided into sub-samples packed in different material meeting conditions for specific tests (e.g. moisture or health). 7. Working Sample: The working sample is the whole of submitted sample or a sub-sample taken from the submitted sample in the laboratory on which one of the quality tests is made. 8. Sub-sample: A sub-sample is a portion of a sample obtained by reducing the sample in a random manner. 9. Duplicate sample: A duplicate sample is another sample obtained for submission from the same composite sample and marked Duplicate sample. 10. Sealed: Sealed means that the container or individual containers in which seed is held are enclosed in such a way that they cannot be opened to gain access to the seed and closed again without either destroying the seal or leaving evidence of tempering. This definition refers to the sealing of seed lots and seed samples.

15

11. Sel f - seal i ng conta iners : The valve-pack bag is a specific type of self sealing container. It is filled through a sleeve-shaped valve which is automatically closed by the completion of filling the bag. 12. Marked/labeled: A container of a seed lot can be considered as marked or labeled when there is a unique identification mark on the container, which defines the seed lot to which the container belongs. All containers of a seed lot must be marked with the same unique seed lot designation (numbers, characters or combination of both). Marking of sub-samples must ensure that there is always an unambiguous link between the seed lot and the sub-samples. 13. Coated seeds: Coated seeds are seeds covered with material that may contain pesticides, fungicides, dyes or other additives. The following types of coated seeds are defined: 14. Seed pellets: More or less spherical units, usually incorporating a single seed with the size and shape of the seed no longer readily evident. 15. Encrusted seed: Units more or less retaining the shape of the seed with the size and weight changed to a measurable extent. 16. Seed granules: Units more or less cylindrical, including types with more than one seed per granule. 17. Seed tapes: Narrow bands of material, such as paper or other degradable material, with seeds spaced randomly, in groups or in a single row. 18. Seed mats: Broad sheets of material, such as paper or other degradable material, with seeds placed in rows, groups or at random throughout the sheets. 19. Treated seed: Seeds with treatments, which have not resulted in a significant change in size, shape or addition to the weight of the original seed. Size of Seed Lot: The quantity of a seed lot has been restricted for each crop based upon statistical calculations to ensure that the sample

16

stays to be representative of the particular seed lot. Ignoring this limit of lot size may lead to disingenuous results. The maximum seed lot weight permitted in respect of various vegetable crops is provided in Table-1. Preconditions for Seed Sampling: 1. Seed lot must be within range of maximum seed lot weight; and larger seed lots must be divided to more number of seed lots to confirm to maximum seed lot weight; and properly demarked and labeled accordingly prior to sampling. 2. Seed lot must be reasonably homogenous; and if there is a doubt in this respect, the seed lot may be well mixed to make it homogenous. The sample drawn from a heterogeneous seed lot is generally not representative of the seed lot. 3. Appropriate record of origin of the seed lot will be required in case of formal certification and labeling; however for seed packing under Truthful Labeling no such record is required. 4. When seeds are in sacks or in small containers, each sack or container should be theoretically and practically accessible for drawing primary samples. Sampling will not be representative if some portions of the seed lot are inaccessible. 5. Seed sample is drawn from a particular seed lot in two stages; first primary samples are drawn from the seed lot in a prescribed manner and then primary samples are mixed and reduced to form Submitted Sample for dispatch to seed testing laboratory. The prescribed quantity of Submitted Sample in respect of vegetable crops is detailed in Table-2. Marking/labeling and sealing of containers: The seed lot shall be in marked/labeled containers which are self-sealing, sealed (or capable of being sealed) or under the control of the seed sampler.

17

Where the seed lot is already marked/labeled and sealed before sampling, the seed sampler must verify marking/labeling and sealing on every container. Otherwise the sampler has to mark/label the containers and must seal every container before the seed lot leaves his/her control. The samplers are personally responsible for the seals, labels and bags supplied to them and it is their duty to ensure that primary, composite or submitted samples shall never be left in the hands of persons not authorized by the seed testing laboratory unless they are sealed in such a way that they cannot be tampered with.

Table- 1: MaximumSeed Lot Weight of Vegetable Crops, subject to a tolerance of 5%

Sr. No . 1 2 3 4 5 6 7 8 9 10 Species Abelmoschus esculentus Allium cepa Beta vulgaris L. (all varieties) Brassica oleracea L. (all varieties) Brassica rapa L. (all varieties) Capsicum annum Citrullus lanatus Coriandrum sativum Cucumis melo (all varieties) Cucumis sativus Crop Okra Onion Beet Cauliflower, Cabbage, etc Turnip, Chinese cabbage Pepper Watermelon Coriander Muskmelon, Long melon Cucumber Maximum Seed Lot Weight (MT) 20 10 20 10 10 10 20 20 10 10 18

11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Cucurbita maxima Cucurbita moschata Cucurbita pepo Cyamopsis tetragonoloba Daucus carota Lactuca sativa Lagenaria siceraria Luffa acutangula Luffa aegyptica Lycopersicon esculentum var. esculentum Momordica charantia Pisum sativum Praecitrullus fistulosus Raphanus sativus Solanum melongena Spinacia oleracea

Pumpkin Red gourd Marrow Cluster bean Carrot Lettuce Bottle gourd Ribbed gourd Sponge gourd tomato Bitter gourd Pea Tinda Radish Eggplant Spinach

20 10 20 20 10 10 10 20 20 10 10 20 10 10 10 10

Note: Seed pel le t s , seed granules , seed tapes or seed mats. The maximum number of seeds that a seed lo t of seed pel l e t s , seed granules , seed tapes or seed mats may conta in i s 1,000,000,000 (10,000 uni ts of 100,000) except that the weight of the seed lo t , inc lud ing the coat ing mater ia l ma not exceed 40,000 kg subject to a to le rance of 5% (42,000 kg) . When seed lo t s ize i s expressed in uni ts the tota l weig of the seed lo t must be given on the ISTA In te rnat i ona l See Lot Cert i f i c a t e . Maximum lot size for treated and encrusted seeds is defined by applying the quantities indicated in Table-1 to the seeds without coating material.

Table-2: Submitted Sample Weight of Vegetable Crops

SPECIES CROP Submitte d Sample (g) 1000

Abelmoschus esculentus

okra

19

Allium cepa Brassica oleracea L. (all varieties) Brassica rapa L. (all varieties) Capsicum annum Citrullus lanatus Praecitrullus fistulosus Cucumis melo Coriandrum sativum Cucumis sativus Cucurbita maxima Cucurbita moschata Cucurbita pepo Cyamopsis tetragonoloba Daucus carota Lagenaria siceraria Lactuca sativa Lycopersicon esculentum var. esculentum Momordica charantia Pisum sativum Raphanus sativus Solanum melongena Spinacia oleracea

onion Cauliflower, Cabbage, etc Turnip, Chinese cabbage pepper watermelon tinda muskmelon coriander cucumber Pumpkin Red gourd marrow Cluster bean carrot Bottle gourd lettuce tomato Bitter gourd pea radish eggplant spinach

80 70 70 150 1000 150 150 500 150 1000 350 1000 800 30 700 700 15 700 1000 300 150 250

Methods for Drawing Primary Seed Samples

A. Seeds in Bulk: Port i ons of seeds shal l

be taken wit a st i ck sampler f rom at leas t the number of posi t i ons ind i ca ted below in Table- 3, selec ted randomly and inc lud ing both vert i ca l and hor i zonta l posi t i ons .

Table-3: Required Intensity of Primary Seed Samples for Seeds in Bulk

20

Sr. No . 1 2 3 4 5

Size of Lot (KG) Up to 50 51-1,500 1,501-3,000 3,001-5,000 5,001-20,000

Number of Positions To be Sampled Not less than 3 Not less than 5 At least 1 for each 300 kg Not less than 10 At least 1 for each 500 kg

B. Seeds in Sacks:

1. When seeds are in sacks they shall be sampled at random and samples are taken from the top, middle and the bottom of each selected sack. The position from which the seeds are taken shall be different from sack to sack and samples shall be drawn from different horizontal positions of the lot. 2. Wherever pract i cab le , seed shal l be sampled with a metal spear , tr i e r or probe wit a sol i d point which shal be of suf f i c i en t length to reach beyond the middle of the sack when inser ted f rom the s ide and shal l have an oval aperture so placed that the ins t rument removes port i ons of seeds of equal volume f rom each part of the sack through which i t trave l s . The ins t rument should be inser ted in to the sack in an upward di rec t i on at an angle of approx imately o 30 to the horizontal, with its aperture downwards until the aperture reaches center of the sack, then rotated to make aperture upwards and withdrawn at a uniform speed. 3. A stick sampler with more than one aperture and transverse partitions can be used through inserting it diagonally in closed position, then opened, gently agitated to fill completely, closed again and withdrawn. 4. Required intensity of primary seed samples for seeds in sacks or small containers is provided below in Table-4. C. Seeds in Small Containers:

21

1. When seeds are in smal l

conta iners ( less than 15 kg) a maximum 100 kg weight of seed i s taken as the basic uni t . Conta iners are combined to form sampl ing uni ts of 100 kg ( fo r example 20 conta iners each of 5 kg form one uni t ) . 2. Containers are properly shaken before drawing primary samples. 3. Samples are drawn at random in accordance with required intensity provided in Table-4. Table- 4: Required Intensity of Primary Seed Samples for Seeds in Sacks or Multiple Containers of 15 kg to 100 kg capacity (inclusively) . Sr. No. of Sacks or No Units of . Containers 1 1 to 5 2 6 to 14 3 4 5 15 to 30 31 to 49 50 or above Numbers to be Sampled Each sack or unit of containers Not less than 5 sack or unit of containers At least 1 for each sack or unit of containers Not less than 10 sack or unit of containers At least 1 in 5 sack or unit of containers

D. Seeds in Processing or Conditioning Machines:

Port i ons of seeds shal l be drawn dur ing seed process ing or condi t i on ing to get ent i re cross sect ion of the seed st ream at regular in te rva l s with sampl ing f requency as provided in Table- 5 below. Table-5: Required Intensity of Primary Seed Samples for Seeds during Seed Processing
22

Sr. Size of Lot No (KG) . 1 Up to 50 2 51-1,500 3 1,501-3,000 4 5 3,001-20,000 20,001 and above

Number of Positions To be Sampled Not less than 3 Not less than 5 At least 1 for each 300 kg, But not less than 5 At least 1 for each 500 kg, but not less than 10 At least 1 for each 700 kg, but not less than 40

E. GENERAL:
1. The quant i t y

of pr imary samples may be reduced to the quant i t y requi red as submit ted sample through mixing and halv ing method with hands. I f the pr imary samples appear uni fo rm they are combined to form the composi te sample, i f not , the sampl ing procedure must be stopped. When pr imary samples are col l ec ted di rec t l y in to one conta iner , the content of th i s conta in shal l be regarded as the composi te sample only i f i t appears uni fo rm, i f not , i t must not be used for obta in ing a submitted sample. 2. Portions of seed for moisture test shall be drawn separately in such a way as to prevent exposure to the environment and packed in air-tight containers for dispatch to the laboratory. 3. Seed samples should be proper ly labe with led the same ident i f i c a t i on as the seed in lo prescr t ibed manner before dispatch to the laboratory .

Preparation of Working Sample in the Laboratory

23

1.

Minimum Weight of Working Sample : Minimum Weight of Working Sample for var ious vegetable crops i s provided below in Table- 6. Obtaining the Working Sample : The submit ted sample f i r s t be thoroughly mixed and then Working Sample i s obta ined ei ther by repeated having or by abstrac t i ng and subsequent ly combining smal l random port i ons .

2.

Storage of Seed Samples

1.

Before Testing: Every ef fo r t must be made to star t test i ng a sample on the day of receip t . I f delay i s unavoidable , the sample shal l be stored in cool wel l vent i l a t ed room in such a way that changes in the qual i t y of seed are minimized.

2.

After Testing:The pr imary aim of storage of samples after test i ng i s to be able to repeat the or ig ina l test carr i ed out on the submitted sample. Therefore , storage condi t i ons should be such that changes in the seed qual i t y tra i t s tested are minimal . E.g . in the cas of pur i t y test or other seed count , the sample should be stored in such a way that the physica l ident i t y i s kept . In the case of germinat ion , viab i l i t y or heal th t in orthodox seeds the sample should be stored under cool and dry condi t i ons . For such tests in reca lc i t r an t and in termediate seeds of trop i ca l and sub- t rop i ca l species , long term storage i s not poss ib le . For such seed of temperate species storab i l i t y i s depending on the fungal status and to some extent whether the seed i s dormant or not . Al l fac to rs perta in i ng to storage need to be determined on a species basis . Protect i on against insects and rodents may be necessary .

24

When a re-test in a different testing laboratory is required, a portion shall be drawn from the stored sample in accordance with prescribed method and submitted to the designated testing laboratory. The remainder shall be retained in store. Table-6: Working Sample Weight of Vegetable Crops
SPECIES CROP Workin g sample (g) 140 8 7 7 15 250 70 70 50 70 700 180 700 80 3 70 70 7 70 900 30 15 25

Abelmoschus esculentus Allium cepa Brassica oleracea L. (all varieties) Brassica rapa L. (all varieties) Capsicum annum Citrullus lanatus Praecitrullus fistulosus Cucumis melo Coriandrum sativum Cucumis sativus Cucurbita maxima Cucurbita moschata Cucurbita pepo Cyamopsis tetragonoloba Daucus carota Lagenaria siceraria Lactuca sativa Lycopersicon esculentum var. esculentum Momordica charantia Pisum sativum Raphanus sativus Solanum melongena Spinacia oleracea

okra onion Cauliflower, Cabbage, etc Turnip, Chinese cabbage pepper watermelon tinda muskmelon coriander cucumber Pumpkin Red gourd marrow Cluster bean carrot Bottle gourd lettuce tomato Bitter gourd pea radish eggplant spinach

25

Mixing and Dividing Procedures

1. Mechanical Divider Method This method is suitable for most kinds of seeds. The apparatus divides a sample into two approximately equal parts. The submitted sample is mixed by passing it through the divider, recombining the two parts and passing the whole sample through a second time and similarly a third time. The sample then is reduced by passing the seed through repeatedly and removing one-half on each occasion. This process of successive halving is continued until a working sample of the required size is obtained Use of compressed air is highly recommended for cleaning mechanical dividers. The dividers described below are examples of suitable equipment. Other devices may be used if it can be demonstrated that they provide an unbiased sub-sample.
a.

Centri fugal Divider (Gamet type) This divider is suitable for all kinds of seed except oilseeds (rapeseed, canola, mustards, flax, etc.) and kinds susceptible to damage (peas, soybeans, etc.) and the extremely chaffy types. This divider makes use of centrifugal force to mix and scatter seeds over the dividing surface. The centrifugal divider tends to give variable results when not carefully operated. i. Preparation of the apparatus: Level the div ider using the adjustab le feet .

26

Check the div ider and four conta iners for clean l i ness . Note that seeds can be t rapped under the spinner and become a source of contaminat ion .

ii.

Sample mixing: Place a conta iner under each spout . Feed the whole sample in to the hopper; when f i l l i n g the hopper, the seed must always be poured centra l l y . After the sample has been poured in to the hopper, the spinner i s operated and the seed passes in to the two conta iners . Turn of f spinner . Ful l conta iners are replaced by empty conta iners . The contents of the two fu l l conta iners are fed centra l l y in to the hopper together , the seed being al l owed to blend as i t f l ows in . The spinner i s operated. The sample mixing procedure i s repeated at leas t once more. Sample reduction: The two fu l l conta iners are replaced by two empty conta iners . The contents of one fu l l conta iner are set aside and the contents of the other conta iner are fed in to the hopper. The spinner i s operated. The success ive halv ing process i s cont inued unt i l the working sample(s ) of not less than the minimum weight (s ) requi red as set out in Table 1 are obta ined. Ensure that the div ider and conta iners are clean after each mixing operat i on .

iii.

b.

Soil divider (r i f f l e divider)

27

This divider is suitable for most kinds of seed, including peas, beans, soybeans, etc., provided the fall of the seed is not such that the seed will be damaged. For round-seeded kinds such as Brass i ca , the rece iv ing pans should be covered to prevent the seeds f rom bouncing out . This divider consists of a hopper with attached channels or ducts, a frame to hold the hopper, four collecting pans and a pouring pan. Ducts or channels lead from the hopper to the collecting pans, alternate ones leading to opposite sides. Riffle dividers are available in different sizes for different sizes of seed. The width and number of channels and spaces are important. The width of the channels must be at least two times the largest diameter of the seed or any possible contaminants being mixed. This apparatus, similar to the centrifugal divider, divides the sample into approximately equal parts. i. Preparation of the apparatus: Place the r i f f l e div ider on a f i rm, leve l clean sur face . Ensure the div ider i s leve l . Ensure that the div ider and the 4 sample col l ec t i on conta iners are clean. Check al l channels , jo in t s and seams of the div ider and col l ec t i on conta iners to ensure there are no seeds or other plant matter present before each use. Two clean empty col l ec t i on conta iners shal l be placed under the channels to receive the mixed seed. Sample mixing: Pour the whole sample in to the div ider by running the seed backwards and forwards along the edge of the div ider so that al l the channels and spaces of the div ider receive an equal amount of seed.
28

ii.

The two fu l l conta iners shal l be rep laced with two clean empty col l ec t i on conta iners . The contents of one fu l l col l ec t i on conta iner shal l be poured in to the div ider by hold ing the long edge of the pan against the long edge of the r i f f l e hopper and then rota t i ng the bottom up so that the seeds pour across al l channels at the same t ime. Repeat the procedure with the other fu l l conta iner . This process of mixing the ent i re submit ted sample shal l be repeated at leas t 2 more t imes before success ive halv ing begins .

iii.

Sample reduction: The contents of one fu l l col l ec t i on conta iner are set aside. Empty col l ec t i on conta iners are placed under each channel , and the contents of the other conta iner i s poured in to the hopper by hold ing the long edge of the pan against the long edge of the r i f f l e hopper and then rotat i ng the bottom up so that the seeds pour across al l channels at the same t ime. The success ive halv ing process i s cont inued unt i l the working sample(s ) of not less than the minimum weight (s ) requi red as set out in Table 1 are obta ined. Ensure that the div ider and col l ec t i on conta iners are clean after each mixing operat i on . Check al l channels of the div ider , the jo i n t s and seams.

2. Hand Mixing or Spoon Method This method should only be used for samples of a single small-seeded species that are smaller than Tr i t i c umspp. , very chaf fy species or uncleaned seed where i t i s demonstrated that one of the mechanica l div iders wi l l not take a representat i ve working sample(s ) . The sample i s poured uni fo rmly over a t ray with a s ide to s ide swinging motion. The receiv i ng pan should be kept leve l . This mixing
29

procedure is repeated a minimum of three times. A tray, a spatula and a spoon with a straight edge are required. After the preliminary mixing, pour the seed evenly over the tray with a side-to-side swing, alternately in one direction and at right angles to it. The depth of the seed in the pan shall not exceed the height of the vertical sides of the spoon. Do not shake the tray thereafter. With the spoon in one hand, the spatula in the other, and using both, remove small portions of seed from not less than five random places on the tray. Sufficient portions of seed are taken to constitute a working sample(s) of the required size as set out in Table-6.

30

PURITY ANALYSIS
1. Objective The objective of purity analysis is to determine (a) the percentage composition by weight of the sample being tested and by inference the composition of the seed lot, and (b) the identity of the var ious species of seeds and iner t part i c l e s const i t u t i ng the sample. 2. Definitions 2.1 Seed: A seed, in laboratory practice, is defined as "a structure which contains at least one ripened ovule with or without accessory parts". In many crop plants, the structure commonly regarded as the seed is botanically a fruit. Thus, in addition to true seeds, the foregoing definition includes florets and caryopses in the Poaceae, achenes, cypselas, schizocarps, mericarps, nutlets, one- and two-seeded pods of small-seeded legumes, seed balls, or portions thereof, in Beta spp. , f ru i t s with enclos ing calyx as in Tetragonia tetragonioides and bulblets as in Poa bulbosa. It also includes coated seed. Very often structures which do not strictly comply with the above definition are included in the pure seed because the analyst cannot tell whether or not the ripened ovule is present. 2.2 Pure Seed:

31

The pure seed shall include seed of the crop kind (or kinds) under analysis which must be named in labelling, and includes small, immature, shrivelled, cracked, insect damaged, diseased, sprouted, or otherwise injured seeds, provided that: In the case of pieces of seeds, any piece which i s la rger than one- hal f the or ig ina l s ize shal l be cons idered pure seed except that seeds of the Fabaceae and Brass i caceae with thei r seedcoats ent i re l y removed shal l be regarded as iner t matter . For separated coty ledons of seeds of the Fabaceae refer to def in i t i o n of Iner t Matter . b. Intact seed units (commonly found dispersal units such as achenes and similar fruits, schizocarps and mericarps with or without perianth and regardless of whether they contain a true seed) shall be considered pure seed unless it is readily apparent that no true seed is present.
a.

(The term "readily apparent" shall be interpreted to mean that the purity analyst should not use a diaphanoscope, stereoscopic microscope, hand lens, pressing or other special equipment or means to detect whether true seeds are present.) c. In the case of the florets and caryopses of Poaceae, pure seed shall consist of: i. Broken florets or free caryopses, provided they are larger than one-half the original size; ii. Entire florets and one-seeded spikelets with an obvious caryopsis containing endosperm,
32

as determined by the use of slight pressure or by examination over light; iii. Floret s of the crop genera Lol i um and Festuca in which the caryopsis is at least one-third the length of the palea as measured from the base of the rachilla; iv. In the case of the Festuca spp., Agropyron cristatum or Agropyron desertorum, attached sterile florets which do not extend to or beyond the tip of the fertile floret shall be left attached and considered part of the pure seed. The length of an awn shall be disregarded when determining the length of a sterile floret; v. Where the uniform blowing method is used, all material of the kind of seed under analysis which remains in the heavy portion after blowing according to the instructions for that kind of seed, not including: Broken florets or free caryopses which are one-half or less than one-half of the original size, Other crop seeds, Weed seeds, Heavy inert matter, In the case of Dactylis glomerata, one-fifth the weight of multiple florets (see section 3.7). vi. Florets with fungus bodies, such as ergot (Claviceps

33

purpurea) , ent i r e l y enclosed with in lemma and palea, and vii. Four- f i f t h s the weight of mult ip l e f l o re t s remain ing in the heavy port i on in the case of Dacty l i s glomerata .

2.3 Other Crop Seeds: Seeds of crop kinds listed in the Procedures and Direct i ons for Seed Cert i f i c a t i on System in Pakis andtan found as contaminants in a sample shal l be cons idered other crop seeds, except that certa in seeds and st ruc tures as descr ibed in the Defin i t i o ns here be cons idered iner t matter . 2.4 Weed Seeds:

Seeds of plants not listed as crop kinds in the Procedures and Direct i ons for Seed Cert i f i c a t i on System in shal Pakis l tan be cons idered weed seeds, except certa in seeds and st ruc tures as descr ibed in the Defin i t i o ns shal l be considere iner t matter . The class i f i c a t i on of seeds as noxious or othe weeds shal l be accord ing to Procedures the and Directions for Seed Certification System in Pakistan. Structures such as the bulblets of Poa bulbosa and Allium vineale and tubers of Cyperus esculentus shall be considered weed seeds. Stolons (including pieces) of various grass species capable of germination shall be treated as weeds (and not as inert matter). 2.5 Inert Matter from the Crop Kind(s) Under Analysis: a. Pieces of broken or damaged seeds: i. ii. One-half the original size or less; Seeds of Fabaceae and Brassicaceae with the seedcoats entirely removed;
34

Separated cotyledons of Fabaceae, irrespective of whether or not the radicle-plumule axis and/or more than half of the seed coat may be attached; iv. Structures in which it is readily apparent that no true seed is present. 2.6 Inert Matter from Weed Plants and Other Crop Plants Found as Contaminants: All broken seeds, florets or free caryopses which are half or less than one-half the original size, including structures which, by visual examination (including dissection and the use of the diaphanoscope) can be definitely demonstrated as falling within the categories listed below. Doubtful structures shall be classed as weed seeds or other crop seeds as the case may be. a. Seeds and structures of Poaceae 1. Florets or free caryopses, with more than one-half the radicle-plumule axis missing, 2. Glumes and empty florets when unattached to fertile florets, 3. Attached ster i l e f l o re t s and basal appendages which must be removed f rom the fer t i l e f l o re t s and considered part of the iner t matter except , in the fo l l ow ing cases: o Attached ster i l e f l o re t s and basal appendages are not removed f rom: Agrost i s spp. ; Alopecurus spp.; Arrhenatherum elatius; Dactylis glomerata; Phalaris arundinacea; Poa spp.; and o Attached sterile florets which do not extend to or beyond the tip of the fertile floret are not
35

iii.

removed from Festuca spp. ; Agropyron cristatum, or Agropyron desertorum. The length of an awn shall be disregarded when determining the length of a sterile floret. 4. Immature florets of Elytrigia repens with a caryopsis less than one-third the length of the palea, measured from the base of the rachilla, 5. Free undamaged caryopses of Agropyron spp. 2 mm or less in length, 6. Florets containing fungus bodies, such as ergot (Claviceps purpurea). b. Seeds of families other than Poaceae Seeds that are devoid of embryo. Seeds of Fabaceae, and Brassicaceae with the seed coat entirely removed. iii. Separated cotyledons of Fabaceae, irrespective of whether or not the radicle-plumule axis and/or more than half of the seed coat may be attached. iv. Empty fruits (seeds) such as occur in the following plant families: Asteraceae, Convolvulaceae, Cyperaceae, Polygonaceae, Solanaceae, etc. v. Seeds of Cuscuta spp. which are either fragile or ashy gray to creamy white in colour. vi. Seeds of Plantago lanceolata which are black, with no brown colour present, whether shrivelled or plump. The colour of questionable seeds should be determined under a magnification of approximately 10x with strong light. i. ii.

36

Dehul led Ambrosia spp. ( invo luc re and per icarp absent) . viii. Ind iv i dua l seeds Juncus of tenuis or other species Juncus of having seeds of a similar size shall be considered inert matter. Clusters or capsules of Juncus spp. shall be left intact, counted and included with the weed seeds. ix. Multiple structures, capsules, pods, heads, etc., are opened, the seeds are removed, and the non-seed material is placed with the inert matter, except as noted in part viii above for Juncus spp..
vii.

2.7 Inert Material other than Seeds: The following materials shall be classed as inert matterin all cases: a. Nematode galls, including galls enveloped by the lemma and palea of grass florets, b. Fungus bodies such as ergot (Claviceps purpurea) and other sclerotia, and smut balls except as defined in section 3.2.2 c.vi, c. Chaff, stems, leaves, stone cells, stones, sand, soil particles, dust, and any other material not seeds, and d. Seeds (crop and weed seeds) in which larvae occupy one-half or more of the seed unit. 3. General Principles The working sample is separated into three components (pure seed, other seeds and inert matter) in ISTA Scheme, into four four components (pure seed, other crop seeds, weed seeds and inert matter) in Canadian Scheme and into five components (pure seed, other varieties seeds, other
37

crop seeds, weed seeds and inert matter ) in Pakis tan i Scheme; and percentage of each part i s determined by weight . Al l species of seed and al l kinds of iner t matter present shal l be ident i f i ed as far as poss ib le and, i f requi r for report i ng , i t s percentage by weight shal l be determined. 4. Apparatus Aids such as transmitted light, sieves and blowers may be used in separating the component parts of the working sample.
5.

Procedure for separation of components

The working sample shall be separated into its component parts based on an examination of each particle in the sample, but in certain cases special procedures are obligatory, such as uni fo rm blowing or s iev ing . The separation of the pure seed must be on such a basis that i t can be made by vis ib l e seed character i s t i c s , mechanica l aids or using pressure without impai r i ng the capaci ty for germinat ion . After separation, each component part and any species of seed or seeds of other varieties or kind of inert matter for which a percentage is to be reported, shall be weighed in grams to the minimum number of decimal places necessary to calculate the percentage to one decimal place; as detailed below in Table-7.

Table-7: Minimum number of decimal places required for weighment of components Weight of Weigh the working sample and its Working Sample components to the following number (in grams) of decimal places

38

Less than 1 1 to 9.999 10 to 99.99 100 to 999.9 1000 or more

4 3 2 1 0

6. Calculation and Expression of Results The percentage by weight of each of the component parts shall be calculated to one decimal place. Percentages must be based on the sum of the weight of the components, not on the original weight of the working sample, but the sum of the weight of the components must be compared with the original weight as a check against loss of material or other error. If a duplicate analys i s i s made on two hal f working samples , the di f f e rence between the two must not exceed the to le rance between dupl i ca te analyses given in TableWhen two or more purity analyses of whole working samples are made, the results must be expressed as a weighted average percentage.

7. Reporting Results The results of a purity analysis shall be given to one decimal place and the percentage of al l components must tota l 100.

39

components of less than 0.05% shall be reported as Traces. If the result for a component is nil, this must be shown as 0.0 in the appropriate space. The Latin names of the species of pure seed, other crop seeds, weed seeds; and kinds of inert matter must be reported on the Analysis Certificate / Report. Percentage of a particular species of other crop seed or weed seed or kind of inert matter, at the request of the sender, must be shown on the Analysis Certificate / Report.

Table- 8. Tolerances for comparing percentage of purity analysis components, to determine i f two tests from same submitted sample are compatible.
Average of two working samples (%) 99.95-100.00 99.90-99.94 99.85-99.89 99.80-99.84 99.75-99.79 99.70-99.74 99.65-99.69 99.60-99.64 99.55-99.59 99.50-99.54 99.40-99.49 0.00-0.04 0.05-0.09 0.10-0.14 0.15-0.19 0.20-0.24 0.25-0.29 0.30-0.34 0.35-0.39 0.40-0.44 0.45-0.49 0.50-0.59 Tolerance of Nonchaffy seeds 0.14 0.23 0.28 0.33 0.36 0.39 0.43 0.46 0.48 0.51 0.54 Tolerance of Chaffy seeds 0.16 0.24 0.30 0.35 0.39 0.42 0.46 0.49 0.52 0.54 0.58

40

99.30-99.39 99.20-99.29 99.10-99.19 99.00-99.09 98.75-98.99 98.50-98.74 98.25-98.49 98.00-98.24 97.75-97.99 97.50-97.74 97.25-97.49 97.00-97.24 96.50-96.99 96.00-96.49 95.50-95.99 95.00-95.49 94.00-94.99 93.00-93.99 92.00-92.99 91.00-91.99 90.00-90.99 88.00-89.99 86.00-87.99 84.00-85.99 82.00-83.99 80.00-81.99 78.00-79.99 76.00-77.99 74.00-75.99

0.60-0.69 0.70-0.79 0.80-0.89 0.90-0.99 1.00-1.24 1.25-1.49 1.50-1.74 1.75-1.99 2.00-2.24 2.25-2.49 2.50-2.74 2.75-2.99 3.00-3.49 3.50-3.99 4.00-4.49 4.50-4.99 5.00-5.99 6.00-6.99 7.00-7.99 8.00-8.99 9.00-9.99 10.00-11.99 12.00-13.99 14.00-15.99 16.00-17.99 18.00-19.99 20.00-21.99 22.00-23.99 24.00-25.99

0.59 0.63 0.67 0.71 0.76 0.84 0.91 0.97 1.02 1.08 1.13 1.18 1.25 1.33 1.41 1.48 1.59 1.72 1.83 1.94 2.04 2.18 2.34 2.49 2.61 2.73 2.83 2.93 3.01

0.63 0.67 0.71 0.75 0.81 0.89 0.97 1.04 1.09 1.15 1.20 1.26 1.33 1.41 1.50 1.57 1.68 1.81 1.93 2.05 2.15 2.30 2.47 2.62 2.76 2.88 2.99 3.09 3.18

41

72.00-73.99 70.00-71.99 65.00-69.99 60.00-64.99 50.00-59.99

26.00-27.99 28.00-29.99 30.00-34.99 35.00-39.99 40.00-49.99

3.09 3.16 3.26 3.37 3.46

3.26 3.33 3.44 3.55 3.65

Taken from Table P11, 5% probability, Handbook of Tolerances (S.R. Miles, Proceedings of the International Seed Testing Association, Volume 28, Number 3).

Determination of Other Seeds by Number

Objective: The objective of the determination is to estimate the number of seeds of other species (for example objectionable weeds or noxious species) required under the notified standards or requested by the sender. Procedure: The determination is made by count and expressed as number of seeds found in the quantity examined. When seeds found cannot be identified with certainity to the species level, it is permitted to report the genus name only.

42

The working sample for determination by count shall be the whole submitted sample or equal to submitted sample if duplicate sample is drawn for the purpose. If a species designated by the sender is difficult to ident i f y , only a minimum one f i f t h of the prescr ibed working sample for counts need be examined for that part i cu l a r species . Calculation and Expression of Results: The result is expressed as the number of seeds belonging to each designated species or category found in actual quantity found. In addition number per unit weight (eg per kilogram) may be calculated. Reporting Results: The actual weight of seed examined, and the Latin name and number of seeds of each species sought and found in this weight, alongwith calculated count per unit weight, shall be reported on the Analysis Certificate / Report.

Verification of Species and Cultivars And Separation of Other Varieties

Objective: The objective is to to determine the the extent to which the submitted seed sample conform to the species (us ing methods not permiss ib l e in pur i t y test ) or cul t i va r cla imed and extent of other species or other cul t i va r contaminants . Precondition for the Test: The determination is valid only if the species or cultivar is stated by the sender of the sample and if an authentic standard sample the species or cultivar is available for
43

comparison. The characters compared may be morpholog ica l , physio log i ca l , cyto log i ca l or chemical . General Principles: The determination is carried out, depending on the species or cultivar in question, on (a) Seeds, (b) Seedlings or more mature plants grown in a laboratory, a glasshouse, a growth chamber or field plots. Normally seeds are compared with seeds from the authentic sample and seedlings and plants are compared with seedlings and plants at the same stage of development grown from the authentic sample contemporaneously, nearby and in identical environmental conditions. Exceptionally, depending on the certainity of the determination (eg ploidy), comparison with the authentic control sample is not necessary. In the case of species or cul t i va r s that are suf f i c i en t l y uni fo rm as to one or more diagnost i c characters (eg in sel f pol l i na ted species ) , a count i s made of the number of seeds, seedl ings or plants that are not genuine. I f the species or cul t i va r i s not suf f i c i en t l y uni fo rm (eg in cross - pol l i na ted species ) , a count i s made of any obvious of f - types and a genera l judgement i s expressed as to the genuineness of the sample under test . Apparatus and Facilities: A determination shall be made only if apparatus, equipment and other facilities are available for the character to be stud ied , and in genera l as fo l l ows ; a) In the laboratory apparatus and reagents for morphological, physiological and cytological

44

examinations, chemical tests and germination of seeds as appropriate. b) In glasshouse and growth chambers provision of controlled environmental conditions adequate to induce the development of diagnostic characters. c) In f i e l d plots cl imat i c , edaphic and cul tu ra l condi t i o to permit normal development of the diagnost i c characters and suf f i c i en t protect i on against pests and diseases . In all cases, the determination shall be made by a specialist familiar with the morphological, physiological or other characters of the species or cultivar in question. Weight of Submitted Samples: Table-9A: Minimum weight of Submitted Samples for Determinat ion of Species / Cult i va r s
Sr. No . 1 2 3 4 Species Laborator y Only (g) Field Plot & Lab (g) 2,000 1,000 500 250

Pisum, Phaseolus , Vic ia , Lup inus Zea, , Glyc ine and other species 1,000 with seeds of similar size Hordium, Avena, Triticum, Secale and other species with seeds of 500 similar size Beta and other spec ies with seeds of 250 s imi la r s ize All other genera with smaller 100 seeds

Examination of Seeds: 1. Working sample: Not less than 400 seeds, taken at random from a sub-sample drawn in accordance with the procedure followed for drawing the working sample. 2. Determinations:

45

a. For morpholog ica l

characters , the seeds shal l be examined with the aid of sui tab le magnify ing apparatus when necessaryBecause . of the s imi la r i t y in appearance among the species of Brass i caand Sinapis, all portions of the Brassica and Sinapis samples must be examined using a stereoscopic microscope, except that for samples of Sinapis alba, a microscope need not be used. b. For color characters , the seeds may be examined under fu l l dayl igh ort l i gh t of l imi ted spect rum, eg ul t rav i o l e t . c. For chemical characteristics, the seeds shall be treated with the appropriate reagent and the reaction of each seed noted. Examination of Seedlings:
1. Working sample: Not less than 400 seeds (or for

plo idy in i t i a l l y 100 with a fur ther 100 i f determinat ion inconc lus i ve ) , taken at random f rom a sub- sample drawn in accordance with the procedure fo l l owed for drawing the working sample. The seeds shal l be germinated in repl i ca tes of not more than 100, on an appropr ia te medium. When the seedl i ngs have reached a sui tab le stage of development , they are examined in whole or in part , with or without fur ther treatment . For a determinat ion of plo idy , root t ip or other t i s sue i s excised and processed for microscopic examinat ion .

2. Determinat ion :

Examination of Plants in Glasshouse or Growth Chamber: 1. Working sample: Sufficient seeds to produce not less than 100 plants, but this number may be reduced in the case of climbing or creeping species. The seeds shall be taken at random from a sub-sample drawn in

46

accordance with the procedure followed for drawing the working sample.
2. Determinat ion :

The seeds shal l be sown in sui tab le conta iners and mainta ined in the envi ronmental condi t i ons necessary for the development of diagnost i c characters . When the plants have reached a sui tab le stage of development, the cr i t i c a l characters shal l be observed on each plant and noted.

Examination of Plants in Field Plots: The submitted sample shall be sown (in whole or in part) as soon as pract i cab le after rec iept . Each sample shall be sown in at least two replicate plots. As insurance against failure the replicates should be situated in different fields or different parts of the same field. The plots may be of any convenient size that will provide enough plants for the determination to be the accuracy required. If the seed is sown in situ, it shall be sown in rows, mechanically if possible. Spacing between rows and between plants shall be sufficient to allow development of the characters to be examined. Both transplanting and thinning are possible source of error and the sowing rate shall be adjusted to produce approximately the same number of plants in the test and control plots. When absolutely necessary, thinning or transplanting of seedlings from elsewhere in the plot is permissible.

Observations shall be made during the whole growing per iod , but part i cu l a r l y at the stages for appearance of diagnost i c characters and deviat i ons f rom the contro l sample recorded. Plants recognizeab le as belonging to another cul t i va r or species or as aberrant (eg spel to i d wheat) shal l counted and recorded. Ei ther an actua l count or an est imate of the number of plants in the plot shal l be made at the t im of plant examinat ion .

47

Calculations and Expression of Results: When not more than 2,000 seeds, seedl ings or plants are examined, the number found to be not genuine i s computed as a percentages without decimals . I f more than 2,000 are examined, the number i s computed as a percentages with one decimal place. In determination of seedlings, the results are expressed as percentage of normal seedlings examined; and abnormal seedlings are excluded from the computation. In case of field plot examination, the number of plants of genuine species / cultivar, other species / cultivars and aberrants shall be calculated as percentages of total number of plants examined.

When characters are measured, the mean and other stat i s t i c s may be calcu la ted . Cult i va r s of cross - fer t i l i z i n g species often show var iab i l i t y of plant character i s t i c s to s a degree that i t i s di f f i c u l t to def ine accurate ly al l of f - ty in such cases, any calcu la t i ons of percentage impur i t i e s sha be supplemented by appropr ia te comments about the genuineness of the test sample. Reporting Results:

For laboratory, glasshouse or growth chamber tests, the number of seeds, seedlings or plants examined shall be stated and the results be reported as the percentage of extraneous seeds or seedlings. If none are found, the results shall be reported as follows: The laboratory (or glsshouse) examination for genuineness of th i s sample revealed noth ing to ind i ca te that the species and cul t i va r stated by the sender i s incor rec t .

48

In case of a fluorescence test of Lol i um, the resu l t s shal l reported as fo l l ows : Of seeds producing normal seedlings, % react positively and % react negatively to ultraviolet light.

be

The result of a field plot examination, whenever possible, shall be reported as percentage of each other species, other cul t i va r s or aberrants found. I f a sample i s found to be a cul t i va r other than that stated by the sender , th i s resu l t s be repor ted . I f the proport i on of plants of other cul t i va r s present in sample exceeds 15%, the repor t shal l state addi t i ona l l y that , The sample cons is t s of a mixture of di f f e ren t cul t i va r s . If none other cultivar found, the results shall be reported as follows: The field plot examination for genuineness of this sample revealed nothing to indicate that the species and cultivar stated by the sender is incorrect.

49

Determination of Seed Moisture Content

Definition: Moisture content of a sample is the loss in weight when it is dried without oxidation, decomposition or the loss of other volatile substances. Sample weight: 100 g for species that have to be ground and 50 g for all other species. When moisture meters are to be used for testing, a larger sample size may be necessary. Sample condit ion: Samples for moisture tests must be received in airtight, moisture proof, and undamaged containers, which must be filled with seed to ensure that as much air as possible has been excluded. The sample should not be used for purity or germination purposes due to the nature of the container and the sub-sampling method. Tests should commence as soon as possible after receipt and exposure of the seed to the ambient air should be kept to a minimum. When the above requirements have not been met, new samples must be requested. Procedure: Either of the following methods may be used for moisture test determinat ion . A. Constant Temperature Oven Method: The determination shall be started as soon as possible after reciept of the sample. For species that do not require
50

grinding, no more than two minutes may elapse from the time the sample is removed from the container in which it was received untill the working sample is enclosed in the drying container and weighed. Weighing: Weighing shall be in grams to three decimal places. Working sample: the determination shall be carried out in duplicate on two independently drawn working samples each of the following weight, depending on the diameter of the containers used: Less than 8 cm diameter: 4 to 5 g 8 cm diameter or larger: 10 g before the working sample is drawn the submitted sample shall be thoroughly mixed without exposure to the atmosphere and working sample should be prepared swiftly within 30 seconds to avoid exposure. Grinding: Larger seeds must be ground before drying unless their high oil content makes them difficult to grind or (particularly in seed of Linum with oi l of a high iod ine number) l i ab l e to gain in weight through oxidat i on . Grinding of fol lowing vegetable species is obligatory: Cit ru l l u s lanatus , Lathyrus spp. , Phaseolus spp. , Pisum sat i vum, Vic ia spp. Pre-drying: I f the species i s one for which gr ind ing i s necessary and the moisture content i s more than 17% (or 10% in the case of Glyc ine max and 13% in the case of Oryza sative), pre-drying is obligatory. Two sub-samples, each weighing at least 25g are placed in weighed containers and then dried to reduce moisture content to less than the limit. After pre-drying the sub-samples are reweighed in their containers to determine the loss in weight. Immediately
51

thereafter the two partly dried sub-samples are separately ground and subjected to following prescribed methods:

Vegetable species for which low constant temperature oven method shall be used: Allium spp., Brassica spp., Capsicum spp., Raphanus sat i vus and Solanum melongena Vegetable species for which high constant temperature oven method shall be used: Asparagus officinalis, Beta vulgaris, Citrullus lanatus, Cucumis spp. , Cucurbi ta spp. , Daucus carota , Lactuca sat i va , Lathyrus spp. , Lycopers i con lycopers i cum, Phaseolus spp. , Pisum sat i vum, Spinac ia oleracea, Vic ia spp. Low constant temperature oven method: The working sample must be evenly distributed over the surface of the container. Weigh the container and its cover before and after filling. Place the container rapidly, on top of its cover, in an oven maintained at a temperature of 1032 oC and dry for 171 hour. The drying period begins at the time the oven returns to the required temperature. At the end of the prescribed period cover the container and place in a desiccator to cool for 30-40 minutes. After cooling Weigh the container with its cover and contents. The relative humidity of the ambient air in the laboratory must be less than 70% when the determination is carried out. High constant temperature oven method: The procedure is same as described above except that the oven is maintained at a temperature of 130-133oC and dried
52

for four hours for Zea mays, two hours for other cereals and one hour for other species (including all vegetable crops). No condition of relative humidity in the laboratory.

Calculation of Results: The moisture content as a percentage by weight shall be calculated to one decimal place by means of the following formula: (M2 M3) X 100 (M2 M1) Where M1 is the weight in grams of the container and its cover, M2 is the weight in grams of the container and its cover and its contents before drying, and M3 is the weight in grams of the container and its cover and its contents after drying. If the material is pre-dried (first stage), the original moisture content of the sample is calculated to one decimal place by means of the following formula: S1 + S2 - S1 X S2 100 Where S1 is the moisture lost in in thhe first stage, and S2 is the moisture lost in in thhe first stage. Tolerance: Take as the result the arithmetic mean of the duplicate determinations carried out on a sample if the difference

53

between the two determinations does not exceed 0.2%. otherwise repeat the determination in duplicate. B. Electric Moisture Meter The electric moisture meter of var ious models can be used i f cal i b ra t i on i s cert i f i e d by a competant agency. Seed test i ng laborato r i es using the Model elect r i c moisture meter must have appropr ia te convers ion charts for the crop kind under test . In addi t i on , they must per iod i ca l l y compare tests with elect r i c moisture meter with constant temperature oven method for standard i za t i on purposes.

Determination of 1,000 Seed Weight

Definition: Weight of 1,000 seeds in grams calculated from the average of 8 replications of 100 seeds drawn manually at random from the working sample; or f rom count ing whole working samle by the machine. The results shall be expressed to the number of decimal places used in purity determinations. Procedure: Working sample: The working sample shall be the entire pure seed fraction of a purity analysis. Counting the entire working sample: Put the whole working sample through the machine, and read vthe number of seeds on the ind i ca to r . After count ing weigh the sample in grams to the same number of decimal places as in the pur i t y analys i s ; then calcu la te the weight of 1,000 seeds accord ing ly .

Counting replicates: From the working sample count out at random, by hand or with a germination counter, eight repl i ca tes , each of 100 seeds. Weigh each rep l i ca te in grams
54

to the same number of decimal places as in the purity analysis. Calculate the variance, standard deviation and coefficient of variation as follows: Variance = n(x2) - (x)2 n(n-1) where x = weight of each replicate in grams n = number of replicates = sum of ________ standard deviation (s) = variance coeffient of variation = (s/x) X 100 where x = mean weight of 100 seeds if the coefficient of variation does not exceed 6.0 for chaffy grass seeds, or 4.0 for other seeds, the results of the determination can be calculated; otherwise count and weigh a further 8 replicatesand calculate the standard deviation for the 16 replicates. Discard any replicate that diverge from the mean by more than twice the standard deviation so calculated.

55

Purity Procedures for Coated Seed

NOTE: Standards and labe l l i n g requi rements for coated seed are not yet not i f i ed under Seed the Act , 1976 . The catagory of the seed or seed mixture must be estab prior l i shed to coating. However, the percentage by weight of seed must appear on the label. Because of the nature of the coated seed product and the procedures for testing, it is not valid to compare percentage by weight values determined by testing de-coated seed, with the standards notified in the rules. Procedures for determining percentages by weight of coated seed are therefore not given here. If necessary for information purposes, the procedures for testing coated seed given in the current Rules for Testing Seeds, published by the Association of Official Seed Analysts (USA), should be used. The following procedure may be used to determine if
56

imported seed meets the minimum standards for foreign seeds. Definition Coated seed is a seed unit covered with any substance which changes the size, shape, or weight of the original seed. Seeds coated with ingredients such as, but not limited to, rhizobia, dyes, and pesticides are excluded. Obtaining the working sample The minimum submitted sample size for samples of coated units to be submitted for a foreign seed determination should be approximately 50,000 units. Due to variation in weight of coating materials, the size or weight of the working sample shall be determined separately for each lot. This weight shall be determined by weighing 100 completely coated units and calculating the weight of 25,000 units for the foreign seed determination. Use the methods described under purity analysis to obtain the working sample. Table-9B: Sample sizes of pelleted seeds in number of pellets

57

Sr. Determinations No. 1 2 3 4 5 6 Purity analysis (including verification of species) Weight determination Germination

Submitted samples not less than 7,500 7,500 7,500

Working Samples not less than 2500 Pure pellet fraction 400 7500 25000 2000

Determination of other seeds 10,000 Determination of other seeds (encrusted seeds and seed granules) Size grading 25,000 10,000

Table-9C: Sample sizes of seeds tapes Sr. Determinations No. 1 Verification of species 2 4 5 Germination 2,500 Purity Analysis (if required) Determination of other seeds 2,500 10,000 400 2,500 7,500 2,500 seeds 100 seeds Submitted Working samples not Samples less than not less than

58

Foreign seed determination To determine the number of foreign seeds (noxious weeds, other weeds and other crops) per unit weight, examine approximately 25,000 units which have been de-coated. Remove the coating material from the seed by washing with water or other solvents such as, but not limited to, vinegar (5% acetic acid) or dilute sodium hydroxide. Use of fine mesh sieves is recommended for this procedure and stirring or shaking the coated units may be necessary to obtain decoated seed. Spread on blotters or filter paper in a shallow container. Air dry overnight at room temperature.

59

The Germination Test

1. Introduction The object of testing for germination is to determine the maximum germination potential of the seed. Testing under field conditions is normally unsatisfactory, as the results cannot be repeated with reliability. Laboratory methods have, therefore, been evolved in which the external conditions are controlled to give the most regular, rapid and complete germination for the majority of samples of a particular species. The conditions have been standardized to enable the test results to be reproduced within limits as near as possible to those determined by random sample variation. This chapter provides the methods and procedures to be used for germination testing for the purpose of grading seed. 2. Methods to be used 2.1 Prescribed methods The laboratory methods prescribed in section 6.2 (Table- 10) shal l be used when germinat ion tests are to be used as the basis for grading seed underSeeds the Regulat i ons , except that modified methods may be used according to section 2.2. 2.2 Modified methods On those exceptional occasions when samples do not respond to the prescribed methods to give the germination potential, an appropriate modified method may be used as follows:

60

a. The analys t

b. c.

d. e.

must be certa in that the prescr i bed methods of Table- 10 do not produce resu l t s which tru l y ref l ec t the maximum germinat ion potent ia l of the sample or samples under test . The modified method must enable the analyst to report germination as defined in section 3.1, i.e. the condition of essential seedling structures must be assessed. The analyst must have reasonable grounds to expect that the method to be used is appropriate for the specific problem at hand and gives reproducible results. Published methods should be used wherever possible. The analyst must be competent in the use of the method. The method used must be clearly indicated on the Report of Analysis.

3. Definit ions 3.1 Seed germination In seed laboratory practice, germination is defined as the emergence and development from the seed embryo of those essential structures which, for the kind of seed under test, are indicative of the ability to produce a useful, mature plant under favorable field conditions. In a laboratory germination test, the plant-producing potential of a sample of seed is evaluated. 3.2 Normal seedlings Seedlings possessing the essential structures that are indicative of their ability to produce useful mature plants under favorable field conditions. Detailed seedling descr ip t i ons are given in sect i on 14. 3.3 Abnormal seedlings All seedlings which cannot be classified as normal seedlings. Detailed descriptions of abnormal seedlings are given in section 14.
61

3.4 Fresh seeds Seeds which have failed to germinate but have imbibed moisture and appear firm, fresh, and capable of germination at the end of the prescribed test period and under the prescribed test conditions. Such seeds may be viable but dormant. 3.5 Dormant seeds Viable seeds, other than hard seeds, which fail to germinate when provided the prescribed germination conditions. 3.6 Hard seeds Seeds which remain hard at the end of the prescribed test period because their impermeable seed coats prevent the absorption of water. See section 12.7. 3.7 Dead seeds Seeds which at the end of the test period are neither hard nor dormant nor have produced any part of a seedling. 4. Preparation of Seeds for Germination Tests 4.1 Source of seeds for germination

a. From the pure seed separation: For germination of grasses, seeds shall be taken, without discrimination, from the pure seed separated in the purity test. b. From the submit ted sample: For germinat ion of al l kinds other than those mentioned in sect i on 4.1.a . , seeds shal l be taken di rec t l y f rom the submit ted sample after carefu l mixing. The seeds for germinat ion shal l be taken without discr iminat i on except that they must be "pure seed" as def ined under pur i t y analys i s . The fo l l ow ing seed st ruc tu res are cons idered as seed uni t s and need not be separated for germinat ion : mult ip l e f l o re t s in oats , ent i r e spike le t s in emmer and spel t , an ent i re schizocarps of the Apiaceae.
62

4.2 Number of seeds for germination Individual kinds of seeds: Four hundred seeds of any kind may be planted in replicates of 100, 50 or 25 seeds as appropr ia te accord ing to s ize of seeds. 5. Germination Conditions 5.1 Planting of seeds Seeds should be adequately and uniformly spaced so that contact of adjacent seeds is avoided. Counting and planting may be done by hand, ensuring seed selection is random and in accordance with the pure seed definitions, or with planting aids provided these do not introduce a bias when the seed is selected. If a vacuum counter is used, the head must be held flat and completely covered with seed before the vacuum is turned on to avoid biased selection of seed. 5.2 Substrata and moisture All Substrata, containers and moistening agents must be non-phytotoxic. New shipments of Substrata must be tested for phytotoxicity according to section 5.6. The Substrata must have a pH value within the range 6.0 to 7.5 when moistened. The substratum must be moist enough to supply the needed moisture to the seeds at all times. Avoid supplying excessive moisture which will restrict aeration of the seeds. Except as provided for those kinds requiring high moisture levels, the substratum should never be so wet that a film of water is formed around the seeds. For most kinds of seeds, blotters or other paper Substrata should not be so wet that by pressing, a film of water forms around the finger. The initial quantity of water to be added will depend on the nature and dimensions of the substratum and also on the size and species of the seed to be tested. The optimum amount should be determined by experiment.

63

Subsequent watering should be avoided wherever possible as it is likely to increase variability between replicates and between tests. Since the rate of evaporation will depend on the relative humidity of the air, it is desirable to keep water in the germination chambers or to provide other means of supplying a relative humidity of approximately 95%. Germination tests should be inspected at frequent intervals to insure that an adequate moisture supply is available at all times.
a. Sand. Sand for

germinat ion tests should be pract i ca l l y f ree of organic matter , solub le sal t s , clay or f i ne s i l t , and should be composed of part i c l e s al l of which wi l l pass through a 2 mm round- holed s ieve, and not more than 25% wi l l pass through a 0.5 mm s ieve. Sand graded at 24- mesh i s sui tab le . To improve the waterhold ing capaci ty of the sand, vermicul i t e may be added. Sand i s used for the fo l l ow ing methods: 1. S ( in sand) . Seeds are placed on a leve l layer of moist sand and covered with a layer of uncompressed sand to a depth of 10 to 20 mm, depending on the s ize of the seed. The quant i t y of water added wi l l depend on the part i c l e s i ze of the sand, the character i s t i c s of the vermicu l i t e when added, and the character i s t i c s of the seeds to be planted. The opt imum amount of water to add should be determined by exper iment . As a guidel i ne , for 24- mesh sand with no vermicul i t e added, add 125 mL water per l i t r e of sand, or add water unt i l the sand can be formed in to a bal l when squeezed in the palm of the hand, the bal l breaking f ree ly when pressed between two f i ngers . 2. TS (top of sand).Seeds are pressed in to the sur face of the sand. No cover ing layer i s added. Moisture leve l s are def ined as fo l l ows : Standard: Two or three drops of water show when conta iner i s t ipped to 45 angle . Light : No f ree water shows when conta iner i s

64

tipped. Heavy: Small pool of water shows when container is tipped.

b. Paper. Blot te r s ,

towels , f i l t e r paper and cel l uco t ton must conform to the genera l speci f i c a t i ons for paper Substra ta as out l i ned in the current ISTA In te rnat i ona l Rules for Seed Test. ing For blot te r tests moisture leve l s are def ined as fo l l ows : Standard: Soak until saturated. Drain until begins to drip. Light: Soak until saturated. Drain thoroughly and press against a dry absorbent surface to remove excess moisture. Heavy: Soak until saturated. Do not drain. Paper is used in the following methods:
1. BB (between blotters). Blot te r s

of an appropr ia te s i ze for the crop kind under analys i s are used, the seeds being spaced on one hal f of the moist blot te r before the other hal f i s fo lded over . In the case of wheat, for example, blot te r s measuring 16x28 cm fo lded to 16x14 cm would be appropr ia te ) . 2. RB (raised blotters) . The blot te r s are prepared as for BB, but to improve aerat i on of the seeds, the top blot te r i s ra i sed f rom the seed by means of corks , via l tops , narrow st r i p s of blot te r , etc . 3. TB (top of blotters)Seeds . are placed on top of standard germinat ion blot te r s or f i l t e r paper . 4. RT (rol led towels).Seeds are evenly spaced on two sheets of standard weight (38 lb ) or a s ing le sheet of heavy weight (76 lb ) germinat ion paper towel , then covered with a s ing le sheet . The towels are ro l l ed and placed in an upr ight posi t i on . The paper towel l i ng should be moistened unt i l i t s

65

wet weight is about three times that of its dry weight. 5. PP (pleated paper). The seeds are placed in a pleated , accord ion- l i ke f i l t e r paper st r i p with 50 pleats , usual l y two to a pleat . This method may be used as an al te rnat i ve where any paper substra tum i s prescr i bed .

c. Soil . Samples may be retes ted in a soi l

mix to conf i rm tests made by other methods, for example test i ng samples which produced seedl ings showing symptoms of phytotox ic i t y when germinated on paper or in sand. Soi l should be a good qual i t y , organic soi l - less pott i ng mix. The soi l should have a pH value with in the range between 6.0 and 7.5. Water should be added unt i l the soi l can be formed in to a bal l when squeezed in the palm of the hand, the bal l breaking f ree ly when pressed between two f i ngers .

5.3 Water The water used to moisten the Substrata shall be reasonably free from organic or inorganic impurities. If the usual water supply is not satisfactory, distilled or de-ionized water may be used. The pH value should be within the range between 6.0 and 7.5. 5.4 Temperature Temperatures are prescribed in Table- 10 and should be determined at the leve l of the seeds on the substra tum. The temperature should be as uni fo rm as poss ib l e throughout the germinat ion or prechi l l chamber. Care should be taken that heat generated by the l i gh t source does not cause the temperature to exceed the temperature leve l prescr ibed . The temperature ind i ca ted should be regarded as a maximum and var ia t i on due to the apparatus should not be more than 2C.
66

Single numerals in the temperature column of Table-9 indicate that the seed should be germinated at a constant temperature. Two numerals separated by a dash indicate that the seed should be germinated with an alternation of temperatures, the test being held at the lower temperature for approximately 16 hours per day, and at the higher temperature for 8 hours per day. When testing seeds that are dormant, it is essential that the change-over of temperature be accomplished in one hour or less. If the tests are not given alternating temperatures over weekends and on public holidays, they may be held at the lower temperatures during such times. When prechilling (rechilling or midchilling) is required for breaking dormancy, a constant temperature of not less than 5C or more than 10C shall be selected, maintained and used as described in section 8.1. 5.5 Light

Seeds of most of the species in Table- 10 wi l l germinate ei ther in l i gh t or in darkness . However, i l l uminat i on i s genera l l y recommended, as better developed seedl ings are produced which are more easi l y evaluated. I f l i gh t i s known to be necessary or benef i c i a l to induce complete germinat ion of any given kind of seed, i t s use i s ind i ca ted in Table- 9 in the columns "Addi t i ona l Direct i ons" . Light should be provided by a cool white fluorescent source for 8 hours in every 24. Where the seeds are germinated at alternating temperatures, they should be illuminated during the high temperature period. Seeds for which light is prescribed in Table-9 should be germinated on top of the substratum, at an intensity of 750 to 1200 lux. Iluminance of seed for which light is not prescribed may be as low as 250 lux. 5.6 Phytotoxicity Testing

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A phytotoxicity test must be conducted to compare a substratum of unknown quality with one in stock of acceptable quality. For this test, seeds of certain species which are known to be sensitive to toxic substances in the substratum are used: e.g. Phleum pratense , Agrostis gigantea, Festuca rubra var. commutata, Allium cepa, Apium graveolens, Cichorium intybus and Lepidium sativum. At least two species must be included in the test. The evaluation is made by comparing the root development of the seedlings of the two species grown on the two sources of substratum. The evaluation should be made on or before the days specified in Table-9 for first count of the species used for the test, because symptoms due to toxic substances are more pronounced at an early stage of root growth. Such symptoms are shortened roots and sometimes discolored root tips, roots raised from the substratum, and root hairs "bunched". In grasses, coleoptiles may be flattened and shortened. 6. Germination Methods 6.1 Outline of germination methods The prescribed methods as given in Table-10 must be used. When alternate methods are indicated, one of them (any combination of substratum and temperature) may be used. If a sample does not respond satisfactorily to the method selected, it may be retested by one or more of the alternate methods or by a modified method as described in section 2.2. The sequence of alternate methods in Table-9 does not indicate any preference. Methods in the "General Requirements" column of Table-10 must be used. Methods in the "Fresh or Dormant Seeds" column may be used when dormant seed is known or suspected to be present. These are described in section 8 and may be applied to the original test, or to retests.

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When a kind of seed for which a method is not given in Table-10 is received for germination, the current ISTA In ternat i ona l Rules for Seed Test or ing AOSA Rules for Testing Seeds are to be consulted for an appropriate method. For kinds for which there is no published method, the method for a closely related species should be followed. The method used must be clearly indicated on the Report of Analysis.

The abbreviations used in Table-10 are defined as follows:

BB - Between blotters (see section 5.2.b.1) RB - Raised blotters (see section 5.2.b.2) TB - Top of blotters (see section 5.2.b.3) RT - Rolled towels (see section 5.2.b.4) PP - Pleated paper (see section 5.2.b.5) S - In sand (see section 5.2.a.1) TS - Top of sand (see section 5.2.a.2) KNO3 - Use solution of potassium nitrate instead of water. (see section 8.2) GA3 - Use solution of gibberellic acid instead of water. (see section 8.4) TZ - Tetrazolium (see section 7.7)

6.2 Table- 10. Germination Methods for Vegetable Crop Species

Kind of Seed Number Substrata of seeds BB; S; RT BB; S; RT BB; S; RT BB; TS; RT TS; BB; RT First Final Temperature Count Count (C) (days) (days) 20 20 20 20-30 20-30 7 7 7 7 7 14 14 14 21 21 Light Prechill Additional Directions General Requirements Lightly covered (sand test) Lightly covered (sand test) Light moisture; Lightly covered (sand test) Additional Directions Fresh/ Dormant Seeds

Allium cepa Onion Allium porrum Leek Allium schoenoprasum Chives Anethum graveolens Dill Anthriscus

69

cerefolium Chervil Anthyllis vulneraria Kidneyvetch

BB; RT

20

10

Apium graveolens Celery and Celeriac Asparagus officinalis Asparagus Beta vulgaris Field, sugar, and garden beet; mangel; swiss chard Brassica spp. Vegetable Brassicas, etc. Capsicum annuum Pepper Carum carvi Caraway Cicer arietinum Chickpea Cichorium endivia Endive

TB

15-25; 20

10

21

Light; water only Presoak 24 hours

Prechill

S; BB; RT

20-30

21

RB; BB; PP

20; 20-30

10

See sections 4.7.3 and 4.12.6.a.

Prechill

TB RB; TB; BB; RT TS S

15-25; 25 20-30 20-30 20-30; 25

4 7 7 4

10 14 14 7

Light Light moisture Light

Prechill Light; KNO3

TS; TB

20-30

10

Light; heavy moisture at first Light; heavy moisture at first

Soak 24 hrs. in H2O; prechill; KNO3 Soak 24 hrs. in H2O; prechill; KNO3; May retest at 30C

Cichorium intybus Chicory Citrullus lanatus var. citroides Citron

TS; TB

20-30

10

100

BB; S; RT

25; 20-30

10

Light moisture

Citrullus lanatus var. lanatus Watermelon Coriandrum sativum Coriander Coronilla varia Crownvetch Cucumis anguria Gherkin Cucumis melo Melon, muskmelon, cantaloupe Cucumis sativus Cucumber Cucurbita pepo, C. maxima, C. moschata Pumpkin, squash and vegetable marrow Cuminum cyminum Cumin

100

BB; S; RT BB; RT BB; RT

25; 20-30 15 20 25; 20-30

4 7 7 4

14 14 14 8

Light moisture

May retest at 30C

100

BB; S; RT

Light moisture

100

BB; S; RT

25; 20-30

10

Light moisture

100

BB; S; RT

25; 20-30

Light moisture

100

BB; S; RT

25; 20-30

Light moisture

BB; S; RT

20-30

Light moisture

70

Cynara scolymus Artichoke Daucus carota Carrot Foeniculum vulgare Fennel Hibiscus esculentus Okra Lactuca sativa Lettuce and celtuce Lathyrus sylvestris Flatpea Lepidium sativum Garden cress Lycopersicon lycopersicum Common tomato Nasturtium officinale Watercress Pastinaca sativa Parsnip

S; BB; RT BB; RT BB; RT S TB S; RT BB; RT BB; TB; RB; RT TB; BB; Cellucotton; Filter paper,

20-30 20-30 20-30 20-30 20; 15 15-25; 20 20 20-30

5 5 7 7

14 14 14 14 7

Lightly covered

Light

Prechill

14 4 4

28 10 14 Light; on saturated absorbent cotton (or 4 layers saturated filter paper) in covered Petri dishes. Light moisture Prechill; may retest at 10-30C Prechill Light; KNO3

20-30

14

BB; TS; RT

20-30

28

Alternate method for Parsnip

20-30; 15-25

10

28

Plant on top of sand, lightly cover seeds with sand, cover test Standard moisture with wet blotters. May prechill 4-5 days; may retest at 10-30C. May retest at 1030C

Petroselinum crispum Parsley Phaseolus coccineus Runner bean

TS; TB

15-25

10

28

100

S; RT

20; 25

10

Phaseolus lunatus Lima bean Phaseolus vulgaris Field and garden bean Physalis pubescens Husk tomato Pimpinella anisum Anise Pisum sativum Field and garden pea Raphanus sativus Radish Solanum melongena Eggplant Spinacia oleracea Spinach Tetragonia

100

S; RT S; RT

20; 25 25

10 7

Heavy moisture first few days. See section 4.7.4

TB TS; BB; RT S; RT BB; RT RB; TB BB; RT BB; RT BB; RT

20-30 20-30 20 20 20-30 15; 10 15

7 7

21 14 7

Light

KNO3

4 7 7 7

7 14 21 21 Light moisture Light moisture Soak fruits overLight, KNO3 Prechill On 21st day,

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tetragonioides New Zealand spinach Trigonella foenum- graecum Fenugreek Vicia faba ssp. faba var. faba Broad bean Vicia faba ssp. faba var. equina Horse bean Vicia faba ssp. minor var. minor Tick or faba bean Vicia pannonica Hungarian vetch Vicia sativa Spring or common vetch Vicia villosa Hairy vetch Vigna unguiculata Cowpea Zea mays Field, sweet and popcorn 100

night (16 hours), air dry 7 hours; plant in scrape fruits and very wet towels. Do test for 7 not re-water unless additional days. later counts exhibit drying out. TB 20-30 5 14 Light Light; See Sec. 4.7.5 Light; See Sec. 4.7.5 Light; See Sec. 4.7.5

20

10

14

Prechill

S; RT

20

10

14

S; RT S; BB; RT S; BB; RT S; BB; RT S; RT S; RT

20 20 20 20 20-30 25

6 5 5 5 5

14 10 10 10 8 7

* 400 seeds to be planted except where otherwise indicated. ** A light intensity of 1000 lux or more may be required to induce complete germination.

7. Special Germination Procedures 7.1 Beta spp. Wash for at least four hours in running water at a temperature of 20 to 25C. If a beet seed washer is not used, the seeds may be soaked for the same period in still water, using at least 250 mL water for each 100 seeds, which must be changed as follows: every 15 minutes for the first hour, then every 30 minutes for the remaining three hours. After soaking is completed, remove the seeds from the water and drain for at least 60 minutes on a dry absorbent surface at a maximum temperature of 25C. Plant on a substrate which has been thoroughly drained to remove all excess water (e.g. stand blotters on edge for at least 1/2 hr after soaking). For multigerm seed, frequent counts must be made (e.g. at

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3, 5, 7 and 10 days) in order to keep track of the seedlings and avoid miscounts. See section 4.12.6.a. 7.2 Phaseolus vulgaris, garden bean If hypocotyl collar rot is observed on seedlings, the sample involved may be retested using a 0.3 to 0.6 percent calcium nitrate solution to presoak the substratum. The solution is prepared by dissolving 3 g (for a 0.3% solution) to 6 g (for a 0.6% solution) of Ca(NO3)2 in 1 litre of water. 7.3 Vicia faba , broad bean The use of a 0.1% calcium nitrate solution in place of water to moisten the substratum prevents the blackening of seedlings. The solution is prepared by dissolving 1 g of Ca(NO3)2 in 1 litre of water. The seeds should be well spaced (e.g. 10 seeds in sand in a 12 X 12 X 4 cm container). The seed must be well covered, with as much sand below as above the seed. 7.4 Coated seeds Germination tests on coated seed units and on de-coated seed shall be conducted in accordance with methods in Table-10 (see definition of coated seed in section on sampling). Coated seeds shall be placed on the substratum in the condition in which they are received without rinsing, soaking, or any other pretreatment which will affect the coating material (except for mixtures, as noted below). Kinds for which soaking or washing is specified in Table-10 (e.g. Beta spp.) shall not be soaked or washed in the case of coated seed. Coated seed units in mixtures which are colour coded or can otherwise be separated by kinds shall be germinated as separate kinds without removing the coating material. Coated seed units in mixtures which cannot be separated by

73

kind without removing the coating material shall have the coating removed in a manner that will not affect the germination capacity of the seeds. The de-coated seeds shall be planted as separate kinds on the same day the coating material is removed. The moisture level of the substratum is important and may depend on the water-absorbing capacity of the coating material. A retest may be necessary before satisfactory germination of the sample is achieved. Symptoms of phytotoxicity may be evident when germinating coated seeds in paper Substrata. In such cases a comparative test or retest in sand or soil may be necessary. 7.5 Tetrazol ium testing

Methods for conducting tetrazolium tests on agricultural seeds are given in the current Handbook on Tetrazo l i um Test ing , publ i shed by the In ternat i ona l Seed Test ing Associa t i on and/or the AOSA Tetrazo l i um Test ing Handbook publ i shed by the Assoc ia t i on of Off i c i a l Seed Analysts . Tetrazo l i um test resu l t s may be used as the basis for grading of fa l l planted cerea l s , but must be conf i rmed by a standard germinat ion test . For Western wheatgrass , a tet razo l i um test i s to be conducted to est imate the percentage dormant seeds (see sect i on 7.2) . For other crop kinds , tet razo l i um test resu l t s are to be used for in fo rmat ion only , not as the basis for grading . Resul t s of tet razo l i um tests are to be reported under "Remarks" or in a sect ion designated for report i ng of tet razo l i um test resu l t s . 7.6 Use of fungicides For grading purposes, seed for germination tests shall not be treated with fungicide in the laboratory (except for peanut, see section 7.1). However, when it is considered necessary to retest because of seed-borne disease present in the sample, or if the results are going to be used for information and not for grading, a suitable fungicide may be applied. The results of the untreated tests are to be reported, with the
74

results of the treated tests being recorded in the 'remarks' section of the Report of Analysis. 8. Treatments for Promoting Germination of Dormant Seeds Except in the case of certain species of the Fabaceae (see section 12.7), dormant or hard seeds are not included when seed is graded in accordance with the germination standards of var ious catagor ies of the Seeds. Therefore , in order to est imate the germinat ion potent ia l of a sample when dormant seeds are present , appropr ia te dormancy- breaking methods must be used. When dormancy is suspected in a sample, or when hard or dormant seeds remain at the end of the test period, the test or retest should include one of the treatments indicated in the "Fresh or Dormant Seeds" column of Table-9 and described in this section. If a treatment is used but is not listed as a method for the kind under test in Table-9, then its use must be indicated on the Report of Analysis. 8.1 Prechilling

Prechilling is the exposure of imbibed seeds to low temperatures before being given the prescribed germination temperature. During the prechilling period, the seeds are held in an imbibed condition, in (or on) the regular germination substratum, at a constant temperature of not less than 5C or more than 10C and this temperature shall be maintained within 2 C. In Table- 10 prechi l l i n g i s ind i ca ted for those kinds of seeds for which exper ience has shown i t to be ef fec t i ve in overcoming dormancy; prechi l l i n g may be t r i ed , however, for other kinds of seeds in which dormancy i s evident . Rechi l l i n g or midchi l l i n g may be used i f many dormant seeds remain at the f i r s t or la te r counts . The prechilling period is usually three to five days, but may be more or less if deemed appropriate. Rechilling is usually

75

two days. The prechilling and rechilling periods are not included in the germination period specified in Table-10. 8.2 Potassium nitrate Instead of water, a 0.2% solution of potassium nitrate, prepared by dissolving 2 g KNO3 in 1 litre of water, is used to saturate the germination substratum at the beginning of the test. Water is used for subsequent moistening. If short roots occur (for example, as may occur in the Poa spp.), the strength of the solution may be reduced to 0.1%. 8.3 Predrying Predrying of seeds having relatively high moisture content is often effective in hastening after-ripening, especially in freshly-harvested cereals. This procedure is particularly useful in years having adverse conditions during the maturing and harvesting season. The period of predrying is not included in the germination period specified in Table-10.
a. Room temperature predrying.

For species where dormancy is naturally of short duration, it is often sufficient to store the sample in a dry place for a short period. Spread the seed in shallow layers in open containers at normal room conditions for a period of up to 7 days. b. Preheating. The replicates for germination are heated at a temperature not exceeding 35C with free air circulation for a period of up to seven days before they are placed under the prescribed germination conditions. In some cases it may be necessary to extend the preheating period. 8.4 Gibberellic acid The use of gibberellic acid (GA3) is listed only for lavender. However, for other crop kinds, occasionally the conventional dormancy breaking techniques are not completely effective

76

and gibberellic acid may be used. The use of gibberellic acid must be reported on the Report of Analysis. The germination substratum is moistened with a 500 ppm (0.05%) solution of GA3, prepared by dissolving 0.5g GA3 in 1 litre of water. When dormancy is weaker 200 ppm may be enough; when it is stronger up to 1000 ppm may be used. When a concentration higher than 800 ppm is required, the GA3 must be dissolved in a phosphate buffer solution. The buffer solution is prepared by dissolving 1.7799 g of Na2HPO4.2H2O and 1.3799 g of NaH2PO4.H2O in 1 litre of distilled water. 8.5 Light For dormant seeds, the light intensity should be approximately 750 to 1250 lux from cool white fluorescent lamps. The tests should be illuminated during at least eight hours in every 24-hour cycle and during the high temperature period when the seeds are germinated at alternating temperatures. Seeds for which light is prescribed should be germinated on top of the substratum. 9. Counts and Duration of Test 9.1 Counts Seedlings may be counted when they have reached a stage of growth at which all essential structures can be evaluated.
a. First counts. The approximate number of days from

planting to the first count is given in Table-9. This is a guideline and deviations are permissible, depending on the development of the seedlings, and whether or not any pretreatments were given. b. Intermediate counts. These may be made at the discretion of the analyst after the seedlings have reached a sufficient stage of growth for all essential structures to be evaluated. Intermediate counts should

77

be conducted if continued growth of seedlings would hamper evaluation at the final count. c. Final counts. The number of days to the f ina l count i s given in the "f i na l count" column of Table- 10. For permiss ib l e deviat i ons , see sect ions 9.2 and 9.3. 9.2 Early termination of tests Test may be terminated only when germination is completed and only clearly abnormal seedlings and clearly dead and decaying seeds remain. 9.3 Extension of tests A test may be extended under the following circumstances: a. If the test has been prechilled or rechilled, the test may be extended by an equivalent number of days (see section 8.1). b. If a test is planted on a day of the week which results in the prescribed final count falling on a weekend or public holiday, the test may be extended to the first working day following. c. If, at the prescribed final count, (i) the number of seeds found to have germinated equals or exceeds the number found on the previous count, indicating a late wave of germination, and ungerminated seeds remain, or (ii) there remains a number of fresh seeds or small seedlings which are difficult to evaluate (the test may be extended no more than one-half the number of days prescribed for final count to a maximum of 5 days). When tests have been extended this must be reported on the Report of Analysis. 10. Calculat ion and Reporting of Germination Results 10.1 Expression of results Results are expressed as percentage by number, calculated to the nearest whole number. The sum of calculation of the
78

percentage of normal and abnormal seedlings and ungerminated seeds must be 100. 10.2 Single tests When a single test is conducted and retesting is not required, the average of the replicates shall be reported as percent germination or, for kinds listed in section 12.7, percent germination plus hard seeds. 10.3 More than one test

When one or more retests are conducted for one of the reasons described in section 11, the result to be reported shall be in accordance with the directions of section 11. When one or more retests or concurrent tests are made by either the same method, or an alternate method listed in Table- 10, for a reason other than descr ibed in sect ion 11, the resu l t s of al l tests with in one to le rance range, as give Table- 12, shal l be averaged and reported as the percentage of germinat ion or germinat ion plus hard seeds (see sect i on 12.7) . When the resu l t s of such tests are not with in to le ran of each other , a th i rd test shal l be conducted and the average of compatib le resu l t s shal l be repor ted . I f the th i rd test fa l l s between the f i r s t two and i s compatib le with both the average of al l three shal l be repor ted . 10.4 Rounding a. The result of a germination test is calculated as the average of two or four 50 seed replicates, two or four 100 seed replicates (sub-replicates of 25 seeds are combined into 50 seed replicates). The percentage is calculated to the nearest whole number (0.5 is taken to the higher figure) except for values between 99.5% and 99.9% which should be dropped to 99%. The sum of the normal and abnormal seedlings and un-germinated seeds must be 100 percent. The percentage of abnormal seedlings, dead seeds, and hard seeds (see section 12.7) is calculated in the same way.
79

1. The percentage of normal seedlings is rounded to the nearest whole number, xx.5 is taken to the higher number (e.g. xx.0 to xx.49 is rounded down to xx; xx.5 and greater is rounded up to xx+1). The value for normal seedlings is not adjusted after this step. 2. Calculate the integer part of the remaining percentages, sum the values obtained. If the sum is 100, the rounding procedure ends, if not continue with the following steps. 3. For the percentage of abnormal seedlings, hard seeds (see section 12.7) and dead seeds: i. Find the value with the greatest decimal part among the remaining percentages and round this percentage to the upper whole number, keep this value as a final result, calculate the integer part of the remaining percentages. ii. Sum the values obtained. iii. If the sum is 100, the procedure ends. If not, continue with another step (i to iii). In case of equal decimal parts, the priority order is abnormal seedlings, hard seeds (see section 12.7) and dead seeds. 11. Retesting In the following cases the germination test shall be repeated using an appropriate method. Refer to section 13 for the tolerance tables and descriptions of how to use them.
a. Retest when dormancy i s

suspected using a dormancy breaking method (see sect ion 4.8 and Table- 9) . Report the best resu l t achieved. Where a dormancy breaking method was used to conduct the in i t i a l test and the same or another dormancy breaking method was used to conduct the retes t , in - to le rance resu l t s should be averaged. b. Retest when the results are not reliable because of phytotoxicity or spread of fungi or bacteria. Report the best result achieved.
80

c. Retest when there are a number of seedlings which are difficult to evaluate. Report the best result achieved. d. Retest when there is evidence of errors in test conditions, seedling evaluation or counting. Report the result of the retest. e. Where the results of a retest do not support the reason/assumption for the retest (e.g. dormancy, test conditions), in tolerance tests should be averaged. f. Retest when the range between rep l i ca tes exceeds the maximum to le ra ted range given in Table- 11. I f the second resu l t i s compatib le with the f i r s t (e .g . the di f f e rence does not exceed the to le rance ind i ca ted in Table- 12) the average of the two tests shal l be reported . I f the second resu l t i s not compatib le with th f i r s t , a th i rd test shal l be conducted. The average of compatib le resu l t s shal l be reported . I f the th i rd test fa l l s between the f i r s t two and i s compatib le with both, the average of al l three tests shal l be reported . 12. Evaluation of Tests 12.1 Seedling evaluation The seedling descriptions of section 14 shall be followed to determine the classification of seedlings as normal or abnormal. Seedlings which have reached a stage when all essential structures can be accurately assessed, and are normal, shall be removed from the test at the first and any subsequent intermediate counts. Badly decayed seedlings should be removed in order to reduce the risk of secondary infection, but abnormal seedlings with other defects should be left on the substratum until the final count. 12.2 Chemically injured seedlings Seeds that have been over-treated with or accidentally exposed to certain pesticides may produce stunted seedlings having short, thickened roots, hypocotyls, epicotyls or
81

coleoptiles. Such seedlings must be classified as "abnormal." If the number of seedlings classified as abnormal because of chemical damage could affect the grade of the sample, then a retest in soil must be conducted. 12.3 Frozen and immature cereals When a sample is characterized by seedlings on the borderline of abnormality as a result of frost damage, or by spindly seedlings from immature seeds, or by these two conditions together, a statement to this effect should be made on the Report of Analysis.

12.4 Seedlings infected with fungi or bacteria Seedlings which are affected by fungi or bacteria to the extent that essential structures are impaired, should be regarded as normal only when it is clearly evident that the parent seed is not the source of infection and it can be determined that all the essential structures are present. Samples difficult to assess due to the presence of fungi or bacteria should be retested, possibly with increased spacing or in sand or soil. Seeds which are obviously dead and mouldy and which may be a source of contamination of healthy seeds should be removed at each count and the number of such dead seeds should be recorded. The presence of disease should be noted on the Report of Analysis, but identification of the disease should only be made by someone with appropriate training. 12.5 Borderline seedlings These are seedlings which are on the borderline between normal and abnormal. If there is only one such seedling, count it as normal. If there is an even number, count half of

82

them as normal. If there is an odd number, count two out of three, three out of five, etc., as normal. 12.6 Two or more seedlings from one seed unit
a. Mult ip l e - seeded uni ts :

New Zealand spinach, Beta spp. , schizocarps of the Apiaceae, mult ip l e seeds of burnet , and seed uni t s of grasses Dactylis ( glomerata, Poa spp. oats, etc.) consisting of multiple florets, shall be regarded as having germinated if they produce one or more normal seedlings. Only one seedling per multiple unit is to be counted. b. Multiple embryos: The development of two or more embryos in one seed, a phenomenon known as polyembryony, occurs occasionally in many kinds of crop seeds and more frequently in a few (e.g. in Kentucky bluegrass). When such seeds produce at least one normal seedling, they are considered to have germinated and are included in the percentage of germination. 12.7 Hard seeds The percentage of hard seeds is to be recorded and added to the percent germination for all legumes listed in Grade Tables VIII, IX and X and for sainfoin, hairy vetch and common vetch of Grade Table II. If fresh seeds of these kinds, or seeds which have just started to germinate are present at the end of the prescribed germination period, remove all hard seeds (record their number) and continue the test no more than one-half the number of days prescribed for final count to a maximum five days. The additional normal seedlings shall be included in the percentage of germination. 13. Germination Tolerances In germination testing, tolerance is defined as the amount by which a result (test or replicate) may differ from another result or from a specification, without it being attributed to a
83

difference in seed quality. The tolerances given here are based on variation resulting from random sampling. The tolerance tables are used in accordance with the principles outlined in sections 10.3 and 11. For crop kinds for which hard seeds are included with percent germination (see section 12.7), tolerances are checked using the normal seedlings plus hard seeds percentage. 13.1 Use of Table- 11: Tolerated differences between repl icates This table gives the maximum range in germination percentage tolerable between replicates, allowing for random sampling variation only at 0.05 probability. A retest must be conducted when the range between replicates exceeds the tolerated range for the average percent germination (see section 11.e.). For tests not exceeding 200 seeds, when less than 100 seeds are planted to a replicate (e.g. 25 or 50-seed replicates), the 50-seed replicate column of Table 6 must be used for deciding whether a retest is necessary. Replicates of less than 50 seeds which were closest together in the germinator must be combined to form 50-seed replicates. Note: To use the 50- seed repl i ca te column, the resu l t s must be converted to percentages before determin ing the to le ra ted range. For tests of 400 seeds, replicates of less than 100 seeds which were closest together in the germinator must be combined to form 100-seed replicates. To find the maximum tolerated range calculate the average percentage of the replicates, to the nearest whole number. Locate the average in columns A or B of the table and read the maximum tolerated range opposite in the appropriate column for 200- or 400-seed tests.
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Example 1. A germination test using 4 x 50 seeds gave replicate counts of 35, 40, 38, and 32 normal seedlings. To determine whether the range between the lowest and the highest result is within the tolerated range for the average percent germination, each replicate result must be expressed as a percentage (i.e. 70, 80, 76 and 64%). The average germination is 73%. The 4 x 50 replicate column of Table 6 shows the maximum tolerated range at 73% to be 23%. The actual range between the highest (80%) and lowest (64%) replicate is 16%, which is within the tolerated range and no retest is therefore necessary. Example 2. A germination test of 2 x 100 seeds gave replicate results of 86 and 73 for an average of 80% (79.5% rounded). To find the tolerated range, enter Table 6 at 80%. The permitted difference in a 200-seed test is 11%; the actual range is 13%. As these results are not within tolerance, the sample must be retested. The retest gave a result of 85%, with all replicates within tolerance, for an average result of 83% (82.5% rounded). From Table-12, the permitted difference between tests is 7%. As the actual range is 5%, the tests are compatible and the average of the two (83%) is reported. Example 3. A germination test using 2 and 50 seeds gave replicate counts of 35 and 45 normal seedlings. To determine whether the range between the lowest and the highest result is within the tolerated range for the average percent germination, each replicate result must be expressed as a percentage (i.e. 70% and 90%). The average germination is 80%. The 2 x 50 replicate column of Table-11 shows the maximum tolerated range at 80% to be 16%. The actual range between the highest (90%) and the lowest (70%) replicate is 20%. The tests are not within tolerance and therefore a retest is necessary. The retest gave a result of 85%, with all
85

replicates within tolerance, for an average result of 83% (82.5% rounded). From Table-12, the permitted difference between tests is 7%; the actual range is 5%, therefore the tests are compatible and the average of the two (83%) is reported.

13.2 Table- 11. Maximumtolerated ranges in germination percentages of repl icates for deciding when to retest.
Average Percent Germination (%)
-A-B-

Number of 50 seeds replicates

2 -C4 -D-

Number of 100 seeds replicates

2 -E4 -F-

99 98 97 96 95 94 93 92 91 90 89 88 87 86 85 84 83 82 81 80

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

4 6 7 8 9 9 10 11 11 12 12 13 13 14 14 14 15 15 15 16

6 8 9 10 11 12 13 14 15 15 16 17 17 18 18 19 19 20 20 20

3 4 5 5 6 7 7 7 8 8 9 9 9 9 10 10 10 10 11 11

4 5 6 7 8 9 9 10 10 11 11 12 12 12 13 13 13 14 14 14

86

79 78 77 76 75 74 73 72 71 70 69 68 67 66 65 64 63 62 61 60 59 58 57 56 55 54 53 52 51

22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

16 16 16 17 17 17 17 17 18 18 18 18 18 18 18 19 19 19 19 19 19 19 19 19 19 19 19 19 19

21 21 22 22 22 22 23 23 23 23 24 24 24 24 24 24 25 25 25 25 25 25 25 25 25 25 25 25 25

11 11 11 12 12 12 12 12 12 12 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 14 14 14 14

15 15 15 15 16 16 16 16 16 16 17 17 17 17 17 17 17 17 17 18 18 18 18 18 18 18 18 18 18

13.3 Use of Table- 12: Tolerated differences between tests

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In accordance with the principles outlined in section 4.10.3, Table 7 indicates the maximum range in germination percentages tolerable between different tests on the same submitted sample, in the same laboratory, allowing for random sampling variation only at 0.05 probabilities. If the results of two tests are found to be compatible, the average of the tests is reported. To calculate the final reported result, the replicate results of all compatible tests are averaged. If the two tests are not compatible, conduct a third test. Report the average of the replicates of the compatible tests (see section 11.f.) Example 1 A germination test using 2 x 100 seeds gives a result of 75%. A retest on 2 x 100 seeds from the same sample gives a germination of 85%. The average of the two tests is 80%. Enter Table-12 at 80% under column E (200-seed tests); the maximum tolerated range is 8. Since the difference between the two tests exceeds this range, a third test must be conducted. If the third test gave a result of 82% it would be compatible with both the first test (average 79%, range 7, tolerance 8) and the second test (average 84%, range 3, tolerance 7), so the average of all three (81%) would be reported. If the third test gave a result of 87%, it would not be compatible with the first test (average 81%, range 12, tolerance 7) but would be compatible with the second test (average 86%, range 2, tolerance 7) and so the average of the second and third tests (86%) would be reported. See section 11.f. Example 2 A germination test using 2 x 50 or 4 x 25 seeds gives a result of 70%. A retest on 2 x 50 or 4 x 25 seeds from the same sample gives a germination of 80%. The average of the two tests is 75%. Enter Table 7 at 75% under column C (100-seed tests) the maximum tolerated range is 12. Since the difference between the two tests does not exceed this
88

range, the average of the tests are reported. To calculate the final reported result, the replicate results of all compatible tests are averaged (see section 11.f.).

13.4 Table- 12. Maximumtolerated differences in germination percentages of tests for deciding which tests to average.
Average Percent Germination (%)
-A-B-

Number of 100 seed tests

2 -C3 -D-

Number of 200 seed tests

2 -E3 -F4 -G-

Number of 400 seed tests

2 -H3 -I4 -J-

99 98 97 96 95 94 93 92 91 90 89 88 87 86 85 84 83 82 81 80 79 78

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

3 4 5 5 6 7 7 7 8 8 9 9 9 9 10 10 10 10 11 11 11 11

4 5 6 7 7 8 8 9 9 10 10 11 11 11 12 12 12 13 13 13 13 14

2 3 3 3 4 4 5 5 5 6 6 6 6 7 7 7 7 7 7 8 8 8

3 3 4 4 5 5 6 6 7 7 7 7 8 8 8 8 9 9 9 9 9 9

3 4 4 5 6 6 6 7 7 8 8 8 8 9 9 9 9 10 10 10 10 10

1 2 2 3 3 3 3 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5

2 2 3 3 3 4 4 4 5 5 5 5 5 6 6 6 6 6 6 6 6 7

2 3 3 3 4 4 4 5 5 5 5 6 6 6 6 6 7 7 7 7 7 7

89

77 76 75 74 73 72 71 70 69 68 67 66 65 64 63 62 61 60 59 58 57 56 55 54 53 52 51

24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

11 12 12 12 12 12 12 12 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 14 14 14 14

14 14 14 14 14 15 15 15 15 15 15 15 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16

8 8 8 8 8 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9

10 10 10 10 10 10 10 10 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11

11 11 11 11 11 11 11 11 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 13 13 13

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7

7 7 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8

7 8 8 8 8 8 8 8 8 8 8 8 8 8 8 9 9 9 9 9 9 9 9 9 9 9 9

Based on 5% probability levels calculated from Table G1, Handbook of Tolerances , S.R. Miles , Proc . In t . Seed Test . Assoc. Vol . 28, No.3, 1963.

14. Seedling Descript ions

90

The seedling descriptions as given in this section are taken from sections 4 through 26 of the Seedl ing Evaluat i on Handbook , publ i shed by the Associa t i on of Off i c i a l Seed Analysts (AOSA) in 1992. The descr ip t i ons here di f f e r f rom the AOSA Handbook in the following: some species have been added or deleted to correspond with the germination methods table (Table-10); the section numbers have been changed; literature references drawings have been deleted; references to the AOSA rules have been deleted or changed to references within this manual where appropriate; directions to report hard seeds in other than the smallseeded Fabaceae have been deleted; and references to the introductory chapters of the Handbook have been deleted. The descriptions and their intent remain unchanged except for the following:
a. Apiaceae : Seedlings of carrot (Daucus carota), celeriac

(Apium graveolens var. rapaceum) and parsnip (Pastinaca sativa) must possess a well-developed primary root (see section 14.21). b. Asteraceae : Seedlings of Lactuca sativa showing any degree of physiological necrosis of the cotyledons shall be classified as abnormal (see section 14.2). c. Brassicaceae : Seedlings of Brassica spp. showing the yellow cotyledon condition shall be classified as abnormal (see section 14.4). In general, the following are considered to be essential structures necessary for the continued development of the seedling (although some structures may not be visible in all species at the time of seedling evaluation):

root system, consisting of primary and/or secondary, seminal or adventitious roots hypocotyl epicotyl cotyledon(s) terminal bud primary leaves

91

Seedlings with defects to these structures, as described in the abnormal seedling descriptions, are judged to be incapable of continued growth. The detailed seedling descriptions are given in sections dealing with individual groups of vegetables. These descriptions assume that test conditions were adequate to allow proper assessment of the essential seedling structures. If it is suspected that the test conditions have contributed to seedling abnormalities or the spread of infection to the point where evaluation is difficult, the sample should be retested under more favourable conditions. The "General Description" for each group of crop kinds describes a seedling without defects. While such a seedling is clearly normal, seedlings with some defects may also be classified as normal, provided the defects do not impair the functioning of the structure. The "Abnormal Seedling Description" is to be followed when judging the severity of defects.

14.1 Aizoaceae (Carpetweed Family) Tetragonia tet ragonio ides , New Zealand spinach General Description Seedling type: Epigea l dicot . Food reserves: Leaf l i ke coty ledons and per i sperm.

Shoot system: The hypocoty l elongates carry ing the coty ledons above the soi l sur face . The epicoty l usual l y does not show any development with in the test per iod . Root system: A pr imary root ; secondary roots may develop with in the test per iod . Abnormal Seedling Description
92

Cotyledons

Less than hal f of the or ig ina l attached. Less than hal f of the or ig ina l necros i s or decay.

coty ledon t i s sue remain in coty ledon t i s sue f ree of

Epicotyl

Miss ing (may be assumed to be present i f are in tac t ) .

coty ledons

Hypocotyl

Deep open cracks extending in to the conduct ing t i s sue . Malformed: such as markedly shortened, cur led or th i ckened. Watery.

Root

None. Weak, stubby or miss ing pr imary root with weak secondary or advent i t i ous roots .

Seedling

One or more essent ia l st ruc tu res impai red as a resu l t decay f rom pr imary in fec t i on . Alb ino .

of

14.2 Asteraceae (Sunflower Family I - Lettuce) Lactuca sat i va , le t tuce General Description Seedling type: Epigea l dicot .

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Food reserves: Coty ledons which expand and become th in , lea f - l i ke and photosynthet i c . Some var ie t i e s develop elongated pet io l es at the base of the coty ledons .

Shoot system: The hypocoty l elongates and carr i es the coty ledons above the soi l sur face . The epicoty l usual l y does not show any development with in the test per iod . Root system: A long pr imary root . Abnormal Seedling Description Cotyledons

Less than hal f of the or ig ina l coty ledon t i s sue remain in attached. Less than hal f of the or ig ina l coty ledon t i s sue f ree of necros i s or decay (see notes 5 and 6) . With any degree of physio log i ca l necros i s (see notes 5 1 and 6) .

Epicotyl

Miss ing (may be assumed to be present i f are in tac t ) . Any degree of necros i s or decay.

coty ledons

Hypocotyl

Deep open cracks extending in to the conduct ing t i s sue . Severely twis ted or gra iny . Watery.

Root

None. Pr imary root t ip blunt , swol len and disco loured . Pr imary root with spl i t s or les i ons .

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Seedling

Swol len coty ledons assoc ia ted with extremely short or vest ig i a l hypocoty l and root . One or more essent ia l st ruc tu res impai red as a resu l t of decay f rom pr imary in fec t i on . Alb ino .

Notes 1. Toxic materials in the substrate will cause short, blunt roots. 2. Seedlings grown on top of white filter paper will be shorter than those on blue blotters. 3. Remove attached seed coats for seedling evaluation. 4. Seedlings with slight dormancy or light sensitivity may be slow to germinate. 5. Physio log i ca l necros i s i s manifes ted on le t tuce coty ledons by sof tened grey, brown, black or reddish areas appear ing adjacent to the midr ib and la te ra l veins . This must not be confused with natura l pigmentat ion of some var ie t i e s , or with insect damage. Physio log i ca l necros i s i s often accompanied by shortened hypocoty l s and roots , with the seed coat f requent ly remain ing attached to the coty ledons. Seedl ings showing any degree of physio log i ca l necros i s should be class i f i e d as abnormal . In "Remarks" ind i ca te the percentage of necrot i c seedl ings ( th i s percentage should inc lude necrot i c seedl ings which are also 2 otherwise abnormal) . 6. Seedlings with extensive physiological necrosis on the cotyledons may be slower in growth than those without such affected areas. Hypocotyl and root length may be affected by other factors such as proximity to light, delayed germination or dormancy.
1

The requi rement to be f ree of any degree of physio log i ca l necros i s di f f e r s f rom the AOSA descr ip t i on , in which only seedl ings with more than 50% of the coty ledons being necrot i c are considered abnormal .
95

Note 5 as i t appears here di f f e r s f rom Note 5 appear ing in the AOSA Seedl ing Evaluat i on Handbook.

14.3 Asteraceae (Sunflower Family) II - Kinds other than lettuce Carthamus t inc to r i u , ssaf f l ower Cichor ium endiv ia , endive Cichorium intybus, chicory Cynara cardunculus, cardoon Cynara scolymus, artichoke Helianthus annuus, sunflower Taraxacum officinale, dandelion Tragopogon porrifolius, salsify General Description Seedling type: Epigeal dicot. Food reserves: Cotyledons which expand and become thin, leaf-like and photosynthetic. Shoot system: The hypocotyl elongates and carries the cotyledons above the soil surface. The epicotyl usually does not show any development within the test period. Root system: A long primary root with secondary roots usually developing within the test period. Abnormal Seedling Description Cotyledons

Less than half of the original cotyledon tissue remaining attached. Less than half of the original cotyledon tissue free of necrosis or decay (see note 7).

Epicotyl
96

Miss ing (may be assumed to be present i f are in tac t ) .

coty ledons

Hypocotyl

Decayed at point of attachment. Deep open cracks extending in to the conduct ing t i s sue . Malformed: such as markedly shortened, cur led or th i ckened. Watery.

Root

None. Weak, stubby or miss ing pr imary root with weak secondary or advent i t i ous roots (see notes 1 and 5) .

Seedling

One or more essent ia l st ruc tu res impai red as a resu l t decay f rom pr imary in fec t i on . Alb ino .

of

Notes 1. Substrate with insufficient moisture may result in slow or abnormal development, bound roots, retarded secondary root development or unshed seed coats. 2. Some seed lots of sunflower will exhibit dormancy if the substrate is on the wet side. 3. Due to the thick, dry seed coat, imbibition may be slow and the subsequent germination erratic. Some seeds may just be starting at the end of the test period and it may be necessary to extend the test as allowed under section 4.9.3. 4. All seeds in this group may exhibit some dormancy and a retest using appropriate dormancy breaking procedures may be necessary. 5. Frequently the root may become bound within the hard seed coat. If left in the test until the final count such

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seedlings may develop secondary roots sufficient to be considered normal. Bound roots are usually not a problem in soil tests since the secondary root development is faster than in artificial media. 6. The hypocotyl may be slow to develop in seedlings with a damaged primary root. 7. Seedlings with unshed seed coats may have decayed cotyledons. The seed coat must be removed for evaluation. 8. For dormant samples of endive, add about 4 mm of water at the beginning of the test and remove excess water after 24 hours.

14.4 Brassicaceae (Mustard Family) Brass i ca spp. , mustards and cabbages etc Crambe . abyss in i ca , crambe Eruca sativa, roquette Lepidium sativum, garden cress Nasturtium officinale, watercress Raphanus sativus, radish Sinapis alba, white mustard General Description Seedling type: Epigeal dicot. Food reserves: Cotyledons which expand and become thin, leaf-like and photosynthetic. In Brassica, Sinapis and Raphanus, the cotyledons are bi-lobed and folded, with the outer cotyledon being larger than the inner. Shoot system: The hypocotyl elongates and carries the cotyledons above the soil surface; the epicotyl usually does not show any development within the test period. Root system: A long primary root. Abnormal Seedling Description

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Cotyledons

Decayed at point of attachment. Less than hal f of the or ig ina l coty ledon t i s sue remain in attached. Less than hal f of the or ig ina l coty ledon t i s sue f ree of necros i s or decay. In seedl ings Brass of i ca , one- hal f or more of the tota l coty ledon t i s sue yel l ow or white , with no green t in t (se 3 note 1) .

Epicotyl

Missing (may be assumed to be present if the cotyledons are intact).

Hypocotyl

Deep open cracks extending into the conducting tissue. Malformed: such as markedly shortened, curled or thickened. Watery.

Root

Weak, stubby or missing primary root (secondary roots will not compensate for a defective primary root).

Seedling

One or more essential structures impaired as a result of decay from primary infection. Albino.

Notes
1. Seedlings of Brass i cawith 50% or more of the total

cotyledonary tissue entirely yellow, with no green tint, are to be classified as abnormal. This requires that the germination test be conducted with light to allow chlorophyll production in the cotyledons. Seedlings
99

which have their seed coats attached at final count should not be considered to be abnormal due to yellow cotyledons. If it is apparent that the sample has a yellow cotyledon problem and there are a significant number of seedlings with their coats attached, then the test should be extended to allow shedding of the coats, or the sample should be retested using higher intensity light. In assessing seedlings with yellow cotyledons, analysts should make a rapid decision by scanning the replicate and removing obviously yellow seedlings, then continuing the evaluation without re-considering the first assessment of yellow. The assessment of yellow cotyledons may be aided by using a colour reference (e.g. the yellow cotyledons of a Brass i ca seedl i ng which has not been exposed to l i gh t ) . I f there i s any hint of green in the yel l ow area, then the seedl ing should not be class i f i ed as abnormal due to the yel l ow coty ledon condi t i on .
3

The evaluat i on of yel l ow coty ledons Brass in i ca di f f e r s f rom the AOSA descr ip t i on , which does not speci f i c a l l y descr ibe th i s condi t i on .

14.5 Chenopodiaceae

(Goosefoot Family)

Beta vulgar i , s beet , sugar beet , mangel , Swiss chard Spinacea oleracea , spinach General Description Seedling type: Epigeal dicot. Food reserves: Leaf-like cotyledons and perisperm. Shoot system: The hypocotyl elongates carrying the cotyledons above the soil surface. The epicotyl usually does not show any development within the test period.

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Root system: A pr imary root ; secondary roots may develop with in the test per iod . Abnormal Seedling Description Cotyledons

Less than hal f of the or ig ina l attached. Less than hal f of the or ig ina l necros i s or decay.

coty ledon t i s sue remain in coty ledon t i s sue f ree of

Epicotyl

Miss ing (may be assumed to be present i f are in tac t ) .

coty ledons

Hypocotyl

Deep open cracks extending in to the conduct ing t i s sue . Malformed: such as markedly shortened, cur led or th i ckened. Watery.

Root

None. Weak, stubby or miss ing pr imary root with weak secondary or advent i t i ous roots .

Seedling

One or more essent ia l st ruc tu res impai red as a resu l t of decay f rom pr imary in fec t i on ( fo r disco loured seedl i ngs of Beta spp. , see note 2) . Alb ino .

Notes
1. See sect ion 4.7.3 for

di rec t i ons to wash samples of Beta pr io r to plant ing . Chemical inh ib i t o r s in the clus te
101

or seed coat may work together with excess water to rob the embryo of oxygen and thus prevent germination. It is important, therefore, to ensure the seeds or clusters are dried before planting. 2. Toxic substances f rom the clus ter Beta s ofmay cause disco lour i ng of the hypocoty l and/or root . Seedl ings which are s l i gh t l y disco loured are to be class i f i e d as normal; however, i f there i s excess ive disco lo ra t i on , retes t in soi l or by washing in running water for 3 hour 3. Frequent counts must be made on multigerm beet since the growing seedlings will separate from the cluster making it difficult to identify its source. Any cluster which produces at least one normal seedling is classified as normal; only one normal seedling per cluster is to be counted.

14.6 Cucurbitaceae (Cucurbit Family) Cit ru l l u s lanatus var .ci t r o i des , citron Citrullus lanatus var. lanatus, watermelon Cucumis melo, muskmelon or cantaloupe Cucumis anguria, gherkin Cucumis sativus, cucumber Cucurbita spp., pumpkin and squash General Description Seedling type: Epigeal dicot. Food reserves: Cotyledons which are large and fleshy; they expand, become photosynthetic and are usually persistent beyond the seedling stage. Shoot system: The hypocotyl elongates and the cotyledons are pulled free of the seed coat, which often adheres to a peg-like appendage at the base of the hypocotyl. The epicotyl usually does not show any development within the test period.
102

Root system: A long pr imary root with numerous secondary roots . Abnormal Seedling Description

Cotyledons

Less than hal f of the or ig ina l coty ledon t i s sue remain in attached. Less than hal f of the or ig ina l coty ledon t i s sue f ree of necros i s or decay (see note 3) .

Epicotyl

Miss ing (can be assumed to be present i f coty ledons are in tac t ) .

the

Hypocotyl

Deep open cracks extending in to the conduct ing t i s sue . Malformed: such as markedly shortened, cur led or th i ckened (see note 2) .

Note: The short, thickened area (or "peg") between the roots and the hypocotyl is a normal development. Root

None. Weak, stubby or miss ing pr imary root , with less than two st rong secondary or advent i t i ous roots .

Seedling

One or more essent ia l st ruc tu res impai red as a resu l t decay f rom pr imary in fec t i on . Alb ino .

of

Notes
103

1. In general, seedling development is best when Substrata are kept on the dry side. Extra moisture may then need to be added at the time of the first count. 2. Samples should be retested in sand or soil if there is evidence of chemical injury (characterized by badly thickened and shortened hypocotyls and roots). Seedlings showing chemical injury symptoms in the retest are to be classified as abnormal. 3. Seedlings with unshed seed coats may have decayed or damaged cotyledons. The seed coat must be removed for evaluation of the cotyledons.

14.7 Fabaceae (Legume Family) I - Large-seeded epigeal, except soybean, peanut, lupine Phaseolus vulgar,i s garden bean and f i e l d bean Phaseolus lunatus , Lima bean Vigna radiata, mung bean Vigna unguiculata, cowpea Notes: For purposes of these rules, a garden bean (Phaseolus vulgaris) variety is defined as one which is grown for its fleshy pod to be eaten. Other beans, including field beans, are defined as those grown for their seeds to be eaten. Beans which are grown for either pod or seed to be eaten are to be considered garden beans, and the requirements for cotyledons apply (see abnormal seedling description). General Description Seedling type: Epigeal dicot. Food reserves: Cotyledons which are large and fleshy; some photosynthesis may occur, but this is a minor function. They shrivel and drop off when the food reserves are depleted.

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Shoot system: The hypocoty l elongates and carr i es the coty ledons above the soi l sur face . The epicoty l elongates causing the terminal bud to emerge f rom between the coty ledons; the pr imary leaves expand rap id l y . Root system: A long pr imary root with secondary roots . Abnormal Seedling Description Cotyledons

Garden bean P ( haseolus vulgar,i s in part ) : o Less than hal f of the or ig ina l coty ledon t i s sue remain ing attached. o Less than hal f of the or ig ina l coty ledon t i s sue f ree of necros i s or decay. Al l others : o Coty ledons are not assessed. Except ion : I f both coty ledons are miss ing and the seedl ing i s genera l l y weak, then the seedl i ng i s considered abnormal .

Epicotyl

Miss ing . Deep, open cracks . Malformed, such as markedly cur led or th ickened. Less than one pr imary lea f . Pr imary leaves too smal l in proport i on to the rest of th seedl ing , usual l y assoc ia ted with vis ib l e defects of , or damage to , the main stem of the epicoty l . Terminal bud miss ing or damaged (see note 8) .

Hypocotyl

Deep open cracks extending in to the conduct ing t i s sue (see notes 1 and 6) . Malformed: such as markedly shortened, cur led or th i ckened. (See also note 3) .

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Root

None. Weak, stubby or miss ing pr imary root with weak secondary or advent i t i ous roots (see note 7) .

Seedling

One or more essent ia l st ruc tu res impai red as the resu l t of decay f rom pr imary in fec t i on (but see note 8) . Alb ino .

Notes 1. Towels rolled too tightly may cause constriction of growing seedlings, resulting in malformation. Tight rolls, often in combination with a mid-test watering, may cause hypocotyl cracking or splitting. 2. Seeds of beans should be well spread out on or in the substrate. In general, the larger the seed, the more space it needs. Tightly concentrated seeds may compete to their detriment for water and for space to expand. 3. Hypocotyl collar rot is a breakdown in hypocotyl tissue characterized by "water-soaking" and collapse of the hypocotyl below the cotyledonary node. The lesion area later becomes discoloured, shrivelled and necrotic. The condition is recognized as a laboratory phenomenon caused by insufficient calcium available to the seedling. If hypocotyl collar rot is observed on seedlings of garden beans, the sample involved shall be retested using a 0.3 to 0.6 % calcium nitrate solution to presoak the substratum (see section 4.7.4). 4. (Note 4 of the AOSA Seedl ing Evaluat i on Handbook deleted . ) 5. (Note 5 of the AOSA Seedl ing Evaluat i on Handbook deleted . ) 6. A healed break in the hypocotyl, sometimes referred to as a "knee," is to be considered an allowable defect.

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7. A seedling with the root bound within a tough seed coat is to be considered normal. 8. If a few seedlings with total or partial decay to the epicotyl are found, they may be classified as normal, provided the hypocotyl and root are normal. The epicotyl on such seedlings usually does not decay when grown in a fairly dry environment and is exposed to light. Retests, preferably in soil or sand, will aid in interpretation of such seedlings. 9. Large-seeded legumes are especially susceptible to threshing or combine damage. Seed which has been mechanically damaged may produce seedlings with damaged primary roots, hypocotyls or epicotyls, or broken or detached cotyledons. Bruised areas are usually necrotic or decayed. Damage at the point of attachment of the cotyledons may be difficult to evaluate if seedlings are removed too early in the test period. 14.8 Fabaceae (Legume Family) II - Large-seeded hypogeal Cicer ar ie t i num , chickpea Lathyrus sylvest , r flat i s pea Lens culinaris, lentil Phaseolus coccineus, scarlet runner bean Pisum sativum, pea (field or garden) Vicia faba, horse bean or broadbean Vicia spp., vetch Vigna angularis, adzuki bean General Description Seedling type: Hypogeal dicot. Food reserves: Cotyledons which are large and fleshy, and remain enclosed within the seed coat beneath the soil surface. They are usually not photosynthetic.

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Shoot system: The epicoty l elongates and carr i es the terminal bud and pr imary leaves above the soi l sur face . The stem bears one or more scale leaves and, pr io r to emergence, i s arched near the apex, causing the terminal bud to be pul l ed through the soi l ; after emergence, the stem st ra ightens . For pract i ca l purposes the hypocoty l i s not discern ib l e and i s not an evaluat i on factor . There are buds the axi l s of each coty ledon and scale lea f but these usual l y remain dormant unless the termina l bud i s ser ious l y damaged. In th i s case, one or more auxi l i a r y buds wi l l star t to develop, forming a secondary epicoty l . Root system: A long pr imary root with secondary roots . Abnormal Seedling Description Cotyledons

Less than hal f of the or ig ina l attached (see notes 6 and 7) . Less than hal f of the or ig ina l necros i s or decay.

coty ledon t i s sue remain in coty ledon t i s sue f ree of

Epicotyl

Miss ing . Less than one pr imary lea f . Malformed stem such as markedly shortened, cur led , or th i ckened. Severely damaged (e .g . termina l bud miss ing or damaged) with only a weak secondary epicoty l develop ing f rom the axi l of a coty ledon or scale lea f . Two weak epicoty l s . Deep, open cracks extending in to the conduct ing t i s sue .

Root

None.

108

Weak, stubby or miss ing pr imary root with weak secondary roots .

Seedling

One or more essent ia l st ruc tu res impai red as a resu l t decay f rom pr imary in fec t i on . Alb ino .

of

Notes 1. There is a greater likelihood of hard seed expression when the substrate does not provide adequate moisture to the seeds throughout the test period. 2. Insufficient moisture will result in apparently disproportionate elongation of the primary root and slow development of the epicotyl. 3. (Note 3 of AOSA Seedl ing Evaluat i on Handbook deleted . ) 4. (Note 4 of AOSA Seedl ing Evaluat i on Handbook deleted . ) 5. Manganese deficiency at the time of seed development may cause a condition known as "marsh spot," characterized by a discoloured brown indentation in the center of the inner surfaces of the cotyledons. Seedlings with this condition are considered normal, provided they are otherwise normal. If the condition causes difficulty in evaluation, then the sample should be retested in soil. 6. Weevil infestation may prevent the development of a normal seedling. Sometimes the cotyledons have been devoured to the extent that no food supply is left for the developing seedling. Such injury can be easily detected by examining the cotyledons. 7. Large-seeded legumes are especially susceptible to threshing or combine damage. Seed which has been mechanically damaged may produce seedlings with damaged primary roots, hypocotyls or epicotyls, or broken or detached cotyledons. Bruised areas are usually necrotic or decayed. Damage at the point of
109

attachment of the cotyledons may be difficult to evaluate if seedlings are removed too early in the test period. 14.9 Fabaceae (Legume Family) III - Small-seeded Anthyl l i s vulnerar , ikidneyvetch a Astraga lus cicer , cicer milkvetch Coronilla varia, crownvetch Lespedeza spp., lespedeza Lotus spp., trefoil Medicago spp., alfalfa, black medick Melilotus spp., sweet clover Onobrychis viciifolia, sainfoin Trifolium spp., clover Trigonella foenum-graecum, fenugreek General Description Seedling type: Epigeal dicot. Food reserves: Cotyledons, which are small and fleshy; they expand, and become photosynthetic. Shoot system: The hypocotyl elongates and carries the cotyledons above the soil surface. The epicotyl usually does not show any development within the test period. Root system: A long tapering primary root, usually with roots hairs. Most of the included species do not normally develop secondary roots within the test period. Abnormal Seedling Description Cotyledons

Less than half of the original cotyledon tissue remaining attached (see note 2). Less than half of the original cotyledon tissue free of necrosis or decay.

110

Epicotyl

Miss ing (may be assumed to be present i f are in tac t ) .

coty ledons

Hypocotyl

Deep open cracks extending in to the conduct ing t i s sue . Malformed: such as markedly shortened, cur led or th i ckened. Watery.

Root

None. Pr imary root stubby ( fo r sweet clover and crownvetch, or for roots bound by the seed coat see note 1) . Spl i t extending in to the hypocoty l .

Seedling

One or more essent ia l st ruc tu res impai red as a resu l t decay f rom pr imary in fec t i on . Alb ino .

of

Notes 1. Stubby roots when germinated on artificial media: a. Sweet clover - The roots of sweet clover may be stubby due to the presence of coumarin in the seed. Since this condition usually does not occur in soil, such seedlings are to be classified as normal. b. Bound by coat - Roots may appear stubby as a result of being bound by the seed coat. Such seedlings are to be classified as normal. c. Crownvetch - Produces phytotoxic effects similar to sweet clover. 2. Breaks at the point of attachment of the cotyledons to the hypocotyl are common in seeds which have been mechanically damaged. It is important that seedlings not be removed during preliminary counts unless
111

development is sufficient to allow the condition of the cotyledons to be determined. If the point of attachment of the cotyledons cannot be seen at the end of the test, the seed coat should be peeled back to determine whether a break has occurred. 3. Mechanical breakage of the seed may result in only vestiges of seedlings with swollen cotyledons and broken, slightly enlarged hypocotyls or radicles. Insect damage may also cause lack of seedling growth. 4. Seedlings of sainfoin which have been "strangled" by growing through the netting of the pod but which are otherwise normal are to be classified as normal. 5. The percentage of hard seeds must be determined at the end of the test period for all genera in this group. Swollen seeds which fail to germinate by the end of the test should be allowed additional days as provided in sections 4.9.3 and 4.12.7. Swollen seeds are an indication of dormancy and can be induced by incorrect temperatures.

14.10 Liliaceae (Lily Family) I - Asparagus Asparagus of f i c i na , l i asparagus s General Description Seedling type: Hypogeal monocot. Food reserves: Endosperm which i s hard, semi- t ransparent and non- starchy ; minor reserves in the coty ledon. The endosperm surrounds the ent i re embryo. Cotyledon: A s ing le cyl i nd r i ca l coty ledon; fo l l ow ing germinat ion al l but the basal end remains embedded in the endosperm to absorb nutr i en ts . Shoot system: The epicoty l elongates and carr i es the terminal bud and pr imary leaves above the soi l sur face . The

112

epicotyl may bear several small scale leaves. A short hypocotyl is barely distinguishable joining the root to the basal end of the cotyledon, which emerges from the seed. Root system: A long s lender pr imary root .

Abnormal Seedling Description Cotyledon

Detached f rom seedl i ng .

Epicotyl

Miss ing . Terminal bud miss ing or damaged. Deep, open cracks . Malformed, such as markedly shortened, cur led , or th i ckened. Spind ly . Watery. (See also note 1)

Hypocotyl

Not evaluated.

Root

No pr imary root . Stubby pr imary root , with weak secondary roots (but see note 3 for ornamental asparagus) .

Seedling

One or more essent ia l st ruc tu res impai red as a resu l t decay f rom pr imary in fec t i on . Alb ino .

of

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Notes 1. Several epicotyls may arise simultaneously and may be considered normal if at least one appears to be vigorous and has a terminal growing point. 2. Some seeds do not conta in an embryo. (Remainder of note 2 of AOSA Seedl ing Evaluat i on Handbook deleted . ) 3. Ornamental asparagus Asparagus ( setaceusand A. densiflorus) has a thickened primary root, in contrast to the long, slender root of the garden asparagus (A. officinalis). 14.11 Liliaceae (Lily Family) II - Onion, leek and chives Allium cepa, onion Allium porrum, leek Allium schoenoprasum, chives

General Description Seedling type: Epigeal monocot. Food reserves: Endosperm which is hard, semi-transparent and non-starchy; minor reserves in the cotyledon. Cotyledon: A single cylindrical cotyledon; following germination the tip remains embedded in the endosperm to absorb nutrients. Shoot system: The cotyledon emerges with the seed coat and endosperm attached to the tip. A sharp bend known as the "knee" forms; continued elongation of the cotyledon on each side of this knee pushes it above the soil surface. The cotyledon tip is pulled from the soil and straightens except for a slight kink which remains at the site of the knee. The first foliage leaf emerges through a slit near the base of the cotyledon, but this does not usually occur during the test period. The hypocotyl is a very short transitional zone between the primary root and the cotyledon.
114

Root system: A long s lender pr imary root with advent i t i ous roots develop ing f rom the hypocoty l . The pr imary root does not develop secondary roots . Abnormal Seedling Description Cotyledon

Short and th ick . Without a def in i t e bend or "knee." Spind ly or watery .

Epicotyl

Not observed dur ing the test per iod .

Hypocotyl

Not evaluated.

Root

No pr imary root . Short , weak or stubby pr imary root .

Seedling

One or more essent ia l st ruc tu res impai red as a resu l t decay f rom pr imary in fec t i on . Alb ino .

of

Notes 1. Excess moisture may cause a delay in germination causing some seed lots to appear dormant. 2. Blotter or towel tests of onion are commonly overcome with fungus. To reduce this problem on a retest, seeds should be spaced farther apart.

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14.12 Malvaceae (Mallow Family) Abelmoscus esculentus , okra General Description Seedling type: Epigea l dicot . Food reserves: Coty ledons, which are much convoluted in the seed; they expand and become th in , lea f - l i ke and photosynthet i c .

Shoot system: The hypocoty l elongates carry ing the coty ledons above the soi l sur face . The epicoty l usual l y does not show any development with in the test per iod . Root system: A pr imary root , with secondary roots usual l y develop ing with in the test per iod . Abnormal Seedling Description Cotyledons

Less than hal f of the or ig ina l attached. Less than hal f of the or ig ina l necros i s or decay.

coty ledon t i s sue remain in coty ledon t i s sue i s f ree

Epicotyl

Miss ing (may be assumed to be present i f are in tac t ) .

coty ledons

Hypocotyl

Deep open cracks or gra iny les i ons extending in to the conduct ing t i s sue . Malformed: such as markedly shortened, cur led or th i ckened.
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Root

None. Weak, stubby or miss ing pr imary root with weak secondary or advent i t i ous roots .

Seedling

One or more essent ia l st ruc tu res impai red as a resu l t decay f rom pr imary in fec t i on . Alb ino .

of

Notes (Notes 1, 2 and 3 of AOSA Seedl ing Evaluat i on Handbook deleted . )

14.13 Miscel laneous Agricultural and Horticultural Apiaceae (Carro t Family ) - anise , caraway, carro t , celery , celer i ac , cherv i l , cor iander , cumin, di l l , fennel , pars ley , parsn ip Boraginaceae(Borage Family) - borage Lamiaceae (Mint Family) - balm, catnip, horehound, hyssop, lavender, rosemary, sage, summer savory, basil, sweet marjoram, thyme Rosaceae (Rose Family) - burnet Solanaceae (Nightshade Family) - eggplant, tomato, husk tomato, pepper, tobacco Valerianaceae (Valerian Family) - cornsalad General Description Seedlings are considered normal if they possess those essential structures that are indicative of their ability to produce a plant under favorable conditions. Abnormal Seedling Description

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Cotyledons

Less than hal f of the or ig ina l attached. Less than hal f of the or ig ina l necros i s or decay.

coty ledon t i s sue remain in coty ledon t i s sue f ree of

Epicotyl

Miss ing (may be assumed to be present i f coty ledons are in tac t ) .

the

Hypocotyl

Malformed: such as markedly shortened, cur led or th i ckened. Deep open cracks extending in to the conduct ing t i s sue . Watery.

Root

None. In kinds other than carrot, celeriac and parsnip: miss ing or stubby pr imary root with weak secondary or advent i t i ous roots . In carrot, celeriac and parsnip: miss ing or stubby pr imary root (even i f secondary roots are present ) .

Biochemical tests for viability

Objectives: The objectives of biochemical tests are :

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1. To make a quick estimate of the viability of seed samples in general and those showing dormancy in particular. 2. In the case of part i cu l a r samples which at the end of germinat ion test reveal a high percentage of dormant seeds, to determine the viab i l i t y of ind iv i dua l dorman seeds or the viab i l i t y of a working sample. Principle: In the topographical tetrazolium test a colorless solution of 2,3,5-tripheny tetrazolium chloride or bromide is used as an indicator to reveal the reduction processes which take place within living cells. The indicator is imbibed by the seed. Within the seed tissues it interacts with the reduction processes of living cells and accepts hydrogen from the dehydrogenases. By hydrogenation of the 2,3,5-triphenyl tetrazolium chloride a red, stable and non-diffusible substance, triphenlyl formazan, is produced in living cells. This makes it possible to distinguish the red-colored living parts of seeds from the colorless dead ones. In addition to completely stained viable seeds and completely unstained non-viable seeds, partially stained seeds may occur. Varying proportions of necrotic tissue are found in different zones of these partially stained seeds. The position and size of the necrotic areas in the embryo and / or endosperm / gametophyte tissue, and not necessarily the intensity of color, determine whether such seeds are classified as viable or non-viable. However, color differences along with tissu3e soundness are to be considered decisive mainly to the extent that they permit recognition and location of sound, weak or dead tissue. Reagent: An aqueous solution of 0.1% of tetrazolium chloride or bromide is used. If the pH of the distilled water available

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does not permit a solution within the range of 6.5-7.5 the solution shall be buffered as described in the Annex. Procedure: Working sample A full test shall be carried out on four replicates of 100 seeds drawn at random from the pure seed fraction of a purity test or on individual seeds that are found dormant at the end of a germination test. Preparation and treatment of the seed The seed shall be prepared in order to facilitate penetration of the tetrazolium solution. The prepared seeds or embryos are then completely immersed in the tetrazolium solution at the temperature and for the period described in the chapter on vegetable crops. At the end of this period the solution is decanted and the seed rinsed with water and examined. At the examination each seed is evaluated as viable or nonviable on the basis of the staining patterns and tissue soundness revealed. Specific directions for preparation, treatment and evaluation of each approved species are given in the chapter on vegetable crops. Calculation and Expression of Results: In testing a sample, the number of seeds considered viable is determined in each replicate. Maximum tolerated ranges for replicate differences are the same as for germination tests (Table-11, 12). The average percentage is calculated to the nearest whole number and reported. Reporting Results:

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The result is reported on the Analysis Report under Other Determinations in the following from: Tetrazolim test:.% of seed are viable. In the case of species of the Fabaceae the following shall be reported additionally: percentage of hard seeds found in the test. Percentage of hard seeds included in the reported percentage of viable seeds. Further details may be given at the discretion of the testing station, eg percentage empty, with larvae, broken or decayed seeds.

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Seed Health Testing

Objective: The objective of a seeds health test is to determine the health status of a seed sample, and by inference that of the seed lot, thus gaining information that can be used to compare the value of different seed lots. Health testing of seed is important for three reasons: 1. Seed-borne inoculums may give rise to progressive disease development in the filed and reduce the commercial value of the crop. 2. Imported seed lots may introduce diseases into new regions. Test to meet quarantine requirements may therefore be necessary. 3. Seed heal th test i ng may eluc idate seedl ing evaluat i on and causes of poor germinat ion or f i e l d estab l i shment and thus supplement germinat ion test i ng . Definitions: Seed health Health of seed refers primarily to the presence or absence of diseases-causing organisms, such as fungi, bacteria and viruses and animal pests, such as eelworms and insects, but physiological conditions such as trace element deficiency may be involved. Incubation

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Maintaining seeds in an environment favorable to the development of pathogens or symptoms. Pretreatment Any physical or chemicals laboratory treatment of the working sample preceding incubation, given solely to facilitate testing. Treatment Any process, physical or chemical, to which a seed lot is submitted, is termed as treatment. Principle: The presence or absence of disease organisms, pests and deleterious physiological conditions specified by the sender is determined and the number of seeds so affected in the sample is estimated as accurately as the method used permits. The determination may be influenced by treatment applied to the seed lot. Therefore, if treatment has been applied, the sender is required to specify the type of treatment and the chemicals used.

Procedure:
Working sample The entire submitted sample, or a portion of it, depending on the test method, may be used as a working sample. Exceptionally, a submitted sample larger than that prescribed may be required and in such cases the sampler shall be instructed accordingly. When a portion of the submitted sample is required as a working sample, the reduction shall be carried out in accordance with the mixing and division methods.
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Normally the working sample shall not be less than 400 pure seeds or an equivalent weight taken from the samples as submitted. Replicates containing a specified number of seeds, if required, shall be taken at random from a sub-sample after through mixing. General directions Different methods of testing are available, varying in sensitivity and reproducibility and in the amount of training and equipment required. The method used will depend on the pathogen or condition to be investigated, the species of the seed, and the purpose of the test. Selection of the method and evaluation of the results requires knowledge and experience of the methods available. In a test the working sample is examined with or without incubation. Growing plants may be examined. 1. Examination without incubation.

( i ) Direct examinat ion : The submitted sample, or a sub-sample from it is examined, with or without a stereoscopic microscope and searched for ergots and other sclerotia, nematode galls, smut-balls, insects, mites and evidence of diseases and pests on seeds or on inert matter, such as fruiting bodies, discoloration and damage. (ii ) Examinat ion of imbibed seeds: The working sample i s immersed in water or other l i qu id to make f ru i t i n g bodies , symptoms or pests more easi l y vis i b l e , or to encourage the l i be ra t i on of spores . After imbib i t i on the seeds are examined ei ther superf i c i a l l y or in terna l l y , preferab ly with a stereoscopic microscope. (iii) Examination of organisms removed by washing:

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The working sample i s immersed in water with a wett ing agent , or in alcohol , and shaken vigorous ly to remove fungal spores , hyphae, nematodes etc . in te rming led with or adher ing to the seeds. The excess l i qu i d i s then removed by f i l t r a t i o n , centr i f ugat i on or evaporat ion and the extr i ca ted mater ia l examined by compound microscope. 2. Examination after incubation. After a specific period of incubation the working sample is examined for the presence of or symptoms of disease organisms, pets and physiological disturbance on or in the seeds and on seedlings. The examination may be superficial, or internal. Three types of media are commonly used. (i) Blotters are used when it is required to grow the pathogens from the seeds or examine the seedlings. The seeds, with or without pretreatment, are spaced before incubation so as to avoid secondary spread of organisms. Light conditions calculated to stimulate speculation or fungi are supplied when appropriate. Germination inhibition by chemical or other means is sometimes desirable. Some pathogens can be identified without magnification, but a stereoscopic microscope is often necessary, or a compound microscope for identifying spores. (ii) Sand, artificial composts and similar media can be used for certain pathogens. The seeds, usually without pretreatment, are sown suitable spaced in the medium so as to avoid secondary spread of organisms and incubated in conditions favorable for symptom expression. (iii) Agar plates are used to obtain identifiable growth of organisms from seeds. Careful sterility precautions are required. The seeds, normally after pretreatment, are spaced on the surface of sterilized agar and incubated. Characteristic colonies on the agar can be identified,

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either macroscopically or microscopically. Lighting is often useful and germination inhibitors may be used.
3. Examination of growing plants.

The growing of plants from seed and examining them for disease symptoms is sometimes the most practicable procedure for determining whether bacteria, fungi or viruses are present in the sample. Seeds from the sample under test may be sown, or inoculum obtained from the sample may be used for infection tests with healthy seedlings or parts of plants. The plants must be protected from accidental infections from elsewhere and conditions may required careful control.

4. Other techniques. Specialized methods involving serological reactions, phageplaque formation, etc., have been developed for some disease organisms and may by used. Calculation and Expression of results: Results are expressed as percentage by number of seeds affected or as number of organisms in the weight of sample examined. Reporting Results The result is reported on prescribed form stating the Latin names of organisms. The results must be accompanied by a statement of the test method used, including any pretreatment applied, and of the amount of the sample or fraction examined.

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The absence of statement concerning the health condition of the seed does not necessarily imply that the health condition is satisfactory.

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Special Instructions for Testing of Seed Samples of Individual Vegetable Crops

Vegetable Crops of Family Solanaceae

(Tomato, Chili, Sweet pepper, Eggplant)
1.

Seed unit: True seed accord ing to botanica l def in i t i o n of seed. Submitted Sample Weight:
128

2.

Capsicum annum (Pepper) = 150 g Solanum melongena (Eggplant ) = 150 g Lycopers i con esculentum var . esculentum (Tomato) = 70 g
3.

Working Sample Weight:

Capsicum annum (Pepper) = 15 g Solanum melongena (Eggplant ) = 15 g Lycopers i con esculentum var . esculentum (Tomato) = 7 g
4.

Purity Analysis:

a. Pure Seed: In tac t seeds, with or without testa , and in the case of pieces of seeds, any piece which i s la rger than one- hal f the or ig ina l s ize shal be cons idered pure seed. b. Other Crop Seeds: Although seed standards do provide space for other crop seed admixture but normal ly such admixture i s not poss ib le . c. Weed Seeds: Although seed standards do provide space for weed seed admixture but normal ly such admixture i s not poss ib le . d. Inert Matter:Seed piecesOne-hal f the or ig ina l s i ze or less , non- l i v i ng mater ia l , nematode gal l s and fungus bodies .

5.

Germination:
Number of seeds Substrata Temperature (C) First Count Final Count Additional Directions Additional Directions -

Kind of Seed

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(days) Capsicum annuum Pepper Lycopersicon lycopersicum Common tomato Solanum melongena Eggplant 400 400 RB; TB; BB; RT BB; TB; RB; RT RB; TB BB; RT 20-30 20-30 7 4

(days) 14 14

General Requirements Light moisture

Fresh/ Dormant Seeds Light; KNO3 Light; KNO3

400

20-30

14

Light moisture

Light, KNO3

Abbreviations: BB - Between blot te r s , RB - Raised blot te rs , TB Top of blot te rs , RT - Rol led towels , PP - Pleated paper, S - In sand TS - Top of sand, KNO3 - Use solution of potassium nitrate instead of water. GA3 - Use solution of gibberellic acid instead of water, TZ Tetrazolium

Seedling Evaluation: Seedlings are considered normal if they possess those essential structures that are indicative of their ability to produce a plant under favorable conditions. Abnormal Seedling Description Cotyledons

Less than hal f of the or ig ina l attached. Less than hal f of the or ig ina l necros i s or decay.

coty ledon t i s sue remain in coty ledon t i s sue f ree of

Epicotyl

Miss ing (may be assumed to be present i f coty ledons are in tac t ) .

the

Hypocotyl
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Malformed: such as markedly shortened, cur led or th i ckened. Deep open cracks extending in to the conduct ing t i s sue . Watery.

Root

None. Mi s s i ng or stubby pr imary root with weak secondary or advent i t i ous roots .

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SEED HEALTH TESTING IN SOLANACEOUS CROPS

6. The main seed-borne tomato pathogens with common names of the diseases they cause Sr. No . 1 2 3 4 5 6 7 8 9 10 Pathogen Al ternar i a solan Soraner i Cladospor ium oxyspor ium Cooke Fusar ium oxysporumSchlecht Glomerre l l a cingulaSpauld ta Phoma destruc t i va Plowr Phytophthora nicotianae B. de Haanvar Rhizocton ia solan Khn i Vert i c i l l i um dahl iKleb ae Corynebacterium michiganense Jenson Viruses Common name Early blight Leaf mold Fusar ium wi l t Anthracnose, ripe rot Fruit/stem rot Buck-eye rot Damping-off, Collar rot Verticillium wilt Bacterial canker, Grand Rapid disease Tobacco Mosaic Virus (TMV), Tobacco Bunchy Top Virus (TBTV), Mosaic, Potato Spindle Tuber Viroid (PSTV)

7. The main seed-borne eggplant pathogens with common names of the diseases they cause Sr. Pathogen No Common name
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. 1 2 3 4 5 6 7 8 9

Al ternar i a al te rnataKeiss le r Col l e to t r i c um melongena Lobik Fusar ium oxysporumSchlecht Phomopsis vexans Harter Rhizocton ia solan Khn i Sclerotinia sclerotiorum de Bar i Vert i c i l l i um albo- atrumReinke and Berth Vert i c i l l i um dahl iKleb ae Viruses

Leaf spot & Root rot Anthracnose Fusar ium wi l t Fruit rot Damping-off Damping-off Verticillium wilt Verticillium wilt Eggplant Mosaic Virus

8. The main seed-borne pepper pathogens with common names of the diseases they cause Sr. Pathogen No . 1 Al ternar i a spp. 2 Cercospora caps ic Heald i 3 4 5 6 7 8 9 Common name

Fruit rot Frog-eye leaf spot, Fruit stem-end rot Col l e to t r i c um piperatum El l . Anthracnose, And Hals t Ripe rot Diaporthe phaseolorum Fruit rot Sacc. Fusar ium solan iSacc. Fusar ium wi l t Gibbere l l a fu j i kuIro toi. Fusar ium wi l t Syn. Fusar ium moni l i f o rme Sheldon Phaeoramular ia caps ic i co l a Leaf mold, Deighton syn. Cecospora Leaf spot capsico laVassil Phytophthora capsic i Phytophthorablight, Fruit Leonian rot Rhizocton ia solan Khn i Damping-off
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10 11

Sclerotinia sclerotiorum de Bar i

Pseudomonas solanacearum E. F. Smith 12 Xanthomonas campestris Bacterial spot of fruit, pv.vesicatoria Dowson stem & leaf, Seedling 13 Viruses Alfalfa Mosaic Virus, Cucumber Mosaic Virus, Tobacco Mosaic Virus, 9. Method for the Detection ofXanthomonas campestris pv. Vesicatoria on Pepper (Capsicum annuum)seed (Adapted f rom ISF) Pathogen: Xanthomonas campestris pv. vesicatoria Note: Recent research has led to proposals for new nomenclature of the pathogen (1, 2). The bacterial populations that cause bacterial spot of pepper (and tomato) are quite diverse and X. c. pv. vesicatoria has been renamed Xanthomonas axonopodis pv. vesicatoria and Xanthomonas vesicatoria. Revision history: Version 2, Jan 2007 Sample and sub-sample size: The recommended minimum sample size is 10,000 seeds, with maximum subsample size of 10,000 seeds. Principle Detection of viable Xanthomonas campestris. pv. vesicatoria bacteria by dilution plating of the seed extract on semi-selective media b) Confirmation of suspected bacterial colonies by a pathogenicity assay
a)

Sclero t i n i rot a , Pink joint, Stem canker Brown rot

Restrictions on use

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a) The test method is suitable for seed that is untreated. b) It is not suitable for seed treated using chemicals or physical techniques with the aim of disinfestation. c) It has not been validated for seed treated with protective chemicals or biological substances. Sensit iv i ty reference: The abi l i t y to recover X. c . pv. vesicatoria on plates is influenced by the presence of other microorganisms. Validation: The method has been in use in the seed industry since 1990.
10. Method for the Detection of Tobamoviruses on

Pepper (Capsicum annuum L.)seed (Adapted from ISF) Pathogen: Tobacco Mosaic Virus (TMV), Tomato Mosaic Virus (ToMV) and Pepper Mild Mottle Virus (PMMoV) Revision history: Version 2, Jan 2007 Sample and sub-sample size The recommended minimum sample size is 3,000 seeds with a maximum sub-sample size of 500 seeds. Principle Detection of infectious virus by inoculation of Nicotiana assay plants that will produce a hypersensitive reaction creating local lesions (3) after infection by tobamoviruses from solanaceous hosts. Nicotiana tabacum cv. Xanthi NN and/or Nicotiana glutinosa can be used as assay plants.

135

Restrictions on use a) This test method is suited for seed that is untreated or seed that has been treated using chemicals with the aim of disinfestation / disinfection provided there no residue is left on it or seed treated by physical processes for disinfestation. b) It is recommended that the seed be checked for traces of the seed treatment that may inhibit the recovery of the virus. This is done by comparing the detection of the virus in two dilution series; one of the treated-seed extract spiked with dilutions of infectious tobamovirus and the second, of an uninfected- and untreated-seed extract also spiked with dilutions of infectious tobamovirus. The dilution series of the untreated seed extract acts as a control. c) The method has not been validated for seeds treated with protective chemicals or biological substances. Sensitivity reference One infested seed in a sub-sample of 500 healthy seeds can be detected with this method. Under improper environmental conditions (i.e. insufficient light or too high temperatures) the sensitivity of the test is extremely reduced (1). Validation The method has been in use in the seed industry for many years and was evaluated in a comparative test organized by ISHI-Veg (2). Method description 1. Extrac t i on of vi rus f rom the seed 1.1 Grind seeds of each sub-sample in seed extraction buffer at a ratio of 4 ml per 100 seeds.

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1.2 Process seed extracts with in an hour after gr ind ing or store at 4 C for maximum of 20 hours . 2. Inocu la t i on of Nicot i ana assay plants Assay plants should have been raised under sufficient light intensity at a temperature of 20 30 C. Choose plants that have 5-7 leaves with high turgor. Do not take flowering or old plants. 2.1 Inoculate each extract on two (nearly) fully expanded leaves of each of two plants, going across the entire surface. _ Do not use the pr imary lea f (o ldes t true lea f ) . 2.1.1 Dust the leaves moderately with carborundum (320 mesh grit powder, Fisher Scientific or equivalent). Wear a protective mask. 2.1.2 Place a drop of inoculum (100-200 ml) onto the leaf. Smear the drop with fingers without applying pressure. _ Work with gloves and change them between samples or clean hands thoroughly between samples by using alkaline soap. 2.1.3 Rinse the plants with tap water a few minutes after inoculation. 2.2 Inoculate positive and negative controls. _ Use a well-characterized positive seed lot or dilutions of purified virus as a positive control. Lesions should be obtained with 1,000- to 10,000-fold dilutions of purified virus stocks of 1 mg/ml. Note that the purified virus should be stored at 4 C and at concentrations higher than 1 mg/ml to preserve infectivity. _ Use seed extraction buffer or extract from virus-free seed lot as a negative control.

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2.3 Incubate the plants for 5-7 days under controlled conditions at 20 30 C and provide at least 12 hours of light. 2.4 Evaluate the inoculated leaves for local lesions (Fig. 1).

_ When a re la t i ve l y high number of loca l les i ons are observed, a comparison with the posi t i ve and negat ive contro l s wi l l readi l y reveal i f at least major i t y of the vi ru les i ons are authent i c .

_ However, in cases where the number of les i ons for a sample i s low i t i s necessary to conf i rm that a les i on was caused by a vi rus in fec t i on and not by an arte fac t ( f rom the mechanica l inocu la t i on , use of pest i c i des , etc . ) . Cut out th suspect les i on , crush i t in a smal l amount of the seed extrac t i on buffer and again inocu la te two leaves of two assay plants . Lesions caused by virus infection contain sufficient amounts of infectious virus to produce multiple lesions in this confirmation test. Buffers _ Use de- ion i zed water . _ Autoc lave buffers at 121 C, 115 psi for 15 minutes . Seed extraction buffer (Phosphate Buffered Saline, PBS) pH 7.2 7.4 per l i ter Sodium chloride NaCl = 8.0 g Disodium hydrogen phosphate Na2HPO4 = 1.15 g Potassium dihydrogen phosphate KH2PO4 = 0.2 g References 1. Dijkstra, J., Bruin, G.C.A., Burgers, A.C., Van Loon, L.C., Ritter, P., Van de Sanden, P.

138

A.C.M. and Wieringa-Brants, D.H. (1977) Systemic infection of some N-gene-carrying Nicot i ana species after inocu la t i on with tobacco mosaic vi rus Netherlands . Journal of Plant Pathology 83: 41-59. 2. Hadas, R. (2000) A report of comparative test for Tobamoviruses in pepper seeds. ISHI-Veg Research Report 12000-pepper tobamo. Nyon, Switzerland: International Seed Federation. 3. Holmes, F.O. (1929) Local lesions in tobacco mosaic. Botanical Gazette (Chicago) 87: 39-55.

Fig. 1. Local lesions on tobacco indicator plants (a) Nicotiana glutinosa, (b) Nicotiana tabacum cv. Xanthi NN

139

140

11. Method for the Detection ofXanthomonas

campestris pv. vesicatoria on Pepper and Tomato seed (STA selective media assay) Note: This method should not be used without permission from Seed Testing of America, Longmont, Co. Sample size: 30,000 seeds Pathogen extraction 1. Randomly select 30,000 seeds; 3 replicates of 10,000 (UNTREATED). 2. Coarse grind the three 10,000 seeds (35 gram) replicates. 3. Transfer the three replications of ground seeds to sterile 500 ml Erlenmeyer flasks and add 120 ml extraction buffer (0.05 M phosphate buffer + 0.05% Tween 20 pH 7.2). Use phosphate buffer + STS for treated seeds). 4. Agitate in flasks for 30 minutes at 180 rpm, ambient temperature. 5. Transfer 3-4 ml supernatant liquid into sterile test tubes (Direct sample). 6. Filter the remaining blended solution through a sterile strainer into a sterile beaker. Pour off 45 ml of the filtered seed extract into centrifuge tubes, one centrifuge tube per replicate. Centrifuge at 10,000 rpm for 10 minutes. 7. Discard supernatant liquid except for ~3 ml, resuspend pellets by mixing well with a vortex mixer for the Concentrate dilution.8. For the final dilution, do a 1/10 serial dilution with cold sterile saline. Dilute form the Direct tube for untreated seed and from the Concentrate tube for treated seeds. Incubation on selective media 1. Plate 100 ul from each dilution onto duplicate plates of MXV and TMB agar media (see recipes), supplemented with antibiotics to reduce the numbers of contaminated bacteria.

141

2. Streak a known XCV culture on media also. 3. Spread the liquid evenly over the media using a flamed Lshaped glass rod and turntable. 4. Incubate samples at 27-30C in the dark 7 to 10 days. Examine after 5, and 7 days for colonies typical of the pathogen. Read the plates according to the following table.

Pathogenicity Test 1. Purify suspected isolates of XCV on YDC agar medium and incubate at 27-30oC for 48 hr to obtain single colonies. 2. After incubation on YDC, transfer a mass of bacteria to sterile distilled water. Adjust the inoculum until the water has an inoculum density of 0.8-0.9. 3. Infiltrate several leaves of susceptible tomato cultivar with inoculum using a sterile syringe. 4. Wound several tomato leaves with a sterile brush or carborundum and swab inoculum over the wounded areas. 5. Incubate plants in a high humidity environment for 24-48 hr. 6. Observe the wounded leaves for symptoms of typical of tomato black spot. Media Preparation mTMB DI water 1 liter Boric acid 0.1g Bacto peptone 10g Potassium bromide 10g
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Calcium chloride 0.25g Agar 15g After autoclaving: Tween 80 10ml (autoclave separately) Cephalexin 65mg (can dissolve in 75% EtOH) 5-fluororacil 12mg (can dissolve in 50% EtOH) Tobramycin 0.2mg (can dissolve in 50% EtOH) 12 Cycloheximide 200mg (can dissolve in 95% methanol) MXV DI water 1 liter Lactose 10g D(+) trehalose 4g Thiobarbituric acid 0.1g Yeast extract 0.5g Ammonium chloride 1.0g Potassium phosphate dibasic 0.8g Potassium phosphate monobasic 0.8g Agar 15 g After autoclaving: Cycloheximide 100mg Bacitracin 100mg Cephalexin 65mg 5-fluorourecil 12mg Tobramycin 0.2mg Neomycin sulfate 10mg YDC DI water 1 liter Yeast extract 10g Dextrose 20g Calcium carbonate 20g Agar 15g References

143

Darrell Maddox, STA Laboratories, 303-651-6417.

12. Method for the Detection ofXanthomonas

campestris pv. vesicatoria on Pepper and Tomato seed (ISHI - Vegetable selective media assay) Source: ISHI - Vegetable Seed Health Testing Manual, (Draft) 2000 Sample size: Minimum number of seeds to be tested is 2000 (protected cultivation in temperate conditions) or 30.000 (outdoor cropping in humid and warm conditions), which will detect with 95 % reliability seed contamination of 0,15 % and 0,01 % respectively. Sample preparation 1. Put every subsample into a sterilized flask or other liquidproof container. 2. Add 0.05 M PB to each flask at a ratio of 3:1 (v:w). 3. Incubate overnight (minimum 14 hrs) at 4C. Serial Dilution 1. Directly from the settled seed wash, serial dilute the supernatant to 10-1 by aseptically pipeting 1.0 ml of the seed wash supernatant into 9 ml of sterile 0.85% NaC1 in a sterile culture tube. Label the dilution with the appropriate sample number.

144

2. From the 10-1 dilution, dilute the supernatant to 10-2 by aseptically pipeting 1.0 ml of 10-1 dilution into 9 ml of sterile 0.85% NaC1 in a sterile culture tube. Label the dilution with the appropriate sample number. 3. Vortex each tube to assure thorough mixing.

Liquid plating : 1. Pipet direct from each sample flask and from the 10-1 and 10-2 dilution, 100 ul on two plates (duplicate plating) of two semi-selective media. 2. With a sterile glass rod spread the 100 ul aliquot evenly over the semi-selective media. 3. Label the plates with the appropriate sample number and dilution factor (direct, 10-1 and 10-2). 4. Also inoculate plates from both media with a known culture of Xcv. Incubation: 1. Incubate the plates in a dark incubator at 26-28C. Examination. 1. Evaluate the plates for suspect Xcv colonies at day 3,5 and 7.14 2. Compare them to the known descriptions of Xcv on the used selective media given below. 3. Record the number of suspect Xcv colonies and background. 4. Purify the suspect colonies by transferring suspect colonies to a general rich medium plates.

145

Additional confirmation tests as ELISA, PCR, IF or biochemical tests are optional. Xcv descript ions on semi-select ive media : Xcv on TMB : 1. Xcv colonies typically appear and can be identified in 4-7 days. 2. Xcv colonies are yellow, slightly mucoid, mounded and round (cicular). 3. Xcv utilizes tween and in 3-7 days a white crystaline halo usually forms around the yellow colony. 4. Some strains of Xcv form only a weak halo, clear halo or only clear the media under the colony (not visible). (NOTE: The white crystaline halo is due to calcium deposits in the clear halo caused by tween utilization.) Xcv on CKTM : 1. Xcv colonies typically appear and can be identified in 4-7 days. 2. Xcv colonis are similar to the colonies on TMB; yellow, mucoid, mounded and round (circular). In 3-7 days a white halo of fine calcium crystals usually forms around the yellow colony. Some strains of Xcv form only a weak halo, clear halo, or only lcear the media under the colony (not visible). Xcv on NA-Starch : 1. No in fo rmat ion prov ided. Check appearance with know Xcv cul tu res . Pathogenicity testing: 1. For best results pepper/tomato seedlings should be at 2-3 true leaves (~3-4 week old seedlings. 2. Purify Xcv volonies (suspects) on a general medium suitable for rapid colony growth (i.e. YDC). Purify a known Xcv volony for a positive control. Incubate the plates for 24 48 hours at 26 - 28C.

146

3. Transfer a mass of the colony aseptically to a culture tube with sterile distilled water. 4. Adjust the inoculum to 108 CFU per mil by visual means (to cloudiness) or optical density methods (0.1 to 0.2 o.d. at 600 nm). 5. Infiltrate a leaf of a susceptible pepper/tomato cultivar with the suspension by gently forcing the liquid into the underside of the leaf using a sterile syringe without a needle and label the leaf with the appropriate sample identification number. 6. Infiltrate a leaf with the positive check and a blank water check (negative control) 7. Incubate the inoculated plants at 90-100% relative humidity and at 27 -32C with 12 Hour light. 8. Observe the plants daily and record the day that the water-soaked lesion appears. Generally, pathogenic Xcv will develop a water-soaked lesion within 48-96 hours. Nonpathogenic bacteria will produce a hypersensitive reaction in + 24 hours or will not develop a lesion. Media and buffers: PBT: 0.05 M Phosphate buffer 0.5% Tween 20 pH 7.2 mTMB : H20 990 ml H3B03 0.1 g Bacto peptone 10.0 g KBr 10.0 g CaC12 0.25 g Agar 15.0 g After autoclaving add: Tween 80 10.0 ml

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Cephalexin 65 mg 5-fluorouracil 12 mg Tobramycin 0.2 mg Cyclohexamide 200 mg YDC : H20 1000 ml Yeast extract 10.0 g CaC03 20.0 g Bacto agar 15.0 g Glucose 20.0 g NA-starch : H20 1000 ml Difco Nutrient agar 23.0 g Soluble Potato starch 12.0 g After autoclaving add: Penicillium G 50 mg 16 CKTM : H20 990 ml Soy peptone 2.0 g Bacto typtone 2.0 g Dextrose 1.0 g L-glutamine 6.0 g L-histidine 1.0 g (NH4)2PH04 0.8 g MgS04.7H20 0.4 g CaC12 0.25 g Pourite (=optional) 1 drop Difco Bacto Agar 15.0 g After autoclaving add: Tween 80 10 ml Cephalexin 65 mg 5-fluorouracil 12 mg

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Tobramycin 0.4 mg Cyclohexamide 100 mg Bacitracin 100 mg Neomycin sulfate 10 mg

13. Method for the Detection ofClavibacter

michiganensis subsp. michiganensis on Tomato seed Revision history: Vers ion 3, Jan 2008 Sample and sub-sample size The recommended minimum sample size is 10,000 seeds, with a maximum sub-sample size of 10,000 seeds. Note: Sub- samples la rger than 5,000 seeds requi re an addi t i ona l concentrat i on step of the extracted bacter i a . Principle o Extrac t i on f rom the seed of externa l l y and in te rna l l y located bacter i a o Iso la t i on of viab C.le m.subsp. michiganensis bacteria by dilution plating seed extract on two different semiselective media o Confirmation of suspected bacterial colonies by a pathogenicity assay Restrictions on use o This test method is suited for seed that is untreated or seed that has been treated using chemicals with the aim of disinfestation / disinfection provided no residue

149

i s le f t on ori tseed t reated by physica l processes for dis in fes ta t i on . o I t i s recommended that the seed be checked for traces of the seed treatment that may inh ib i t the recovery of the bacter ium. This i s done by comparing the recover ies of the bacter ium in two di lu t i on ser ies ; one of the treated- seed extract spiked C. with m.subsp. michiganensis and the second, of an uninfected- and untreated-seed extract also spiked with C. m. subsp. michiganensis. The dilution series of the untreated seed extract acts as a control. o The method has not been validated for seed treated with protective chemicals or biological substances. Sensitivity reference The ability to recover C. m. subsp. michiganensis on plates is influenced by the presence of other microorganisms. In case C. m. subsp. michiganensis is not detected, the test should be considered not sensitive enough for the particular subsample if, for both media used, more than 50 % of the surface of the plates corresponding to the most concentrated extract is covered by colonies of other microorganisms. Retesting a new sample is recommended after (further) treatment/disinfestation of the seed lot. Validation Test commonly in use by the seed industry for many years.

150

Method description 1. Extraction of bacteria from the seed 1.1. Put every sub-sample into a sterile stomacher bag. Add sterile seed extraction buffer to each bag at a ratio of 4 ml of seed extract buffer to 1 g of seed (v:w). Incubate overnight (minimum 14 hours) at 4C, and macerate for at least 7 min in a stomacher machine. 2. Isolation on semi-selective media 2.1. Taking further steps into account, filter coarse particles from the required volume of extract using a filter bag (Bagfilter or Bagpage from Interscience, France, or an extraction bag with synthetic intermediate layer from Bioreba, Switzerland or Neogen Europe, Scotland). (If a stomacher bag with filter is used this is automatically achieved.) Prepare a 10-fold dilution series (to 10-2) of the seed extract in sterile seed extraction buffer. For sub-samples larger than 5,000 seeds also prepare a 10-fold concentrated extract: 2.1.1. Centrifuge at least 2 ml of the filtered extract for 5 minutes at 5,000 g. Carefully remove the supernatant and resuspend the pellet in 1/10 of the original centrifuged volume of sterile seed extraction buffer. Prepare a 10-fold dilution series of a suspension of a pure culture of a known C. m . susbp. michiganens is reference strain in sterile seed extraction buffer 2.2. Spread-plate 0.1 ml of the concentrated (if applicable), the undiluted and diluted extracts onto plates each of the two semi-selective media, D2ANX and either SCM or mSCM. Spread-plate 0.1 ml for

151

each dilution of the reference strains giving at least one plate within a range of 30-300 colonies per plate on each of the semi-selective media (reference plates for section 2.4). 2.3. Incubate plates in the dark at 26-28 C for 10 days. Check plates at 5, 7 and 10 days. 2.4 Check recovery and morphology of the C. m. subsp. michiganensis reference strain on both media. Examine the sample plates for the presence of the colonies with typical C. m. subsp. michiganensis morphology by comparing them with the reference strain. Record the number of suspected colonies as well as other colonies and indicate when overgrowth of C. m. subsp. michiganensis by the other colonies could have occurred (see Sensitivity reference).
o After 5-7 days of incubation on D2ANX, C. m.

subsp. michiganensis colonies are yellow, mucoid and convex.

o After 710 days of incubation on SCM, C. m. subsp.

michiganensis colonies are translucent grey, mucoid, eventually often irregularly shaped, with internal black flecks.
o After 710 days of incubation on mSCM, C. m.

subsp. michiganensis colonies are translucent beige, mucoid, eventually often irregularly shaped, with yellow/orange internal flecks. o The colony size and color can differ within a sample. The morphology and colors of Cmm strains may vary considerably on SCM and mSCM.

152

2.5 If present, select at least 5 suspected colonies per medium per sub-sample for further identification on YDC (1) media. 3. Identification by morphology on YDC medium 3.1. Transfer selected suspected colonies as well as the reference strains onto YDC medium. Incubate YDC plates at 26 - 28 C for 2-3 days. 3.2. Determine whether transferred colonies have typical C. m. subsp. michiganensis morphology by comparing them with the reference strains and record which of the isolates are still suspected to be C. m. subsp. michiganensis. On YDC C. m. subsp. michiganensis is yellow, raised and mucoid in appearance. It should be noted that in this stage of the test (plating of pure isolates) no separate colonies are cultured so that the round shape is not always visible. 3.3. If present, select suspected isolates for further identification using the pathogenicity assay. The selection should reflect the distribution of suspected colonies over the initial sub-samples and/or semiselective media. 4. Identification by pathogenicity assay 4.1. Grow seedlings of a known susceptible tomato cultivar (e.g. Moneymaker) under suitable conditions until 2-3 true leaves have developed (about 3-4 weeks after sowing). 4.2. Dip a sterile toothpick directly in a suspected colony on YDC medium and inoculate two tomato seedlings by stabbing the toothpick into the stem between the

153

cotyledons and the first true leaf. Use the sharp (rather than blunt) end of the toothpick to avoid damage while introducing inoculum. Include a reference C. m. subsp. michiganensis strain as a positive control and do a mock inoculation with a clean toothpick as a negative control. 4.3. Incubate the inoculated plants at 25-32 C with at least 8 hours light. 4.4. Observe the plants for wilting symptoms after 2-3 weeks and compare with the positive control. Typical symptoms caused by Cmm are canker formation at the site of inoculation, yellowing and marginal necrosis, and wilting of true leaves. Buffers and media o Use de-ionized water. o Autoclave buffers and media at 121 C, 115 psi for 15 minutes. o Antibiotic (units/mg) activity is critical for the recovery of C. m. subsp. michiganensis. The purity of an antibiotic, and therefore its activity, can vary from batch to batch. o Antibiotics are not stable in time. Therefore, add antibiotics to the media at a relatively low temperature (< 50 C) and store plates before use in polythene bags at 4 C in the dark. Use plates within a month to maintain the selectivity of the media. Seed extraction buffer (pH 7.4) per liter

154

Na 2HPO4 KH2PO4 Tween 20 Na2S2O3 1

1

7.75 g 1.65 g 0.2 ml 0.5 g

recommended when seeds have been treated with hypochlor i t e

D2ANX per l i ter (2) Yeast extract Casein hydrolysate Glucose MgSO4.7H2O NH4Cl Trizma base Agar Nalidixic acid (salt) 1
[sodium sal t , NaOH ]

10 mg/ml in

0.1

2.0 g 4.0 g 10.0 g 0.3 g 1.0 g 1.2 g 15 g 28 mg 10 mg 100 mg

Polymyxin sulfate1 Cycloheximide1

1

[10 mg/ml in distilled water] [200 mg/ml in absolute methanol]

add afte r autoc lav ing

SCM per l i ter (3) Yeast extract Sucrose H3BO3 (boric acid) MgSO4.7H2O K2HPO4 KH2PO4 Agar Nalidixic acid 1 Nicotinic acid
1

0.1 g 10.0 g 1.5 g 0.25 g 2.0 g 1.5 g 15 g 30 mg 100 mg

sodium salt, 10 mg/ml in 0.1 M NaOH 20 mg/ml in distilled water

155

Potassium tellurite Cyclohex imide1

1

1 ml 200 mg

1 % Chapman tellurite solution Difco 200 mg/ml in absolute methanol

add afte r autoc lav ing

YDC (Yeast extract - dextrose - CaCO3 Agar) per liter (1, 5)

Yeast extract D-glucose (dextrose) CaCO3 Agar mSCM per liter (4)
Yeast extract Mannose H3BO3 (boric acid) MgSO4.7H2O K2HPO4 KH2PO4 Agar Nalidixic acid (salt)

10.0 20.0 20.0 15.0

g g g g

30 mg 100 mg 200 mg

Nicotinic acid (free acid)

1

Cycloheximide
1

0.1 g 10.0 g 1.5 g 0.25 g 2.0 g 1.5 g 15 g sodium salt, 10 mg/ml in 0.1 M NaOH 20 mg/ml in distilled water 200 mg/ml in absolute methanol

add afte r autoc lav ing

References
1. Schaad, N.W. Jones, J.B. and W. Chun (eds) (2001) Laboratory Guide for Identification of Plant Pathogenic Bacteria, Third Edition. St Paul, USA: American Phytopathological Society Press. 2. Chun, W.C.C. (1982) Identification and detection of Corynebacterium michiganense in tomato seed using the indirect enzyme-linked immunosorbent assay. M.S. Thesis, University of Hawaii.

156

3. Fatmi, W. and N.W. Schaad (1988) Semiselective agar medium for isolation of Clavibacter michiganense subsp. michiganense from tomato seed. Phytopathology 78: 121-126. 4. Waters, C.M. and H.A. Bolkan (1992) An improved semi-selective medium for detecting Clavibacter michiganensis subsp. michiganensis in tomato seeds. (Abstr.) Phytopathology 82: 1072. 5. Wilson, E.E., Zeitoun, F.M. and D.L. Fredrickson (1967) Bacterial phloem canker, a new disease of Persian walnut trees. Phytopathology 57: 618-621.

14. Method for the Detection of Tobamoviruses on

Tomato seed (Adapted f rom ISF) Crop : Tomato Lycopers ( i con esculentum L. ) Pathogen : Tobacco Mosaic Virus (TMV) and Tomato Mosaic Virus (ToMV) Revision history : Vers ion 2, Jan 2007 Sample and sub-sample size The recommended minimum sample size is 3,000 seeds with a maximum sub-sample size of 500 seeds. Principle

Detection of infectious virus by inoculation of Nicot i ana assay plants that wi l l produce a hypersens i t i v e react i on creat ing loca l les i ons (3) after in fec t i on by tobamovirus f rom solanaceous hosts Nicotiana . tabacum cv. Xanthi NN and/or Nicotiana glutinosa can be used as assay plants. Restrictions on use This test method is suited for seed that is untreated or seed that has been treated using chemicals with the aim of disinfestation / disinfection provided there no residue is left on it or seed treated by physical processes for disinfestation.
157

It is recommended that the seed be checked for traces of the seed treatment that may inhibit the recovery of the virus. This is done by comparing the detection of the virus in two dilution series; one of the treated-seed extract spiked with dilutions of infectious tobamovirus and the second, of an uninfected- and untreated-seed extract also spiked with dilutions of infectious tobamovirus. The dilution series of the untreated seed extract acts as a control. The method has not been validated for seeds treated with protective chemicals or biological substances. Sensitivity reference One infested seed in a sub-sample of 500 healthy seeds can be detected with this method. Under improper environmental conditions (i.e. insufficient light or too high temperatures) the sensitivity of the test is extremely reduced (1). Validation The method has been in use in the seed industry for many years and was evaluated in a comparative test organized by ISHI-Veg (2). Method description 1. Extraction of virus from the seed 1.1 Grind seeds of each sub- sample in seed extrac t i on buffer at a rat i o of 4 ml per 100 seeds.

Process seed extracts with in an hour after gr ind ing or stor at 4C for maximum of 20 hours . 2. Inoculation of Nicotiana assay plants

158

Assay plants should have been ra i sed under suf f i c i en t l i gh in tens i t y at a temperature of 20 30 C. Choose plants having 5- 7 leaves with high turgor . Do not take f l ower ing o old plants .

2.1 Inocu la te each extract on two (near l y ) fu l l y expanded leaves of each of two plants , going across the ent i re sur face Do not use the pr imary lea f (o ldes t t rue lea f ) . 2.1.1 Dust the leaves moderate ly with carborundum (320 mesh gr i t powder, Fisher Scient i f i c or equiva lent ) . Wear protect i ve mask 2.1.2 Place a drop of inocu lum (100- 200 l) onto the leaf. Smear the drop with fingers without applying pressure. Work with gloves and change them between samples or clean hands thoroughly between samples by using alkaline soap. 2.1.3 Rinse the plants with tap water a few minutes after inoculation. 2.2 Inoculate positive and negative controls. Use a well-characterized positive seed lot or dilutions of purified virus as a positive control. Lesions should be obtained with 1,000- to 10,000-fold dilutions of purified virus stocks of 1 mg/ml. Note that the purified virus should be stored at 4 C and at concentrations higher than 1 mg/ml to preserve infectivity. Use seed extraction buffer or extract from virus-free seed lot as a negative control.

159

2.3 Incubate the plants for 5- 7 days under contro l l ed condi t i ons at 20 30 C and prov ide at leas t 12 hours of l i gh t .

2.4 Evaluate the inocu la ted leaves for loca l les i ons (F ig . 1) When a re la t i ve l y high number of loca l les i ons are observed, a comparison with the posi t i ve and negat ive contro l s wi l l readi l y reveal i f at least a major i t y of the v les i ons are authent i c .

However, in cases where the number of les i ons for a sample i s low i t i s necessary to conf i rm that a les i on was caused by a vi rus in fec t i on and not by an art i f a c t ( f rom the mechanica l inocu la t i on , use of pest i c i des , etc . ) . Cut out th suspect les i on , crush i t in a smal l amount of the seed extrac t i on buffer and inocu la te two leaves of two assay plants again . Les ions caused by vi rus in fec t i on conta in suf f i c i en t amounts of in fec t i ous vi rus to produce mult ip l e les i ons in th i s conf i rmat ion test . Buffers o Use de- ion i zed water . o Autoc lave buffer s at 121 C, 115 psi for 15 minute s. Seed extraction buffer (Phosphate Buffered Saline, PBS) pH 7.2 7.4 per liter Sodium chloride NaCl = 8.0 g Disodium hydrogen phosphate Na2HPO4 =1.15 g Potassium dihydrogen phosphate KH2PO4 = 0.2 g References
1. Dijkstra, J., Bruin, G.C.A., Burgers, A.C., Van Loon, L.C., Ritter, P., Van de Sanden, P. A.C.M. and Wieringa-Brants, D.H. (1977) Systemic infection of some N-gene-carrying Nicot iana spec ies afte r inocu la t i on with tobacco mosaic vi rus Netherlands . Journal of Plant Pathology 83 : 41-59.

160

2. Hadas, R. (1999) A report of comparative test for Tobamoviruses in tomato seeds. ISHI-Veg Research Report 1-1999-Tomato-Tobamo. Nyon, Switzerland: International Seed Federation. 3. Holmes, F.O. (1929) Local lesions in tobacco mosaic. Botanical Gazette (Chicago) 87 : 39- 55. Fig. 1. Local les i ons on tobacco ind ica to r plants Nicot (a) iana glut inosa , (b) Nicotiana tabacum cv. Xanthi NN

15. Method for the Detection of Pepino Mosaic Virus

on Tomato seed (Adapted f rom ISF) Revision history : Vers ion 2, Jan 2007 Sample and sub-sample size The recommended minimum sample size is 3,000 seeds with a maximum sub-sample size of 250 seeds. Principle

161

Detect ion of in fec t i ous Pepino Mosaic Virus inocu la t i on of suscept Nicot ib l e i ana benthamiana assay plants followed by ELISA of the inoculated assay plants in order to confirm infection by PepMV

by

o Optionally, seed extracts can be pre-screened with ELISA. If ELISA detects no virus in the seed extracts, the test is finished. If, however, the ELISA on seed extracts is positive a bioassay is necessary to determine whether infectious PepMV is present, as ELISA detects both infectious and non-infectious virus particles. Restrictions on use o This test method is suitable for seed that is untreated, or seed that has been treated using chemicals with the aim of disinfestation / disinfection prov ided no res idue i s le f t on , or i t seed treated by physical processes for disinfestations. o It is recommended that the seed be checked for traces of the seed treatment that may inhibit the recovery of the virus. This is done by comparing the detection of the virus in two dilution series; one of the treated-seed extract spiked with dilutions of infectious PepMV virus and the second, of an uninfected- and untreated-seed extract also spiked with dilutions of infectious PepMV. The dilution series of the untreated seed extract act as a control. o The method has not been validated for seed treated with protective chemicals or biological substances. Sensitivity reference o The ELISA has been shown to reproducibly detect single PepMV infested seeds in test samples in a comparative test with 6 participating laboratories (1). It should be noted that infested seeds (a single one being

162

detectable in a sample) rarely lead to infected seedlings. Given this low transmission rate (less than 1 in 1000 (1)), a sample size of 3000 seeds is considered to be appropriate. Validation o Using a di lu t i on ser ies of systemica l l y in fec ted leaves heal thy seed extracts , 10,000- fo ld di lu t i ons were reproduc ib ly detected in both ELISA and bioassay. Method description 1. Extraction of virus from the seed 1.1 Grind seeds of each sub-sample in a 10 ml seed extraction buffer. Process extracts immediately after grinding or store at 4C for a maximum of 20 hours. Do not freeze. Note that if the extracts are to be used for the bioassay after ELISA, the ELISA must be completed within 20 h after extraction. 2. (Opt iona l ) ELISA 2.1 Run a double-antibody-sandwich (DAS-)ELISA (2, 3) on the extracts. The source of antiserum is critical. It is recommended that the performance of other antisera be verified against the antiserum supplied by PRI (http://www.plant.wageningenur.nl/default.asp?section=products). 3 . Inocu lat ion of Nicot iana bentha miana assay plants Assay plants should have 4-7 (nearly) fully expanded leaves, should not yet have started flowering, and
163

should have been raised under sufficient light intensity at an average temperature of 20 25C. Assay plants should have good turgor at the time of inoculation (Fig. 1). 3.1 Inoculate each extract on the two youngest (nearly) fully expanded leaves of two plants, going across the whole surface. Do not use the primary leaf (oldest true leaf). 3.1.1 Dust the leaves moderate ly with carborundum (320 mesh gr i t powder, Fisher Scient i f i c or equiva lent ) . Wear a protect i ve mask. 3.1.2 Place a drop of inocu lum (100- 200 l ) onto the lea f . Smear the drop with f i ngers without apply ing pressure . Work with gloves and change them between samples or clean hands thoroughly between samples by using alkaline soap. 3.1.3 Rinse the plants with tap water a few minutes after inocu la t i on . 3.2 In order to allow the assay plants to become systemically infected, incubate them for at least 14 days under controlled conditions at 25 5 C and with at least 12 hours of light per day. 3.3 Determine infection of the assay plants by PepMV using ELISA. 3.3.1 For each sub sample, sample and pool lea f mater ia l f rom both assay plants making sure that the pooled leaves weigh 0.20.5 g. Select leaves that have expanded dur ing the preceding week (not the inocu la ted leaves) .
164

Process samples immediately, store at 4 C for at most 48 hours or freeze until use. If the samples were frozen, process them as soon as they have thawed. 3.3.2 Grind each pooled lea f sample in 10 - 12 ml ELISA extract i on buffer . Process extracts immediately after grinding, store at 4C for a maximum of 24 hours or freeze unti l use. 3.3.3 Run a DAS-ELISA (2 , 3) on the extracts . The source of ant i se rum i s cr i t i c a l . I t i s recommended that the performance of other ant i sera be ver i f i ed against the ant i se rum suppl i ed by PRI (ht tp : / /www.plant .wageningenur .n l / de fau l t . a sp?sect i on=products ) . Notes o N. benthamianai s a systemic host for al l PepMV st ra ins tested .N. benthamiana is preferred over tomato as an assay plant because the systemic movement of the virus in tomato can be erratic. Furthermore, leaves of N. benthamiana are more easily inoculated. o Although PepMV infection of N. benthamiana usually results in conspicuous symptoms this is not the case at all times, and symptoms can be caused by other factors than PepMV. Therefore, ELISA of assay plants is required. Buffers o Use de-ionized water. o Autoclave buffers at 121 C, 115 psi for 15 minutes.
165

Seed extraction buffer after preparat i on ) Sodium chloride NaCl Disodium hydrogen phosphate dodecahydrate Na2HPO4.12H2O Potassium dihydrogen phosphate KH2PO4 Potassium chloride KCl Sodium sulphite Na2SO3 (add after autoclaving) References

per l i ter (use 8.0 g 2.9 g

with in

24 hours

0.2 g 0.2 g 1g

1. Krinkels, M. (2001) Pepino mosaic virus causes sticky problem. Prophyta, The Annual May 2001: 30-33. 2. Albrechtsen, S.E. (2006) Testing methods for seed-transmitted viruses: principles and protocols. Wallingford, UK: CABI Publishing. 3. Clark, M.F. and A.N. Adams (1977) Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. Journa l of General Viro logy 34 : 475- 483

Vegetable Crops of Family Cucurbitaceae

(Melons, Gourds, Squashes and Pumpkins)
1.

Seed unit: True seed accord ing to botanica l def in i t i o n of seed. Submitted Sample Weight: Cit ru l l u s lanatus (Watermelon) = 1,000 g Praec i t r u l l u s f i s t u l o (T sus inda)= 150 g Cucumis melo (Muskmelon) = 150 g
166

2.

Cucumis sat i vus(Cucumber) = 150 g Cucurbi ta maxima (Pumpkin) = 1,000 g Cucurbi ta moschata (Red gourd) = 350 g Cucurbi ta pepo (Marrow) = 1,000 g Lagenar ia s icera r(Bot i a t l e gourd)= 700 g Momordica charant ia (B i t t e r gourd)= 700 g
3.

Working Sample Weight:

Cit ru l l u s lanatus (Watermelon) = 250 g Praec i t r u l l u s f i s t u (T lo inda)= sus 70 g Cucumis melo (Muskmelon) = 70 g Cucumis sat i vus(Cucumber) = 70 g Cucurbi ta maxima (Pumpkin) = 700 g Cucurbi ta moschata (Red gourd) = 180 g Cucurbi ta pepo (Marrow) = 700 g Lagenar ia s icera r(Bot i a t l e gourd)= 70 g Momordica charant (B iai t t e r gourd)= 70 g 4. Purity Analysis:

a. Pure Seed: In tac t seeds, with or without testa , and in the case of pieces of seeds, any piece which i s la rger than one- hal f the or ig ina l s ize shal be cons idered pure seed. b. Other Crop Seeds: Although seed standards do provide space for other crop seed admixture but normal ly such admixture i s not poss ib le . c. Weed Seeds: Although seed standards do provide space for weed seed admixture but normal ly such admixture i s not poss ib le . d. Inert Matter:Seed piecesOne-hal f the or ig ina l s i ze or less , non- l i v i ng mater ia l , nematode gal l s and fungus bodies .

167

5. Germination:
Number Temperature Substrata of seeds (C) First Count (days) Final Count (days) Additional Directions General Requirements Additional Directions Fresh/ Dormant Seeds May retest at 30C May retest at 30C

Kind of Seed

Citrullus lanatus var. citroides Citron

100

BB; S; RT

25; 20-30

10

Light moisture

Citrullus lanatus var. lanatus Watermelon Cucumis anguria Gherkin Cucumis melo Melon, muskmelon, cantaloupe Cucumis sativus Cucumber Cucurbita pepo, C. maxima, C. moschata Pumpkin, squash and vegetable marrow

100 100 100 100

BB; S; RT BB; S; RT BB; S; RT BB; S; RT

25; 20-30 25; 20-30 25; 20-30 25; 20-30

4 4 4 4

14 8 10 7

Light moisture Light moisture Light moisture Light moisture

100

BB; S; RT

25; 20-30

Light moisture

Lagenaria siceraria Bottle gourd Momordica Charantia Bitter gourd

100 100

BB; S; RT BB; S; RT

25; 20-30 25; 20-30

4 4

7 7

Light moisture Light moisture

Abbreviations: BB - Between blot te r s , sand.

RT - Rol led towels , S - In

Seedling Evaluation: Seedlings are considered normal if they possess those essential structures that are indicative of their ability to produce a plant under favorable conditions.
168

General Description Seedling type: Epigea l dicot . Food reserves: Coty ledons which are la rge and f l eshy ; they expand, become photosynthet i c and are usual l y pers i s ten t beyond the seedl ing stage. Shoot system: The hypocoty l elongates and the coty ledons are pul led f ree of the seed coat , which often adheres to a peg- l i ke appendage at the base of the hypocoty l . The epicoty l usual l y does not show any development with in the test per iod . Root system: A long pr imary root with numerous secondary roots . Abnormal Seedling Description

Cotyledons

Less than hal f of the or ig ina l coty ledon t i s sue remain in attached. Less than hal f of the or ig ina l coty ledon t i s sue f ree of necros i s or decay (see note 3) .

Epicotyl

Miss ing (can be assumed to be present i f coty ledons are in tac t ) .

the

Hypocotyl

Deep open cracks extending in to the conduct ing t i s sue . Malformed: such as markedly shortened, cur led or th i ckened (see note 2) .

Note: The short, thickened area (or "peg") between the roots and the hypocotyl is a normal development.
169

Root

None. Weak, stubby or miss ing pr imary root , with less than two st rong secondary or advent i t i ous roots .

Seedling

One or more essent ia l st ruc tu res impai red as a resu l t decay f rom pr imary in fec t i on . Alb ino .

of

Notes 4. In general, seedling development is best when Substrata are kept on the dry side. Extra moisture may then need to be added at the time of the first count. 5. Samples should be retested in sand or soil if there is evidence of chemical injury (characterized by badly thickened and shortened hypocotyls and roots). Seedlings showing chemical injury symptoms in the retest are to be classified as abnormal. 6. Seedlings with unshed seed coats may have decayed or damaged cotyledons. The seed coat must be removed for evaluation of the cotyledons.

SEED HEALTH TESTING IN CUCURBITACEAE CROPS

170

1.

The main seed-borne Watermelon pathogens with common names of the diseases they cause Pathogen
C. orbiculare (Berk, and Mont.)Arx. syn. Colletotrichum lagenarium (Pass.) Ell. and Halst. Didymella bryoniae (Auersw.) Reh m. syns. Mycosphaerella m elonis (Pass.) Chiu and W alker, and Phyllostica Fusarium oxysporum Schlecht ex f.sp. niveum (E.F.Sm.) Snyder and Hansen syn. F. citml l i Taub. Pyth ium aphanidermatum(Edson) Fi t zp . Pseudomonas sp. Virus

Sr. No .
1 2

Common Names
Anthracnose Gummy stem blight, black rot Wilt Fruit & Seed rot Seed rot Squash mosaic virus

3 4 5 6 2.

Sr. No.
1

The main seed-borne Cucumber pathogens with common names of the diseases they cause Pathogen Common Names
Alte rnar ia cucumerina (E l l . and Ev.) El l i o t t syn. A. brass icae (Berk . ) Sacc. nigrescens f. Peglion Cladospor ium cucumerinum Ell. and Arth. Col le to t r i c hum lagenar ium (Pass.) Ell. and Halst. Corynespora cass i i c o (Berk, la and Curt.) Wei syns. Cercospora melonis Cooke and Helminthospor ium cass i i c oBerk l a and Curt. Didymella bryoniae (Fucke l ) Rehm syn.Mycosphaerel l a melonis (Pass . ) Chiu and WalkerM. ci t ru l l i n (C. a O. Smith) Grossenb Fusar ium oxysporumSchlecht. ex. Fr. Pseudomonas lachrymans (E , F. Smith and Bryan) Carsner Viruses Leaf spot / blight

2 3 4

Scab, gum m osis Anthracnose Cercospora leaf spot

5 6 7

Leaf spot, black Fusarium wil t Cucumber angular leaf spot Cucumber green mottle mosaic virus 171

8 3.

The main seed-borne Melon pathogens with common names of the diseases they cause Pathogen Common Names

syn. Cucumis virus 2 Cucumber mosaic virus

Sr. No .
1 2 3 4

Cladospor ium cucumerinum Scab El l . and Arth. Col le to t r i c hum lagenar ium Anthracnose (Pass . ) El l . and Halst . Fusar ium oxysporum Fusarium wil t Schlecht . ex. Fr . Pleospora herbarum (Pers . ex Fr . ) Rabenh. , syn. Stemphyl ium botryosum Wallr . Viruses Leaf spot Cucumber mosaic vi rus (syn. Cucumis virus) Melon mosaic virus Musk- melon mosaic virus (syn. Marmor melonis) Squash mosaic virus , Tobacco r ingspot virus

4.

The main seed-borne Squash pathogens with common names of the diseases they cause Pathogen Common Names

Sr. No.
1 2 3 4 5 6 7

Alternaria spp. Leaf and stem spot Cladospor ium Scab cucumerinum El l . and Arth . Fusarium foot rot Fusarium solani (Mart . ) Sacc. f . spCucurbi . tae Snyder and Hansen Sclerot in i a scle ro t i o rum Watery soft rot - stem (L ib . ) de Bary Stemphyl ium spp. Leaf and stem spot Xanthomonas cucurbi tae Bacter ia l leaf spot (Bryan) Dowson Cucumber mosaic virus (syn. Cucumis virus 1) Musk- melon Viruses mosaic virus Prunus necrot i c r ing- spot vi rus (syn. Peach r ingspot

172

Vegetable Crops of Family Brassicaceae

(Cauliflower,Cabbages, Turnip, Radish)
6.

Seed unit: True seed accord ing to botanica l def in i t i o n of seed. Submitted Sample Weight: Brass i ca oleracea L. (all varieties) (Cabbages) = 70 g Brass i ca rapa L. (a l l var ie t i e s ) (Turnip, Chinese cabbage) = 70 g Raphanus sat i vus (Radish) = 300 g

7.

8.

Working Sample Weight: Brass i ca oleracea L. (all varieties) (Cabbages) = 7 g Brass i ca rapa L. (a l l var ie t i e s ) (Turnip, Chinese cabbage) = 7g Raphanus sat i vus (Radish) = 30 g

9. Purity Analysis:

a. Pure Seed: In tac t seeds, with or part i a l l y removed testa , and in the case of pieces of seeds, any piece which i s la rger than one- hal f the or ig ina l s i ze shal l be cons idered pure seed. b. Other Crop Seeds: Al l other species and other botanica l var ie t i e s of same species not i f i ed as crop. c. Weed Seeds: Al l other species and other botanica l var ie t i e s of same species not i f i ed as weeds.
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d. Inert Matter:Seed with seed- coat ent i re l y removed and seed pieces One-hal f the or ig ina l s i ze or less , non- l i v i ng mater ia l , nematode gal l s and fungus bodies . 10. Germination:
Number Temperature Substrata of seeds (C) First Count (days) Final Count (days) Additional Directions General Requirements Light Additional Directions Fresh/ Dormant Seeds Prechill

Kind of Seed

Brassica spp. Vegetable Brassicas, etc. Raphanus sativus Radish

TB BB; RT

15-25; 25 20

4 4

10 7

Abbreviations: BB - Between blot te r s , of blot te.rs

RT - Rol led TB towels - Top ,

Seedling Evaluation: Seedlings are considered normal if they possess those essential structures that are indicative of their ability to produce a plant under favorable conditions. General Description Seedling type: Epigea l dicot . Food reserves: Coty ledons which expand and become th in , lea f - l i ke and photosynthet iBrass c . Ini ca , Sinapis and Raphanus, the cotyledons are bi-lobed and folded, with the outer cotyledon being larger than the inner. Shoot system: The hypocotyl elongates and carries the cotyledons above the soil surface; the epicotyl usually does not show any development within the test period. Root system: A long primary root.

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Abnormal Seedling Description Cotyledons

Decayed at point of attachment. Less than hal f of the or ig ina l coty ledon t i s sue remain in attached. Less than hal f of the or ig ina l coty ledon t i s sue f ree of necros i s or decay. In seedl ings Brass of i ca , one- hal f or more of the tota l coty ledon t i s sue yel l ow or white , with no green t in t (se 3 note 1) .

Epicotyl

Missing (may be assumed to be present if the cotyledons are intact).

Hypocotyl

Deep open cracks extending into the conducting tissue. Malformed: such as markedly shortened, curled or thickened. Watery.

Root

Weak, stubby or missing primary root (secondary roots will not compensate for a defective primary root).

Seedling

One or more essential structures impaired as a result of decay from primary infection. Albino.

Notes
2. Seedlings of Brass i cawith 50% or more of the total

cotyledonary tissue entirely yellow, with no green tint, are to be classified as abnormal. This requires that the
175

germination test be conducted with light to allow chlorophyll production in the cotyledons. Seedlings which have their seed coats attached at final count should not be considered to be abnormal due to yellow cotyledons. If it is apparent that the sample has a yellow cotyledon problem and there are a significant number of seedlings with their coats attached, then the test should be extended to allow shedding of the coats, or the sample should be retested using higher intensity light. In assessing seedlings with yellow cotyledons, analysts should make a rapid decision by scanning the replicate and removing obviously yellow seedlings, then continuing the evaluation without re-considering the first assessment of yellow. The assessment of yellow cotyledons may be aided by using a colour reference (e.g. the yellow cotyledons of a Brass i ca seedl i ng which has not been exposed to l i gh t ) . I f there i s any hint of green in the yel l ow area, then the seedl ing should not be class i f i ed as abnormal due to the yel l ow coty ledon condi t i on .
3

The evaluat i on of yel l ow coty ledons Brass in i ca di f f e r s f rom the AOSA descr ip t i on , which does not speci f i c a l l y descr ibe th i s condi t i on .

176

SEED HEALTH TESTING IN BRASSICACEAE CROPS

5.

The main seed-borne Brass ica spp. pathogens with common names of the diseases they cause Pathogen
Alternaria brassicae (Berk.) Sacc. Alternaria brassic ico la (Schw.) Wilts. , syns. A. oleracea Milbrai th, A. circ inans (Berk, and Curt . ) Bol le Ascochyta oleracea J . W. El l i s Leptosphaeria maculans (Desm.) Ces. and de Not. , syns. Phoma l i ngam (Tode ex Fr. ) Desm., Plenodomus l ingani (Tode ex Hohnel

Sr. No .
1 2 3 4

Common Names
Grey leaf spot, Black spot, wirestem Grey leaf spot , Black wirestem Leaf spot Dry rot , black leg,

6.

The main seed-borne Radish pathogens with common names of the diseases they cause Pathogen Common Names

Sr. No.

177

1 2 3 4 5 6 7

Alternaria brassicae (Berk.) Sacc. Alternaria brassic ico la (Schw.) Alternar ia raphani Groves and Skolko , syn. A. matthio laeNeergaard Col le tot r i chum higgins ianum Sacc.

Grey leaf spot Black leaf spot Leaf spot

Anthracnose, leaf spot Leptosphaer ia maculans (Desm.) Ces. and de Black leg Not. , syns. Plenodomus l i ngam (Todc ex Fr. ) Hohnel Phoma lingam (Tode ex Fr.) Rhizoctonia solani Kuhn Damping off, canker Xanthomonas vesicatoria (Doidge) Dowson Bacter ia l spot var. raphani (White) Star and

Detection of Phoma l ingam on Brassica spp.

[ISTA Validated Method]
Published by: International Seed Testing Association (ISTA), Bassersdorf, Switzerland, 2008

Pathogen : Phoma l i ngam (Tode ex Fr . ) Desm. syn Plenodomus lingam (Tode ex Fr.) Hohn perfect state Leptosphaeria maculans (Tode ex Fr.) Ces. & de Not. Prepared by: ISTA-PDC Method Validation Sub-committee Revision History:Version 1.0 November 19, 2001 Revised 19.11.2001 J. Sheppard, V. Cockerell Reprinted 2003 Version 1.1 2008-01-01 Treated seed revised; Reporting results revised Submitted by: ISTA-PDC Method Validation Sub-committee Background This method was originally published in the ISTA Handbook of Seed Health Testing in November 1964 as S.3. No. 31. The method was incorporated into the newly revised Annexe to Chapter 7 in 2002 from the 1999 edition of the ISTA Rules.
178

The method was reviewed by the ISTA-Seed Health Committee in 2006 (Cockerell & Koenraadt, 2007) with the recommendation to accept for a further five years. Summary of Validation Study Studied in International Comparative Testing 1959, 1978, 1981, 1983 Safety Precautions Ensure you are familiar with hazard data and take appropriate safety precautions, especially during preparation of media, autoclaving and weighing out of ingredients. It is assumed that this procedure is being carried out in microbiological laboratory by persons familiar with the principles of Good Laboratory Practice, Good Microbiological Practice, and aseptic technique. Dispose of all waste materials in an appropriate way (e.g. autoclave, disinfect) and in accordance with local safety regulations. Treated Seed This method has not been validated for the determination of Phoma l i ngam on t reated seed. Seed treatments may af fec t the perfo rmance of the method. (Definition of treatment: any process, physical, biological or chemical, to which a seed lot is subjected, including seed coatings. See 7.2.3) Materials Reference Material - The use of reference cul tu res or other appropr ia te mater ia l i s recommended when ever poss ib le . Media - Blot te r s ( f i l t e r equiva lent paper) , e.g . Whatman No 1 or

179

2,4 Dichlorophenoxyacetate (2,4D) - Sodium sal t 0.2% solu t i on Petri dishes - When sowing densi ty i s given by a number of seeds per Petr i dish , a diameter of 90 mm i s assumed. Incubator - Capable of operat ing in the range 20 2C. To st imulate sporu la t i on , al te rnat i ng 12 h per iods of darkness and near- ul t rav i o l e t l i gh t (NUV) dur ing incubat ion are recommended. The recommended source i s the black l i gh t fluorescent lamp (peak at 360 nm) but daylight fluorescent tubes are satisfactory. Sample Preparation The test is carried out on a working sample of 1000 pure seeds as described in Section 7.4.1 of the ISTA Rules. Method 1. Blot te r Place three pieces of filter paper (Whatman No. 1 or equivalent) in each Petri dish and add 5 mL of a 0.2% solution of the sodium salt of 2,4 dichlorophenoxyacetic acid (2,4-D) to inhibit seed germination. Pour of the excess 2,4-D solution, rinse the seeds in sterile water and place 50 in each dish. 2. Pret reatment: None. 3. Incubat ion Incubate for 11 days at 20 C with alternating cycles of 12 h light and 12 h darkness. 4. Examinat ion After 6 days, examine at 25 magnification for loose growing silver white mycelium (Fig. 1) and pycnidial primordia of Phoma l i ngam on the seed and substrate. After 11 days, make a second examination for pycnidia on infected seeds and on the filter paper near infected seeds.
180

Seeds from which pycnidia of Phoma l i ngam have developed are recorded as in fec ted . Pycnidia are relatively large, about 250 m, with papilla, sometimes developed as a neck (Figs. 2, 3). The ubiquitous saprophyte Phoma herbarum Westend occurs also on Brass i ca seed (F ig . 4) , but has smal le r pycnid ia formed super f i c i a l l y on the seed coat , not papi l l a t e , with white yel l ow or pink but not purple (amethyst ) exudate. General Methods (common to many test procedures) 1. Checking tolerances Tolerances provide a means of assessing whether or not the variation in result within or between tests is sufficiently wide as to raise doubts about the accuracy of the results. Suitable tolerances, which can be applied to most direct seed health tests, can be found in Tables 5B of Chapter 5 of the ISTA Rules, or in the Handbook of Tolerances and Measures of Prec i s i on for Seed Test by ing S.R. Miles (Proceedings of the International Seed Testing Association 28 (1963) No 3, p 644). 2. Reporting Results The result of a seed health test should indicate the scientific name of the pathogen detected and the test method used. When reported on an ISTA International Seed Analysis Certificate, results are entered under Other Determinations. Quality Assurance Crit ical Control Points: None listed

181

Fig-1: Seed in blotter test showing white mycelium and pycnidia with amethyst coloured spore exudate.

References The following references are extracted from the ISTA Handbook on Seed Health Testing, Working Sheet No. 31, 1964. Boerema, G. H., (1964): Phoma herbarum Westend. , the type species of the form genus Phoma Sacc. Persoonia 3, 9-16. Boerema, G. H. & Van Kesteren, H. A., (1964): The nomenclature of two fungi parasitizing brassica. Persoonia, 3, 17-28.

182

Fig-2: Infected seedling in blotter test showing pycnidia on the hypocotyls and cotyledons.

183

Fig. 3a and 3b. Pycnidia on seed

Fig. 4. Phoma herbarum on seed

184

Detection of Xanthomonas campestris on Brassica spp. [ISTA Validated Method] Crop: Brass i caspp. (brocco l i , caul i f l ower , oi l seed rape)

pv. campestris

cabbage, calabrese, canola ,

Pathogen: Xanthomonas campestr i s pv. campestris (black rot) Prepared by: Roberts, S.J.1 and Koenraadt, H.2 1Plant Health Solut i ons , 20 Beauchamp Road, Warwick, CV34 5NU, UK. E-mail: s.roberts@planthealth.co.uk 2Naktuinbouw, P.O. Box 40, 2370 AA Roelofarendsveen, The Netherlands. E-mail: h.koenraadt@naktuinbouw.nl Revision History: Version 1.0, 13 May 2003. Version 2.0, 06 August 2004. Version 3.0, 05 July 2006. Background This method is based on methods originally published by Franken et al. (1991) and in the 2nd edition of Working Sheet No. 50 in the ISTA Handbook of Seed Health Testing (Schaad and Franken, 1996). Compared to the 2nd edition of Working Sheet No. 50, this version incorporates a number of modifications resulting from comparative tests in 13 laboratories (Koenraadt et al., 2004), a study done in a single laboratory (Roberts et al., 2004), and experience of routine testing in a number of laboratories. Summary of modifications: no fungicides used in extraction buffer; NSCAA medium replaced by mCS20ABN; no centrifugation step after 5 min; continuous shaking instead of static incubation; only one plate of each medium per dilution;
185

removal of check for antagonistic bacteria; minor changes to media preparation; simplified pathogenicity test method; removal of IF and direct plating assays; changes to format and layout. This method differs from that originally proposed in the validation report (Roberts and Koenraadt, 2003) by the omission of a centrifugation step, which may theoretically give a reduced analytical sensitivity (Roberts et al . ,2004) . Users of th i s method should be aware that the values quoted for analyt i ca l sens i t i v i t y (detect i on l imi t s ) theoret i ca l ; in pract i ce the actua l leve l of sens i t i v i t y achieved wi l l vary with the background leve l of saprophytes . Details of how to use centrifugation with this method and other methods will be included in the ISTA Seed Health Handbook which is currently in preparation. Validation Studies Roberts, S.J. and Koenraadt, H. (2003) Copies are available: by E-mail from ista.office@ista.ch; by mail from the ISTA Secretariat. Reproducibility: dispersion = 1 Repeatability: dispersion = 1 Detection limits: 15 cfu/ml (theoretical, P=0.95) Safety Precautions Ensure you are familiar with hazard data and take appropriate safety precautions, especially during preparation of media, autoclaving, and weighing out of ingredients. It is assumed that this procedure is being carried out in a microbiological laboratory by persons familiar with the principles of Good Laboratory Practice, Good Microbiological Practice, and aseptic technique. Dispose of all waste materials in an appropriate way (e.g. autoclave, disinfect) and in accordance with local health, environmental and safety regulations. Treated Seed

186

Seed treatments may affect the performance of this test. It must only be performed on untreated seed. Note. Brass i ca seed subjected to a physica l treatment , example, hot water i s regarded as treated seed. Sample and sub-sample size The sample (total number of seeds tested) and sub-sample size to be tested depends on the desired tolerance standard (maximum acceptable percentage of seeds infested) and detection limit (theoretical minimum number of pathogen propagules per seed which can be detected), in any case the maximum sub-sample size should be 10 000 seeds. A full discussion of these aspects can be found in Geng et al . (1987) , Roberts et al. (1993) and Roberts (1999). Materials
Reference material Plates of FS medium Plates of mCS20ABN medium Plates of YDC Conical flasks Dilution bottles 70% ethanol Incubator Balance pH meter Automatic pipettes Brassica seedlings Known strain of Xanthomonas campestr i s pv. campestris or standardised reference material. 9.0 cm Petri dishes (3 plates of each medium per sub-sample + controls) 9.0 cm Petri dishes (3 plates of each medium per sub-sample + controls) for sub-culture (at least 1 per sub-sample). of sterile saline (0.85% NaCl) plus Tween 20 (0.02% - 20l per 100 ml) for soaking of seeds (10 ml per 1000 seeds). containing 4.5 ml of sterile saline (2 per sub-sample). Other volumes may be acceptable, see General Methods for disinfection of surfaces, equipment operating at 28-30C capable of weighing to the nearest 0.001 g capable of being read to the nearest 0.01 pH unit check accuracy and precision regularly susceptible to all races of the pathogen (e.g. B. oleracea cv. Wirosa) for 187

for

pathogenicity test Orbital shaker Sterile pipette tips Sterile bent glass rods

Sample Preparation 1. This can be done in advance of the assay. 2. It is vital to exclude any possibility of cross-contamination between seed samples, it is therefore essential to disinfect all surfaces, containers, hands, etc. both before and after handling each sample. This can achieved by swabbing/spraying equipment and gloved hands with 70% ethanol. 3. If the submitted sample is received in several packets, these should be combined by emptying into a new, clean polythene bag and mixing by hand to give a composite sample. 4. Count the number of seeds in a known weight. Estimate the Thousand Seed Weight (TSW) as:

5. Based on the TSW, weigh out sub-samples of the required size into new, clean polythene bags. Method [Critical control points are indicated by CCP ] 1. Extrac t i on 1.1. Suspend seeds in pre-chilled (2-4C) sterile saline plus Tween 20 (0.02% v/v) in a conical flask. The volume of saline should be adjusted according to the number of seeds used (10 ml per 1,000 seeds). 1.2. Shake for 2.5 h at room temperature (20-25C) on an orbital shaker set at 100-125 rpm. 2. Di lu t i on and plat i ng
188

2.1. Shake the flasks to mix just before dilution. 2.2. Prepare two serial tenfold dilutions from the seed extract. Pipette 0.5 ml of the extract into 4.5 ml of sterile saline and vortex to mix (101 dilution). Pipette 0.5 ml of the 101 dilution into another 4.5 ml of sterile saline and vortex to mix (102 dilution) (see General Methods). 2.3. Pipette 100 l of each dilution and the un-diluted seed extract onto plates of each of the selective media (FS, mCS20ABN) and spread over the surface with a sterile bent glass rod (see General Methods). 2.4. Incubate plates at 28-30C and examine after 3-4 d. 3. Posi t i ve contro l (cu l tu re or reference mater ia l ) 3.1. Prepare a suspension of a known strain of X. campestr i s pv. campestr i s in sterile saline or reconstitute standardized reference material according to the suppliers instructions. 3.2. Dilute sufficiently to obtain dilutions containing approx. 102 to 104 cfu/ml. This may require up to seven ten-fold dilutions from a turbid suspension. 3.3. Pipette 100 l of appropriate dilutions onto plates of each of the selective media (FS, mCS20ABN) and spread over the surface with a sterile bent glass rod. 3.4. Incubate plates with the sample plates. 4. Ster i l i t y check 4.1. Prepare a dilution series from a sample of the extraction medium (i.e., saline plus Tween 20), containing no seeds, and plate on each of the media as for samples. 5. Examinat ion of the plates 5.1. Examine sterility check and positive control plates (CCP ). 5.2. Examine the sample plates for the presence of typical X. campestr i s pv. campestr i s (Xcc) colonies by comparison with the positive control plates. 5.3. On FS after 3-4 d, Xcc colonies are small, pale green, mucoid and surrounded by a zone of starch hydrolysis. This zone appears as a halo that may be easier to see with a black background (Fig. 1a). Colonies may show marked

189

variation in size and may be visible on FS after 3 d; if not, incubate for an additional day. 5.4. After 3-4 d on mCS20ABN, Xcc colon ies are pale yel l ow, mucoid and surrounded by a zone of starch hydro lys i s (F ig 1b) . Colonies may show marked var ia t i on in s ize . Depending on the number of colon ies present , i t may be easier to evaluate plates after 3 d, before coalescence of starch hydro lys i s zones which can make i t more di f f i c u l t to ident i f y suspect colon ies . 5.5. Incubation of the plates at 4C for several hours before recording may result in sharper zones of starch hydrolysis with some starch sources. 5.6. Record the number of suspect and other colonies (see General Methods). 6. Conf i rmat ion / i dent i f i c a t i o n of suspect colon ies 6.1. Sub-culture suspect colonies to sectored plates of YDC. To avoid the potential for cross-contamination of isolates, use a new sectored plate for each sub-sample. The precise numbers of colonies sub-cultured will depend on the number and variability of suspect colonies on the plate: if present, at least six colonies should be sub-cultured per sub-sample (CCP ) . 6.2. Sub-culture the positive control isolate to a sectored plate for comparison. 6.3. Incubate sectored plates for 24-48 h at 28-30C. 6.4. Compare appearance of growth with positive control. On YDC X. campestr i s pv. campestris colonies are pale yellow and mucoid/fluidal (Fig 2). 6.5. Confirm the identity of isolates by pathogenicity on Brassica seedlings of known susceptibility (CCP ). 6.6. Record results for each colony sub-cultured. 7. Pathogenicity 7.1. Grow seedlings of a Brassica cultivar known to be susceptible to all races of X. campestris pv. campestris (e.g. cabbage cv. Wirosa, see Vicente et al., 2001) in small pots or modules until at least 3-4 true leaf stage.

190

7.2. Scrape a small amount of bacterial growth directly from a 24-48 h YDC culture (e.g. sectored plate) with a sterile cocktail stick or insect pin. 7.3. Inoculate six of the major veins at a point near the leaf edges on the two youngest leaves by stabbing with the cocktail stick or insect pin. 7.4. The number of plants which should be inoculated will depend on the variability of the cultivar and experience of the operator, but 1-3 plants per isolate should usually be sufficient. It is better to inoculate more isolates with 1 plant per isolate than fewer isolates with 3 plants per isolate. 7.5. Inoculate with the positive control isolate and stab with a sterile cocktail stick or insect pin as a negative control (CCP ) . 7.6. Grow on plants at 20-25C. 7.7 Examine plants for the appearance of typical progressive V-shaped, yellow/necrotic lesions with blackened veins after 10-14 d. (See Fig. 3). Symptoms may be visible earlier depending on temperature and the aggressiveness of the isolate. Compare with positive control (CCP ) . I t i s important to discr iminate between the progress ive les i ons caused by the vascula r pathogen X. campestr i s pv. campestris and the limited dark necrotic lesions at the inoculation site caused by leaf spot Xanthomonas (often classified as either X. c. pv. armoraciae or X. c. pv. raphani (see Kamoun et al. 1992; Alvarez et al., 1994; Tamura et al., 1994; Vicente et al., 2001; Roberts et al., 2004).

191

Fig- 1: Plates of FS (a) and mCS20ABN (b) media afte r 4 d incubat ion at 30C showing typica l colon ies Xanthomonas of campestr i s pv. campestris surrounded by zones of starch hydrolysis.

Fig- 2: Typical pale yellow mucoid/fluidal growth of Xanthomonas campestris pv. campestris isolates on a sectored plate of YDC after 48 h at 30C.

192

Fig- 3: Leaves of a cabbage seedl ings cv. Wirosa, 10 d (at 22C) afte r inocula t i on : (a) ster i l e contro Xanthomonas l , (b) campestr i s pv. campestris Race 1, (c) Race 3, (d) Race 5, (e) leaf spot Xanthomonas.

General Methods (common to many test procedures) 1. Preparat i on of ten- fo ld di lu t i on ser ies Each dilution should be prepared by pipetting 0.5 ml ( 5%) from a well-mixed seed extract or previous dilution into a universal bottle (screw-capped) or similar containing 4.5 ml ( 2%) of sterile diluent and then vortexing to mix prior to the next dilution step. A new sterile pipette tip should be used for each dilution step. Pipettes should be checked regularly for accuracy and precision and re-calibrated as necessary. It is acceptable to prepare ten-fold dilutions using other volumes provided that the laboratory can demonstrate that the required accuracy and precision can be achieved 2. Plating of dilutions. This should be done as soon as possible after dilutions have been prepared and certainly within 30 min. Working from the highest (most dilute) dilution to the undiluted extract, 0.1 ml is pipetted onto the centre of a surface-dry, labelled agar plate. The liquid should then be spread evenly over the entire surface of the medium with a bent glass rod. If care is taken to work from the highest to the lowest dilution (or undiluted extract) a single pipette tip and a single bent glass rod can be used for each sample. Ensure that all liquid has been absorbed by the agar before inverting and incubating plates. If necessary allow plates to dry under a sterile airflow in a microbiological safety cabinet or laminar flow hood.

3. Recording of dilution plates Record the results for al l di lu t i on plates . The most accurate est imate of bacter i a l numbers should be obta ined f rom spread plates with tota l number between 30 and 300 colon ies . However th i s may be fur ther compl ica ted depending on the re la t i ve numbers of suspect pathogen and other colon ies . In order to minimise ef fo r t , star t record ing with the highest di lu t i on (most di lu te ) and count the number
193

of suspect and the number of other colonies. If the total number of colonies on a plate greatly exceeds 300 there is little value in trying to make a precise count if a more reliable count has already been obtained from a more dilute plate, in which case it is sufficient to record the number of colonies as m (many) if they are still separate or c (confluent) if they have run together. 4. Sectored Plates Using a laboratory marker pen draw lines on the base of a standard 9 cm plate (Petri dish) to divide it into six equal sectors: Sub-culture single colonies from dilution plates and make a single zigzagged streak within a single sector on the plate. Take care to leave sufficient space between each isolate to ensure the growth does not coalesce. Thus six suspect colonies can be sub-cultured to each sectored plate. Separate plates should be used for each sample/sub-sample. If the purity of sub-cultured isolates is doubtful, they should be further streaked out on whole plates. 5. Reporting Results The result of a seed health test should indicate the scientific name of the pathogen and the test method used. When reported on an ISTA Certificate, results are entered under Other Determinat ions . In the case of a negative result (pathogen not detected in any sub-samples), the results should be reported in terms of the tolerance standard and detection limit. The tolerance standard depends on the total number of seeds tested, n, and is approximately 3/n (P=0.95) (see Roberts et al .1993); , the detect i on l imi t per sub- sample i s equal to the detect i on l imi t per ml mult ip l i ed by the volume of extract . In the case of a positive result, the report should indicate the mean number of pathogen propagules (cfu) per seed and either the number of positive sub-samples out of the total number tested and the sample size or the maximum likelihood estimate of the proportion of infested seeds.

194

Quality Assurance General A record should be kept of the date and results of pipette calibration checks. It is essential that operators have received appropriate training and use automatic pipettes correctly. Critical Control Points [Identified by CCP in the methods] Dilution plates prepared from positive control isolate(s) or reference material, should give single colonies with typical morphology (Step 5.1). The numbers of colonies on dilution plates prepared from the positive control isolate(s) or reference material should be similar on both media (Step 5.1). Numbers of bacteria on dilution plates should be consistent with the dilution (i.e. should decrease approx. ten-fold with each dilution) (Step 5.1). There should be no growth on dilution plates prepared as a sterility check (Step 5.1). Due to the potential for non-pathogenic isolates to be present in seedlots together with pathogenic isolates, it is essential to sub-culture (Step 6.1), if present, at least the minimum number of suspect colonies specified (six per subsample) and to test all Xanthomonas - l i ke sub- cul tu red i so l a tes for pathogenic i t y (Step 6.5) . Positive control isolates should be included in every pathogenicity test (Step 7.5). The positive control isolate should give typical symptoms in pathogenicity test (Step 7.7). The source of starch used in the selective media is critical for observation of starch hydrolysis. Verify that each new batch of starch gives clear zones of hydrolysis with reference

195

cultures of X. campestr i s pv. campestris (FS and mCS20ABN media). The activity (units/mg) of some antibiotics may vary between batches. It may be necessary to adjust the weight or volume added to ensure that the final number of units per liter of medium is consistent (FS and mCS20ABN media). The activity of neomycin against some strains of Xcc is known to be affected by pH. It is essential that the pH of the medium is less than 6.6 (mCS20ABN medium, Step 3) Preparation of sterile saliner Compound Sodium chloride (NaCl) Distilled/ de-ionised water Preparation g/l 8.5 1000 ml g/500 ml 4.25 500 ml

1. Weigh out all ingredients into a suitable container. 2. Add 1000 ml (or 500 ml) of distilled/de-ionised water. 3. Dissolve and dispense into final containers. 4. Autoclave at 121C, 115 psi for 15 min. 5. For extraction of seeds, add 20 l of sterile Tween 20 per 100 ml after autoclaving Storage Provided containers are tightly closed, may be stored for several months before use. Preparation of mCS20ABN agar medium Note. This medium is based on, but not identical to, the medium of Chang et al. (1991), from which it differs in starch source and concentration, cycloheximide concentration and solution media for antibiotics. The amounts of phosphate salts have also been adjusted to achieve the correct pH without further adjustment

196

Compound Soya Peptone (Oxoid L44) Bacto Tryptone (Difco Bacto) KH2PO4 (NH4)2HPO4 MgSO4.7H2O L-Glutamine (Sigma G-3126) L-Histidine (Sigma H8000) D-Glucose (Dextrose) Distilled/de-ionised water Soluble starch (Aldrich No 17,993-0) (CCP ) Bacto Agar (Difco) Cycloheximidea (200 mg/ml 70% EtOH) Neomycinb (40 mg/ml in 20% EtOH) Bacitracinc (100 mg/ml in 50% EtOH) Preparation

g/l 2.0 2.0 1.59 0.33 0.4 6.0 1.0 1.0 1000 ml 25.0 15.0 200 mg (1 ml) 40 mg (1 ml) 100 mg (1 ml)

g/ 500 ml 1.0 1.0 0.79 0.17 0.2 3.0 0.5 0.5 500 ml 12.5 7.5 100 mg (0.5 ml) 20 mg (0.5 ml) 50 mg (0.5 ml)

a, b, c Added after autoclaving

1. Weigh out all ingredients except agar, starch and antibiotics into a suitable container. 2. Add 1000 ml (or 500 ml) of distilled/de-ionised water. 3. Dissolve and check pH which should be 6.5, adjust if necessary (important, CCP ) . 4. Add starch and agar then steam to dissolve. 5. Autoclave at 121C, 115 psi for 15 min. 6. Prepare antibiotic solutions if necessary.

197

7. Allow medium to cool to approx. 50C and add antibiotic and methionine solutions. 8. Mix thoroughly but gently by inversion/swirling to avoid air bubbles and pour plates (22 ml per 9.0 cm plate). 9. Leave plates to dry in a laminar flow bench or similar before use. Antibiotics (amounts for guidance only, CCP ) a. Dissolve 2.0 g cycloheximide (Sigma C-7698) in 10 ml 70% ethanol. Add 1 ml/l (0.5 ml/500 ml). b. Dissolve 400 mg neomycin (Sigma N-1876) in 10 ml 20% ethanol. Add 1 ml/l (0.5 ml/500 ml). c. Dissolve 1.0 g bacitracin (Sigma B-0125, 66k units/g) in 10 ml 50% ethanol. Add 1 m/l (0.5 ml/500 ml). Storage Store prepared plates inverted in polythene bags at 4C and use within two weeks of preparation to ensure activity of antibiotics. Depending on the source of starch, pre-storage in the refrigerator for several days before use may result in more easily visible zones of starch hydrolysis. Preparation of FS agar medium

Note. A number of di f f e ren t vers ions of th i s medium have been publ i shed; for the sake of consis tency , the rec ipe give below i s the one refer red to (Schaad, 1989) in the previou working sheet . Compound g/l Bacto Agar 15.0 Soluble starch (Aldrich No 10.0 17,993-0) (CCP ) Bacto yeast extract 0.1 (Difco) K2HPO4 0.8 KH2PO4 0.8 MgSO4.7H2O 0.1 g/ 500 ml 7.5 5.0 0.05 0.4 0.4 0.05

198

Methyl Green (1% aq.) Distilled/de-ionized water Cycloheximidea (200 mg/ml 70% EtOH) D-methionineb (3 mg/ml 50% EtOH) Pyridoxine-HClc (1 mg/ml 50% EtOH) Cephalexind (50 mg/ml 70% EtOH) Gentamycine (1 mg/ml H2O) Trimethoprimf (10 mg/ml 70% EtOH) Preparation

1.5 ml 1000 ml 200 mg (1 ml) 3 mg (1 ml) 1 mg (1 ml) 50 mg (1 ml) 0.4 mg (0.4 ml) 30 mg (3 ml)

0.75 ml 500 ml 100 mg (0.5 ml) 1.5 mg (0.5 ml) 0.5 mg (0.5 ml) 25 mg (0.5 ml) 0.2 mg (0.2 ml) 15 mg (1.5 ml)

a, b, c, d, e, f Added after autoclaving

1. Weigh out all ingredients except antibiotics and methionine into a suitable container. 2. Add 1000 ml (or 500 ml) of distilled/de-ionised water. 3. Steam to dissolve. 4. Autoclave at 121C, 115 psi for 15 min. 5. Prepare antibiotic and methionine solutions and filter sterilise as appropriate 6. Allow medium to cool to approx. 50C before adding antibiotic and methionine solutions. 7. Mix the molten medium gently to avoid air bubbles and pour plates (22 ml per 9.0 cm plate). 8. Leave plates to dry in a laminar flow bench or similar before use. Antibiotics (amounts for guidance only, CCP ) a. Dissolve 2 g cycloheximide in 10 ml 70% ethanol. Add 1 ml/l (0.5 ml/500 ml). b. Dissolve 60 mg D-methionine in 10 ml distilled water, then add 10 ml ethanol. Add 1 ml/l (0.5 ml/500 ml). c. Dissolve 20 mg pyridoxine in 20 ml 50% ethanol. Add 1 ml/l (0.5 ml/500 ml).

199

d. Dissolve 500 mg cephalexin in 10 ml 70% ethanol. Add 1 ml/l (0.5 ml/500 ml). e. Dissolve 10 mg gentamycin in 10 ml distilled water, filter sterilize. Add 0.4 ml/l (0.2 ml/500 ml). f. Dissolve/suspend 200 mg trimethoprim in 20 ml 70% ethanol. As the chemical may not dissolve fully it may be necessary to vortex the suspension immediately before adding it to the medium (3 ml/l or 1.5 ml/500 ml). Storage Store prepared plates inverted in polythene bags at 4C and use within two weeks of preparation to ensure activity of antibiotics. Depending on the source of starch, pre-storage in the refrigerator for several days before use may result in more easily visible zones of starch hydrolysis. Preparation of Yeast Dextrose Chalk (YDC) agar medium Compound Bacto Agar Yeast Extract CaCO3 (light powder) D-Glucose (Dextrose) Distilled/ de-ionized water Preparation 1. Weigh out all ingredients into a suitable oversize container (i.e. 250 ml of medium in a 500 ml bottle/flask) to allow swirling of medium just before pouring. 2. Add 1000 ml (or 500 ml) of distilled/de-ionised water. 3. Steam to dissolve. 4. Autoclave at 121C, 115 psi for 15 min. 5. Allow medium to cool to approx. 50C . g/l 15.0 10.0 20.0 20.0 1000 ml g/500 ml 7.5 5.0 10.0 10.0 500 ml

200

6. Swirl to ensure even distribution of CaCO3 and avoid air bubbles, and pour plates (22 ml per 9.0 cm plate). 7. Leave plates to dry in a laminar flow bench or similar before use. Storage Store prepared plates inverted in polythene bags at room temperature. Prepared plates can be stored for several months provided they do not dry out. References
Alvarez, A.M., Benedict, A.A., Mizumoto, C.Y., Hunter, J.E. and Gabriel, D.W. (1994) Serological, pathological, and genetic diversity among strains of Xanthomonas campestr i s in fec t i ng cruc i f e r sPhytopathology . 84 , 1449-1457. Chang, C.J., Donaldson, R., Crowley, M. and Pinnow, D. (1991) A new semi-selective medium for the isolation of Xanthomonas campestris pv. campestris from crucifer seed. Phytopathology 81 , 449-453. Geng, S., Campbell, R.N., Carter, M. and Hills, M. (1987) Quality control programs for seedborne pathogens. Plant Disease 67 , 236-242. Kamoun, S., Kamdar, H.V., Tola, E. and Kado, C.I. (1992) Incompatible interactions between crucifers and Xanthomonas campestris involve a vascular hypersensitive response: role of the hrpX locus. Molecular Plant-Microbe Interactions 5, 22-33. Koenraadt, H., van Bilsen, J.G.P.M. and Roberts, S.J. (2004) Comparative test for the detection of Xanthomonas campestris pv. campestris in Brassica seeds. Seed Science and Technology (in press). Roberts, S.J. (1999) Thresholds, standards, tests, transmission and risks. In: Proceedings of 3rd ISTA Seed Health Symposium, Ames, Iowa, USA, 16-19 August 1999. pp. 20-24. ISTA, Zurich, Switzerland.

201

Roberts, S.J., Brough, J., Everett, B. and Redstone, S. (2004) Extraction methods for Xanthomonas campestr i s pv. campestris from brassica seed. Seed Science and Technology 32 , 439-453. Roberts, S.J. and Koenraadt, H. (2003) ISTA-PDC Technical report: Revised method for detection of Xanthomonas campestris pv. campestris in Brassica seed. ISTA Method Validation Reports 1, 19. Roberts, S.J., Phelps, K., Taylor, J.D. and Ridout, M.S. (1993) Design and interpretation of seed health assays. In: Sheppard, J.W., (Ed.) Proceedings of the First ISTA Plant Disease Committee Symposium on Seed Health Testing, Ottawa, Canada. pp. 115125. Agriculture Canada, Ottawa, Canada. Schaad, N.W. (1989) Detection of Xanthomonas campestris pv. campestris in Crucifers. In: Saettler, A.W., Schaad, N.W. and Roth, D.A., (Eds.) Detection of bacteria in seeds and other planting material, pp. 68-75. American Phytopathological Society, St. Paul, Minnesota, USA. Schaad, N.W. and Franken, A.A.J.M. (1996) ISTA Handbook on Seed Health Testing Working Sheet No 50 (2nd Ed): Xanthomonas campestris pv. campestris. ISTA, Zurich, Switzerland. Tamura, K., Takikawa, Y., Tsuyumu, S. and Goto, M. (1994) Bacterial spot of crucifers caused by Xanthomonas campestris pv. raphani. Annals of the Phytopathological Society of Japan 60 , 281-287. Vicente, J.G., Conway, J., Roberts, S.J. and Taylor, J.D. (2001) Identification and origin of Xanthomonas campestris pv. campestris races and related pathovars. Phytopathology 91 , 492499.

Method for the Detection of Xanthomonas campestris pv. campestris in disinfested seed of Brassica spp. [Adapted from ISF]

202

Crop : Brass i caspp. (brocco l i , caul i f l ower , oi l seed rape)

cabbage, calabrese, canola ,

Pathogen : Xanthomonas campestr i s pv. campestris (X.c. pv. campestris) Revision history : Version 1, January 2008 Sample and sub-sample size The recommended minimum sample size is 30,000 seeds with a maximum sub-sample size of 10,000 seeds. Background To facilitate the detection of internally located propagules of Xanthomonas campestris pv. campestris that may have survived a seed disinfestation treatment, seed must be ground to release surviving propagules. The test method for untreated Brassica seed published as method 7-019 in the ISTA rules (1) involves only extraction by soaking. An adaptation of this method involving wet grinding of the seed has been found to strongly enhance the detection of Xcc in disinfested seed and is described here. Other changes to the original method for untreated seed are: buffering of the soaking solution, a larger ratio of buffer to seed, concentration of the extract by centrifugation and a longer incubation time of the plated extracts. Changes with respect to method 7-019 are in the Method Description below. The paragraph numbers refer to method 7-019 in the ISTA rules. It is emphasized that the method for untreated seeds remains unchanged. For treated seeds, in cases when they are infested with this pathogen, the Xanthomonas bacteria are located on the seed surface and in the funiculus of the seed from which they are readily released by soaking. Restrictions on use

203

o This test i s sui tab le for seed that has been treated using chemicals with the aim of dis in fes prov ta t i on ided no res idue i s le f t on the, or seed seed treated by physical processes for disinfestation. o It has not been validated for seed treated with protective chemicals or biological substances. Sensitivity reference The ability to recover X.c . pv. campestr i s on plates can be influenced by the presence of other micro-organisms and inhibiting factors in the ground seed extract. Validation This method allows the detection of Xanthomonas campestr i s pv. campestr i s in samples of treated seeds. Dry grinding the seeds followed by the addition of an extraction buffer has not been found to be adequate. Soaking followed by wet grinding is a prerequisite. Comparative tests have shown higher numbers of positive subsamples using a buffered extraction solution when compared to a non-buffered solution (saline). A drop in pH is consistently measured when Brassica seeds are ground. Buffering compensates for this pH reduction. This method has been in use in the seed industry since 2006 and will be validated by the ISTA method validation procedure in 2008. Additional materials o Grinding equipment o Centrifuge able to provide a centrifugal force of 5,000 g

204

Method description Note Only the changes with respect to method 7- 019 in the ISTA Rules are descr ibed below. The paragraph numbers refer to those used in method 7- 019 and, i f not otherwise stated , should be used ins tead of those used in 7- 019. 1. Extraction 1.1. Suspend each sub-sample in PBS (0.05M phosphate) plus Tween 20 (0.02% v/v) in a conical flask. The volume of PBS should be adjusted according to the number of seeds used (25 ml per 1,000 seeds). 1.2. Shake for 2.5 h at room temperature (20-25C) on an orbital shaker set at 100-125 rpm. 1.3. Grind the seeds with a suitable grinder such as an Ultra Turrax T25 with S25N-25G dispersion tool. Grind till all seeds are completely ground. This point should have been reached in at most 2 minutes of grinding. If not, select alternative grinding equipment. Note Run the gr inder in hot water and/or 70% ethanol and subsequent ly in ster i l e water to prevent any cross contaminat ion between sub- samples. To achieve complete ster i l i z a t i o n between samples the S25N-25G dispers i on too l has to be autoc laved or disassembled and the parts immersed in 70% ethanol . 2. Dilution, concentration and plating 2.1. Filter coarse particles from the extract, using a bag filter model P 400 ml (Interscience, France), universal extraction bag model with synthetic intermediate

205

layer (Bioreba, Switzerland) or filter extraction bags (Neogen Europe, Scotland). 2.2. Transfer 2,5 ml of filtrate to a tube and keep the samples on ice. 2.3. Prepare a tenfold dilution from the filtered seed extract. Pipette 0.5 ml of the extract into 4.5 ml of sterile PBS (0.05M phosphate) plus Tween 20 (0.02% v/v) and vortex to mix (101 dilution). 2.4. Prepare a tenfold concentrated extract (101 concentration) by centrifugation of the remaining 2 ml sample for 5 minutes at 5,000 g. Carefully remove the supernatant and re-suspend the pellet in 200 l of sterile PBS (0.05M phosphate) plus Tween 20 (0.02% v/v). 2.5. Pipette 100 l of the 101 dilution, the un-diluted extract and the 101 concentration onto two plates of each of the selective media (mFS, mCS20ABN) and spread over the surface with a sterile bent glass rod. 2.6. Incubate plates at 28-30C and examine after 4-6 d. Note: This is a longer incubation time than is required when testing untreated seed and than is required for the positive control.
Continue with step 3 and subsequent steps in ISTA Method 7-019

Preparation of sterile PBS 0.05 M plus Tween 20 (0.02% v/v) NaCl 8,0 g/L Na2HPO4 5,75 g/L KH2PO4 1,0 g/L Tween 20 0,2 ml/L pH 7,2-7,4 Preparation of mCS20ABN agar medium
206

Described in ISTA 7-019. Preparation of mFS agar medium This medium differs from FS medium described in ISTA 7-019 in the following ingredients: o Solub le starch 25,0 g/L ( in stead of 10,0 g/L) o Gentamycin 0,0 mg/L ( in stead of 0,4 mg/L) Reference
1. Roberts ,

S. J . and Koenraadt , H (2005) . Detect i on of Xanthomonas campestr i s pv. campestris on Brassica spp. Method description 7-019 (2005). ISTA International Rules for Seed Testing, Annex to Chapter 7 Seed Health Testing Methods 2003.

207

Vegetable Crops of Family Fabaceae

(Peas and Beans)
1.

Seed unit: True seed accord ing to botanica l def in i t i o n of seed. Submitted Sample Weight:

2.

Cyamopsis tet ragonoloba L. (Cluster bean) = 800 g Pisum sat i vumL. (Peas) = 1,000 g Lathyrus sat i vus L. (Ye l l ow pea)1,000 = g Vigna unguicu la ta L. (Cowpea) 1,000 = g Trigonella foenum-graecum (Fenugreek Methe) 1,000 g =
3.

Working Sample Weight:

Cyamopsis tet ragonoloba L. (Cluster bean) = 80 g Pisum sat i vumL. (Peas) = 900 g Lathyrus sat i vus L. (Ye l l ow pea) =0 45 g Vigna unguicu la ta L. (Cowpea) = 0 40 g Trigonella foenum-graecum (Fenugreek Methe) 0 g = 45

4. Purity Analysis:
208

a. Pure Seed: In tac t seeds, with or part i a l l y removed testa , and in the case of pieces of seeds, any piece which i s la rger than one- hal f the or ig ina l s i ze shal l be cons idered pure seed. b. Other Crop Seeds: Al l other species and other botanica l var ie t i e s of same species not i f i ed as crop. c. Weed Seeds: Al l other species and other botanica l var ie t i e s of same species not i f i ed as weeds. d. Inert Matter:Seed with seed- coat ent i re l y removed and seed pieces One-hal f the or ig ina l s i ze or less , non- l i v i ng mater ia l , nematode gal l s and fungus bodies Separated . cotyledons of Fabaceae, irrespective of whether or not the radicle-plumule axis and/or more than half of the seed coat may be attached;

5.

Germination Methods:
Number Temperature Substrata of seeds (C) First Count (days) 5 5 7 5 5 5 Final Count (days) 14 14 10 8 14 8 Additional Directions General Requirements Heavy moisture first few days. Light Additional Directions Fresh/ Dormant Seeds -

Kind of Seed

Cyamopsis tetragonoloba
Lathyrus sativus Phaseolus lunatus Lima bean Pisum sativum Field and garden pea Trigonella foenumgraecum Fenugreek Vigna unguiculata Cowpea

400 400 400 400 400 400

BP BP; S S; RT S; RT; BP TB S; RT

20-30 20 20; 25 20 20-30 20-30

Abbreviations: BP - Between peppers , RT - Rol led towels S - Sand , .

209

7.2 Phaseolus vulgaris, garden bean If hypocotyl collar rot is observed on seedlings, the sample involved may be retested using a 0.3 to 0.6 percent calcium nitrate solution to presoak the substratum. The solution is prepared by dissolving 3 g (for a 0.3% solution) to 6 g (for a 0.6% solution) of Ca(NO3)2 in 1 litre of water. 7.3 Vicia faba , broad bean The use of a 0.1% calcium nitrate solution in place of water to moisten the substratum prevents the blackening of seedlings. The solution is prepared by dissolving 1 g of Ca(NO3)2 in 1 litre of water. The seeds should be well spaced (e.g. 10 seeds in sand in a 12 X 12 X 4 cm container). The seed must be well covered, with as much sand below as above the seed. Seedling Evaluation: Seedlings are considered normal if they possess those essential structures that are indicative of their ability to produce a plant under favorable conditions. General Description 14.7 Fabaceae (Legume Family) I - Large-seeded epigeal, except soybean, peanut, lupine Phaseolus vulgar,i s garden bean and field bean Phaseolus lunatus , Lima bean Vigna radiata, mung bean Vigna unguiculata, cowpea Notes: For purposes of these rules, a garden bean (Phaseolus vulgaris) variety is defined as one which is grown for its fleshy pod to be eaten. Other beans, including field beans, are defined as those grown for their seeds to be eaten. Beans which are grown for either pod or seed to be eaten are to be considered garden beans, and the
210

requirements for cotyledons apply (see abnormal seedling description). General Description Seedling type: Epigea l dicot . Food reserves: Coty ledons which are la rge and f l eshy ; some photosynthes i s may occur , but th i s i s a minor funct i on . They shr ive l and drop of f when the food reserves are depleted . Shoot system: The hypocoty l elongates and carr i es the coty ledons above the soi l sur face . The epicoty l elongates causing the terminal bud to emerge f rom between the coty ledons; the pr imary leaves expand rap id l y . Root system: A long pr imary root with secondary roots . Abnormal Seedling Description Cotyledons

Garden bean P ( haseolus vulgar,i s in part ) : o Less than hal f of the or ig ina l coty ledon t i s sue remain ing attached. o Less than hal f of the or ig ina l coty ledon t i s sue f ree of necros i s or decay. Al l others : o Coty ledons are not assessed. Except ion : I f both coty ledons are miss ing and the seedl ing i s genera l l y weak, then the seedl i ng i s considered abnormal .

Epicotyl

Miss ing . Deep, open cracks . Malformed, such as markedly cur led or th ickened. Less than one pr imary lea f .

211

Pr imary leaves too smal l in proport i on to the rest of th seedl ing , usual l y assoc ia ted with vis ib l e defects of , or damage to , the main stem of the epicoty l . Terminal bud miss ing or damaged (see note 8) .

Hypocotyl

Deep open cracks extending in to the conduct ing t i s sue (see notes 1 and 6) . Malformed: such as markedly shortened, cur led or th i ckened. (See also note 3) .

Root

None. Weak, stubby or miss ing pr imary root with weak secondary or advent i t i ous roots (see note 7) .

Seedling

One or more essent ia l st ruc tu res impai red as the resu l t of decay f rom pr imary in fec t i on (but see note 8) . Alb ino .

Notes 10. Towels rolled too tightly may cause constriction of growing seedlings, resulting in malformation. Tight rolls, often in combination with a mid-test watering, may cause hypocotyl cracking or splitting. 11. Seeds of beans should be well spread out on or in the substrate. In general, the larger the seed, the more space it needs. Tightly concentrated seeds may compete to their detriment for water and for space to expand. 12. Hypocotyl collar rot is a breakdown in hypocotyl tissue characterized by "water-soaking" and collapse of the hypocotyl below the cotyledonary node. The lesion area later becomes discoloured, shrivelled and necrotic.

212

The condition is recognized as a laboratory phenomenon caused by insufficient calcium available to the seedling. If hypocotyl collar rot is observed on seedlings of garden beans, the sample involved shall be retested using a 0.3 to 0.6 % calcium nitrate solution to presoak the substratum (see section 4.7.4). 13. (Note 4 of the AOSA Seedl ing Evaluat i on Handbook deleted . ) 14. (Note 5 of the AOSA Seedl ing Evaluat i on Handbook deleted . ) 15. A healed break in the hypocotyl, sometimes referred to as a "knee," is to be considered an allowable defect. 16. A seedling with the root bound within a tough seed coat is to be considered normal. 17. If a few seedlings with total or partial decay to the epicotyl are found, they may be classified as normal, provided the hypocotyl and root are normal. The epicotyl on such seedlings usually does not decay when grown in a fairly dry environment and is exposed to light. Retests, preferably in soil or sand, will aid in interpretation of such seedlings. 18. Large-seeded legumes are especially susceptible to threshing or combine damage. Seed which has been mechanically damaged may produce seedlings with damaged primary roots, hypocotyls or epicotyls, or broken or detached cotyledons. Bruised areas are usually necrotic or decayed. Damage at the point of attachment of the cotyledons may be difficult to evaluate if seedlings are removed too early in the test period. 14.8 Fabaceae (Legume Family) II - Large-seeded hypogeal Cicer ar ie t i num , chickpea Lathyrus sylvest , r flat i s pea Lens culinaris, lentil Phaseolus coccineus, scarlet runner bean Pisum sativum, pea (field or garden)
213

Vic ia faba , horse bean or broadbean Vic iaspp., vetch Vigna angularis, adzuki bean General Description Seedling type: Hypogeal dicot. Food reserves: Cotyledons which are large and fleshy, and remain enclosed within the seed coat beneath the soil surface. They are usually not photosynthetic. Shoot system: The epicotyl elongates and carries the terminal bud and primary leaves above the soil surface. The stem bears one or more scale leaves and, prior to emergence, is arched near the apex, causing the terminal bud to be pulled through the soil; after emergence, the stem straightens. For practical purposes the hypocotyl is not discernible and is not an evaluation factor. There are buds in the axils of each cotyledon and scale leaf but these usually remain dormant unless the terminal bud is seriously damaged. In this case, one or more auxiliary buds will start to develop, forming a secondary epicotyl. Root system: A long primary root with secondary roots. Abnormal Seedling Description Cotyledons

Less than half of the original cotyledon tissue remaining attached (see notes 6 and 7). Less than half of the original cotyledon tissue free of necrosis or decay.

Epicotyl

Missing. Less than one primary leaf. Malformed stem such as markedly shortened, curled, or thickened.
214

Severely damaged (e .g . termina l bud miss ing or damaged) with only a weak secondary epicoty l develop ing f rom the axi l of a coty ledon or scale lea f . Two weak epicoty l s . Deep, open cracks extending in to the conduct ing t i s sue .

Root

None. Weak, stubby or miss ing pr imary root with weak secondary roots .

Seedling

One or more essent ia l st ruc tu res impai red as a resu l t decay f rom pr imary in fec t i on . Alb ino .

of

Notes 8. There is a greater likelihood of hard seed expression when the substrate does not provide adequate moisture to the seeds throughout the test period. 9. Insufficient moisture will result in apparently disproportionate elongation of the primary root and slow development of the epicotyl. 10. (Note 3 of AOSA Seedl ing Evaluat i on Handbook deleted . ) 11. (Note 4 of AOSA Seedl ing Evaluat i on Handbook deleted . ) 12. Manganese deficiency at the time of seed development may cause a condition known as "marsh spot," characterized by a discoloured brown indentation in the center of the inner surfaces of the cotyledons. Seedlings with this condition are considered normal, provided they are otherwise normal. If the condition causes difficulty in evaluation, then the sample should be retested in soil.

215

13. Weevil infestation may prevent the development of a normal seedling. Sometimes the cotyledons have been devoured to the extent that no food supply is left for the developing seedling. Such injury can be easily detected by examining the cotyledons. 14. Large-seeded legumes are especially susceptible to threshing or combine damage. Seed which has been mechanically damaged may produce seedlings with damaged primary roots, hypocotyls or epicotyls, or broken or detached cotyledons. Bruised areas are usually necrotic or decayed. Damage at the point of attachment of the cotyledons may be difficult to evaluate if seedlings are removed too early in the test period. 14.9 Fabaceae (Legume Family) III - Small-seeded Anthyl l i s vulnerar , ikidneyvetch a Astraga lus cicer , cicer milkvetch Coronilla varia, crownvetch Lespedeza spp., lespedeza Lotus spp., trefoil Medicago spp., alfalfa, black medick Melilotus spp., sweet clover Onobrychis viciifolia, sainfoin Trifolium spp., clover Trigonella foenum-graecum, fenugreek General Description Seedling type: Epigeal dicot. Food reserves: Cotyledons, which are small and fleshy; they expand, and become photosynthetic. Shoot system: The hypocotyl elongates and carries the cotyledons above the soil surface. The epicotyl usually does not show any development within the test period.

216

Root system: A long taper ing pr imary root , usual l y with roots hai rs . Most of the inc luded species do not normal ly develop secondary roots with in the test per iod . Abnormal Seedling Description Cotyledons

Less than hal f of the or ig ina l attached (see note 2) . Less than hal f of the or ig ina l necros i s or decay.

coty ledon t i s sue remain in coty ledon t i s sue f ree of

Epicotyl

Miss ing (may be assumed to be present i f are in tac t ) .

coty ledons

Hypocotyl

Deep open cracks extending in to the conduct ing t i s sue . Malformed: such as markedly shortened, cur led or th i ckened. Watery.

Root

None. Pr imary root stubby ( fo r sweet clover and crownvetch, or for roots bound by the seed coat see note 1) . Spl i t extending in to the hypocoty l .

Seedling

One or more essent ia l st ruc tu res impai red as a resu l t decay f rom pr imary in fec t i on . Alb ino .

of

Notes
217

1. Stubby roots when germinated on artificial media: a. Sweet clover - The roots of sweet clover may be stubby due to the presence of coumarin in the seed. Since this condition usually does not occur in soil, such seedlings are to be classified as normal. b. Bound by coat - Roots may appear stubby as a result of being bound by the seed coat. Such seedlings are to be classified as normal. c. Crownvetch - Produces phytotoxic effects similar to sweet clover.

2. Breaks at the point of attachment of the cotyledons to the hypocotyl are common in seeds which have been mechanically damaged. It is important that seedlings not be removed during preliminary counts unless development is sufficient to allow the condition of the cotyledons to be determined. If the point of attachment of the cotyledons cannot be seen at the end of the test, the seed coat should be peeled back to determine whether a break has occurred. 3. Mechanical breakage of the seed may result in only vestiges of seedlings with swollen cotyledons and broken, slightly enlarged hypocotyls or radicles. Insect damage may also cause lack of seedling growth. 4. Seedlings of sainfoin which have been "strangled" by growing through the netting of the pod but which are otherwise normal are to be classified as normal. 5. The percentage of hard seeds must be determined at the end of the test period for all genera in this group. Swollen seeds which fail to germinate by the end of the test should be allowed additional days as provided in sections 4.9.3 and 4.12.7. Swollen seeds are an indication of dormancy and can be induced by incorrect temperatures.

218

SEED HEALTH TESTING IN LEGUME VEGETABLES

219

1.

The main seed-borne pathogens of peas with common names of the diseases they cause Pathogen
Ascochyta pisLib i . Botryt i s cinerea Pers . ex Pers . Cladospor ium cladospor iodes (Fresen. ) de Vries f.sp. pis i co l a (Snyder) de Vries Col le to t r i chum pis Pat. i Erys iphe pis DC. i ex St . Am. Fusarium oxysporum Schlecht . ex Fr . f . sp pis . i (van Hall) Snyd. and Hans Mycosphaerel l a pinodes (Berk , and Blox . )Vesterg . , syn. Ascochyta pinodes Jones Peronospora viciae (Berk.) Gasp., syn. P. pisi (dc Bary) Syd. Phoma medicaginis Malbr. and Roum. var. pinodella Jones, syn. Ascochyta pinodella Jones Pleospora herbarum (Pcrs. ex Fr.) Rabenh., syn. Stemphylium botryosum Wallr. Rhizoctonia solani Kuhn Sclerotinia sclerotiomm (Lib.) de Bary Septoria pisi West Pseudomonas phaseolicola (Burkholder) Dowson, syn. Pseudomonas medicaginis Sackett var. phaseolicola (Stapp and Kotte) Dowson Pseudomonas pisi Sackett Xanthomonas rubefacines (Burr) Magrou and Prevot Viruses

Sr. No .
1 2 3 4 5 6 7 8 9 10 11 12 13 14

Common Names
Leaf and pod spot Grey mould, pod rot White mould, leaf and stem spot Anthracnose Powdery mildew Wilt Foot rot, leaf spot, black spot Downy mildew Foot rot , col l ar rot Foot rot Damping- off , Stem rot stem rot

Leaf blotch, Septoria blotch Bacter ia l bl ight

15 16 17

Bacter ia l bl ight Purple spot Pea early browning virus, Pea enation virus, syn. Pisum virus Pea mild mosaic Pea mosaic virus Pea seed- borne mosaic virus (syns. pea f izz le - top virus and pea leaf rol l i ng

2.

The main seed-borne pathogens of Phaseolus spp. with common names of the diseases they cause

220

Sr. No .
1 2 3

Pathogen
Ascochyta SPP . Aspergi l l u s sp. Botryt i s cinerea Pers . ex Pers .

Common Names
Leaf spot Baldhead and snakehead of seedl ings Grey mould, pod rot Leaf Blotch

Cercospora sp. 4 5 6 7 8 9 10 11 12 13 14 15 16

Col le to t r i c hum l i ndemuthianum (Sacc . and Anthracnose Magn.) Bri . and Cav. C. C. phaseolomm Tak. Anthracnose of Adzuke bean C. truncatum(Schw.) Andrus and Moore Stem anthracnose of l ima bean Diaporthe phaseolorum (Cook and El l . ) Pod and stem Sacc. var. Sojae (Lehm.) Wehm. blight of lima bean Els inoe phaseol i Jenkins Scab of l ima Fusar ium oxysporum Schlecht . ex Fr . Yel l ows and wil t f . sp . phaseol i Kendr. and Snyd. of P. vulgar is F. solani (Mart . ) Sacc. f .sp . Root rot phaseol i (Burkh. ) Snyd. and Hans. Macrophomina phaseol ina(Tass i . ) Goid. , Ashy stem bl ight syns. M. phaseol i(Maubl.) Ashby and charcoal rot Rhizoctonia bataticola (Taub). Briton-Jones Sderotium bataticola Taub. Phaeoisariopsis griseola (Sacc.) Ferraris, syn. Angular leaf spot of Isariopsis griseola Sacc. P. vulgaris Phytophthora phaseoli Thaxter Downy mildew col l ar rot of l ima bean Pleospora herbamm (Pers. ex Fr.) Rabenh. Red nose, syn. Stemphylium botryosum Wallr. leaf spot Rhizoctonia solani Kiihn Sclerotinia sderotiorum (Lib.) de Bary Damping- off , stem canker Sclerotinia wilt, stern rot , watery soft rot , white mould Rust Bacter ia l wil t Brown stem Halo bl ight , grease spot

17 18 19 20

Uromyces appendiculatus (Pers.) Unger Corynebacterium ftaccumfadens (Hedges) Dowson Corynebacterium spp. Pseudomonas phaseolicola (Burkholder) Dowson, syn. P. medicaginis Sackett var. phaseolicola (Burkholder ) Stapp and

221

21 22 23

Pseudomonas syr ingaevan Hal l Xanthomonas phaseol i (E. F. Smith) Dowson X. phaseol i (E. F. Smith) Dowson vzr . j uscans (Burkholder) Starr and Burkholder Viruses

Bacter ia l brown spot of P. lunatus and P. vulgar i s Com mon bacter ia lbl igh t of P. vulgaris Fuscous bl ight P. of vulgar i s Bean common mosaic virus (syn. bean mosaic virus) of P. lunatus and P. vulgar i s Black grain leaf cr ink l e Virus , Broad bean mottle vi rus of Cherry leaf ro l l vi rus of P. vulgar i s , Cucumber mosaic vi rus of P. aureus Runner bean mosaic vi rus

24

3.

The main seed-borne Broad bean pathogens with common names of the diseases they cause Pathogen Common Names

Sr. No

222

.
1 2 3 4 5 6 7

Ascochyta fabae Spcg. Leaf and pod spot Botryt i s fabae Sardina Chocolate spot Col le to t r i c hum l i ndemuthianum Anthracnose (Sacc . and Magn.) Brios i and Fusar ium spp. Fusar ium wil t Pleospora herbarum (Pers . ex. Fr .Net ) blotch Rabenh. , syn. Stemphyl ium botryosum Wall r . Uromyces vidae- fabae (Pers . ) Rust Schroet . , syn. Uromyces fabae (Grev. ) dc Bary Pseudomonas fabae (Yu) Burkholder Viruses Bean yel l ow mosaic vi rus Broad bean mild mosaic vi rus (syn. bean seed pattern vi rus ) Broad bean sta in vi rus , Echtes ackerbohnenmosaik Vi rus (syn. broad bean true mosaic virus) , Broad bean wil t vi rus , Pea seed- borne mosaic Virus (syn. pea f i z z l e top vi rus ) Dity lenchus dipsac (Kiihn) i Filipjev Stem eelworm

Detection of Ascochyta pisi on Pisum sativum (Pea)

223

[ISTA Validated Method] Crop : Pisum sat i vum (Pea) Pathogen: Ascochyta pisLib i . Prepared by: ISTA- PDC Method Val ida t i on Sub- committee Revision History:Vers ion 1.0 Ju l y 13, 2000 Revised 19.11.2001 J. Sheppard, V. Cockerell Reprinted 2003 Version 1.1 2008-01-01 Treated seed revised; Reporting results revised Submitted by: ISTA- PDC Method Val ida t i on Sub- committee Background This method was originally published in the ISTA Handbook of Seed Health Testing in November 1964 as S.3. No. 16 revised 1987 by P.D. Hewett, Official Seed Testing Station for England and Wales, Cambridge, United Kingdom. The method was incorporated into the newly revised Annexe to Chapter 7 in 2002 from the 1999 edition of the ISTA Rules. The method was reviewed by the ISTA-Seed Health Committee in 2006 (Cockerell & Koenraadt, 2007) with the recommendation to accept for a further five years. Studied in International Comparative Testing: 1960, 1966, 1967, 1968- 71, 1973- 1975. Summary of Validation Studies Agar tests detect approximately 50% more infection by Ascochyta spp. than blot te r tests (Anselme and Champion, 1962; Tempe, 1968). International comparative tests (Hewett, 1987) showed that of over 350 results obtained by

224

experienced stations, 95% fell within tolerance limits used for germination tests. Safety Precautions Ensure you are familiar with hazard data and take appropriate safety precautions, especially during preparation of media, autoclaving and weighing out of ingredients. It is assumed that this procedure is being carried out in microbiological laboratory by persons familiar with the principles of Good Laboratory Practice, Good Microbiological Practice, and aseptic technique. Dispose of all waste materials in an appropriate way (e.g. autoclave, disinfect) and in accordance with local safety regulations. Treated Seed This method has not been validated for the determination of Ascochyta pison i t reated seed. Seed t reatments may af fec t the perfo rmance of the method. (Definition of treatment: any process, physical, biological or chemical, to which a seed lot is subjected, including seed coatings. See 7.2.3) Materials Reference Material - The use of reference cul tu res or other appropr ia te material is recommended when ever possible. Media - Malt Agar or Potato Dextrose Agar. Sodium hypochlorite solution - (1% avai l ab le chor ine) for seed dis in fec t i on . Petri dishes - When sowing densi ty i s given by a number of seeds per Petri dish, a diameter of 90 mm is assumed.

225

Incubator - Capable of operat ing in the range 20 2 C. Sample Preparation The test is carried out on a working sample of 400 seeds as described in Section 7.4.1 of the ISTA Rules as appropriate. Method 1. Pret reatment 10 minutes in a 1% (available chlorine) sodium hypochlorite solution followed by draining. 2. Agar Method Malt or potato dextrose agar. Place 10 seeds on the agar surface in each Petri dish. 3. Incubat ion 7 days at 20 C in darkness. 4. Examinat ion After 7 days examine each seed by naked eye for abundant white mycelium which often covers infected seeds (Fig. 1). Doubtful colonies may be confirmed by the presence of wavy hyphae at the edge of the colony when examined at 25 magnification. Colony diameter typically 20-30 mm, occasionally smaller or incompletely surrounding the seed. Reverse of colonies medium to dark orange-brown centrally, opaque and even, becoming lighter in color towards the edge of the colony. Gelatinous-looking orange-brown pycnidia often present (although only sometimes clearly visible), particularly where seed touches agar. Under STM at 2025 magnification, using both transmitted and incident light, hyphae are curled, often several running together, typically with moisture drops (although these evaporate easily) (Fig. 2). Very limited growths from some seeds may only be seen if dishes tilted to get lighting at best angle, or under STM examination or after extended incubation. Pycnidia are up to 250 m in diameter. Spores, hyaline, cylindric, of slightly curved with rounded ends, 1-septate, slightly constricted at septum, mostly 12 4.5 m (Punithalingam, and Holliday, 1972).

226

Notes : Samples frequently bear A. pinodes (Mycosphaerella pinodes (Berk. & Blox.) Westerg.) and, occasionally, A. pinodella (Phoma medicaginis Malbr. & Roum. var. pinodella (Jones) Boerema). These pathogens differ markedly from A. pisi in their colony and mycelial characters and in spore morphology (see CMI descriptions Nos. 340 and 518, respectively). General Methods (common to many test procedures) 1. Checking Tolerances Tolerances provide a means of assessing whether or not the variation in result within or between tests is sufficiently wide as to raise doubts about the accuracy of the results. Suitable tolerances, which can be applied to most direct seed health tests, can be found in Tables 5B of Chapter 5 of the ISTA Rules, or in the Handbook of Tolerances and Measures of Precision for Seed Testing by S.R. Miles (Proceedings of the International Seed Testing Association 28 (1963) No 3, p 644). 2. Reporting Results The result of a seed health test should indicate the scientific name of the pathogen detected and the test method used. When reported on an ISTA International Seed Analysis Certificate, results are entered under Other Determinations. Preparation of Media and Solutions 1. Sodium Hypochlorite Solution

227

Sodium hypochlorite for pretreatment of seed can be prepared from commercial bleach diluted to 1% available chlorine. The concentration of chlorine in commercial bleach varies considerably. Use the formula: Vstock = Vfinal Cfinal / Cstock (where V= volume and C= % available chlorine) to calculate the volume of commercial bleach stock solution required to prepare sodium hypochlorite solutions for use in seed pretreatment. To prepare a 1 L solution of sodium hypochlorite containing 1% chlorine from a stock of commercial bleach containing 12% available chlorine: Vstock = Vfinal Cfinal / Cstock Vstock = 1 1/12 = 0.083 Thus add 83 mL of the 12% stock to 917 mL water. 2. Malt Agar Compound
1 Malt Agar

g/L According instructions 1000 mL to manufacturer's

Distilled/de-ionized water

1 CCP Malt agar const i t uents should be equiva lent to those of the fo l l ow ing manufacturers Difco , USA or Oxoid, UK Preparation 1. Weigh out ingredients into a suitable autoclavable container. 2. Add 1000 mL of distilled/de-ionized water.
228

3. Dissolve powdered Malt Agar in distilled/de-ionized water by stirring. 4. Autoclave at 15 p.s.i. and 121 C for 15 min. 5. Allow agar to cool to approx. 50 C. 6. Pour 1522 mL of molten agar into 90 mm Petri plates and allow to solidify before use. Storage: Prepared plates may be stored at 4 C for up to 6 weeks.

4. Potato Dextrose Agar Compound

1 Potato Dextrose Agar

g/L According instructions 1000 mL to manufacturer's

Distilled/de-ionized water

1 CCP Potato Dextrose Agar const i t uents should equiva lent to those of the fo l l ow ing manufacturers USA or Oxoid, UK Preparation 1. Weigh out ingredients into a suitable autoclavable container. 2. Add 1000 (or 500) mL of distilled/de-ionized water. 3. Dissolve powdered PDA in distilled/de-ionized water by stirring. 4. Autoclave at 15 p.s.i. and 121 C for 15 min. 5. Allow agar to cool to approx. 50 C.
229

be Difco ,

6. Pour 1522 mL of molten agar into 90 mm Petri plates and allow to solidify before use. Storage Prepared plates may be stored at 4 C for up to 6 weeks. Quality Assurance Critical Control Points Where the wording of the original Working Sheet suggests that an action is critical this has been marked with CCP . References The following references are extracted from the ISTA Handbook on Seed Health Testing, Working Sheet No. 16, P. D. Hewett, 1987. Anselme, C. and Champion, R. (1962). Lanalyse sanitaire des semences de pois. Proceedings of the International Seed Testing Association, 27, 829-842. Hewett, P.D. (1979). Pretreatment in seed health testing. 2. Duration of hypochlorite pretreatment in the agar plate test for Ascochyta spp. Seed Science and Technology, 7, 83- 85 Hewett, P.D. (1987). Detection of seed-borne Ascochyta pis i Lib . and test agreement with in and between laboratories. Seed Science and Technology, 15, 271-283. Leach, C.M. (1962). The quantitative and qualitative relationship of ultra violet and visible radiation to the induction of reproduction in Ascochyta pis . i Canadian Journa l of Botany, 40, 1577-1602.

230

Punithalingam, E. and Holliday, P. (1972). C.M.I. Descriptions of pathogenic fungi and bacteria No. 334. Commonwealth Mycological Institute, Kew. Tempe, J. de (1968). The quantitative evaluation of seedborne pathogenic infection. Proceedings of the International Seed Testing Association 33, 573-581.

Fig. 1. Colonies of A. pis , i face ( le f t ) and reverse ( r i gh t ) , f rom tes t on PDA, fo l l ow ing hypochlor i t e pret reatment. Incubat ion for 7 days at 21C in darkness

231

Fig. 2. Typical appearance of hyphae of A. pis , i test condi t i ons as for Fig . 1.

Detection of Colletotr ichum l indemuthianum on Phaseolus vulgaris (Bean) [ISTA Validated Method] Crop: Phaseolus vulgar i(Bean) s Pathogen: Col le to t r i c hum l i ndemuthianum (Sacc . Br ios i et Cav. & Magn.)

Prepared by: ISTA- PDC Method Val ida t i on Sub- committee Revision History: Vers ion 1.0 November 19, 2001 Revised 19.11.2001 J. Sheppard, V. Cockerell Reprinted 2003 Version 1.1 2008-01-01 Treated seed revised; Reporting results revised Submitted by: ISTA- PDC Method Val ida t i on Sub- committee

232

Background This method was originally published in the ISTA Handbook of Seed Health Testing in 1981 as Working Sheet No. 45 prepared by C. Anselme and R. Champion, La Minire, France. The method was incorporated into the newly revised Annexe to Chapter 7 in 2002 from the 1999 edition of the ISTA Rules. The method was reviewed by the ISTA-Seed Health Committee in 2006 (Cockerell & Koenraadt, 2007) with the recommendation to accept for a further five years. Studied in ISTA Comparative Tests:1962 Safety Precautions Ensure you are familiar with hazard data and take appropriate safety precautions, especially during preparation of media, autoclaving and weighing out of ingredients. It is assumed that this procedure is being carried out in microbiological laboratory by persons familiar with the principles of Good Laboratory Practice, Good Microbiological Practice, and aseptic technique. Dispose of all waste materials in an appropriate way (e.g. autoclave, disinfect) and in accordance with local safety regulations. Treated seed This method has not been validated for the determination of Col le to t r i c hum l i ndemuthianum on treated seed. Seed t reatments may af fec t the performance of the method. (Definition of treatment: any process, physical, biological or chemical, to which a seed lot is subjected, including seed coatings. See 7.2.3) Materials Reference Material The use of reference cultures or other appropriate material is
233

recommended when ever possible. Media Sodium hypochlorite solution Incubator Paper towelling. (1% available chorine) for seed disinfection. Capable of operating in the range 20 2 C.

Sample Preparation The test is carried out on a working sample of 400 seeds as described in Section 7.4.1 of the ISTA Rules. Method 1. Pret reatment Seeds are submerged in a solution of 1% (available chlorine) sodium hypochlorite for 10 min and allowed to drain. 2. Between Paper (BP) Spread the seeds in replicates of 50 on double sheets of paper towelling 350 x 450 mm which have been soaked in water. Cover seeds with one sheet of paper towelling soaked in water. Fold the paper twice lengthways and cover it with a sheet of polythene to maintain the moisture during incubation. 3. Incubat ion 7 days at 20 C in darkness 4. Examinat ion After 7 days remove the seed coats and examine the cotyledons by naked eye for black depressed areas with well delimited outlines (Fig. 2). Check each spot for the presence of acervuli with or without dark brown setae using 25 magnification (Fig. 3). The septate setae measure approx. 6 m x 100 m. The pale orange acervuli contain cylindrical, hyaline conidia with rounded ends containing one or two guttulaes. Conidia measure 2.55.5 m 1120 m
234

(Mordue, 1971; Kulshrestha, Mathur, and Neergaard, 1976). The use of a high-power microscope (magnification 200) is sometimes necessary (Fig. 3). Small spots may require longer incubation for development of acervuli. General Methods (common to many test procedures) 1. Checking tolerances Tolerances provide a means of assessing whether or not the variation in result within or between tests is sufficiently wide as to raise doubts about the accuracy of the results. Suitable tolerances, which can be applied to most direct seed health tests, can be found in Tables 5B of Chapter 5 of the ISTA Rules, or in the Handbook of Tolerances and Measures of Prec i s i on for Seed Test by ing S.R. Miles Proceedings ( of the International Seed Testing Association 28 (1963) No 3, p 644). 2. Reporting Results The result of a seed health test should indicate the scientific name of the pathogen detected and the test method used. When reported on an ISTA International Seed Analysis Certificate, results are entered under Other Determinations. Preparation of Media and Solutions 1. Sodium Hypochlorite Solution Sodium hypochlorite for pretreatment of seed can be prepared from commercial bleach diluted to 1% available chlorine. The concentration of chlorine in commercial bleach varies considerably. Use the formula Vstock = Vfinal x Cfinal /Cstock (where V = volume and C = % available chlorine) to calculate the volume of commercial bleach stock solution required to prepare sodium hypochlorite solutions for use in seed pretreatment. To prepare a 1 L solution of sodium hypochlorite containing 1% chlorine from a stock of commercial bleach containing 12% available chlorine:
235

Vstock = Vf i na l x Cfinal /Cstock Vstock = 1 1/12 = 0.083 Thus add 83 mL of the 12% stock to 917 mL water. Quality Assurance Critical Control Points None listed References The following references are extracted from the ISTA Handbook on Seed Health Testing, Working Sheet No. 45, C. Anselme and R. Champion, 1981. Champion, R. (1969). Quelques parasites importants transmis par les semences. Identification au laboratoire, Agriculture, 322, 3 8. Kulshrestha, D.D., Mathur, S.B. and Neergaard, P. (1976). Identification of seed borne species of Colletotrichum. Friesia 11, 116-125. Mordue,J.E.M. (1971). C.M.I. Description of pathogenic fungi and bacteria No. 316. Commonwealth Mycological Institute, Kew. International Seed Testing Association, Committee on Plant Diseases (1963) Report on the Fifth International Conference (workshop) on seed pathology sponsored by the International Seed Testing Association convened by the ISTA Committee on Plant Diseases. Bundesanstalt fur Pflanzenbau and Samenprfung in Wien 17th to 23rd September, 1962, p. 23. Copenhagen. Mimeographed.

236

Fig-1: Dry, white bean seeds showing symptoms

Fig-2: Cotyledons of seedlings after 7 days incubation with seed coats removed, showing black areas with well defined outline.

237

Fig- 3: Acervul i setae. x 150 .

with cyl i nd r i c ,

hyal ine conid ia and dark brown septate

Detection of Pea Early- Browning Virus and Pea Seedborne Mosaic Virus on Pisum sativum [ISTA Validated Method] Crop: Pisum sat i vum L. Pathogen: Pea Ear ly - Browning Virus and Pea Seed- borne Mosaic Virus Prepared by: Koenraadt , H.M.S. 1 and Remeeus, P.M.2Naktuinbouw, PO Box 40, 2370 AA Roelofarendsveen, The Netherlands. 1E-mail: h.koenraadt@naktuinbouw.nl2Email: p.remeeus@naktuinbouw.nl Sponsored by: ISF, ISHI-Vegetables Committee Revision history:Version 1.0, 1 February 2007.

238

Background Pea Early-Browning Virus (PEBV) and Pea Seed-borne Mosaic Virus (PSbMV) are seed-transmissible viruses of the pea, and therefore the detection of these viruses in pea seeds is an important tool in control strategies. The enzyme-linked immunosorbent assay (ELISA) is widely used for the detection of plant viruses (Clark and Adams, 1977), and ELISA methods have been described for the detection of PEBV and PSbMV (Hamilton and Nichols, 1978; Van Vuurde and Maat, 1985, Maury et al., 1987). The test method described here was designed using this information and twenty years of laboratory experience, and evaluated in a comparative test. The method, using ground seed and a DAS-ELISA, can be used to simultaneously detect PSbMV and PEBV in a single extract. Note that the extract is tested in two microtitre plates, one for PEBV and one for PSbMV. The theoretical detection limit is one seed in 100. To ensure a 95% probability that infestations of 0.15% or higher are detected, it is necessary to test 20 subsamples of 100 seeds. Validation studies Koenraadt, H.M.S. and Remeeus, P.M. (2007) Copies are available by e-mail from ista.office@ista.ch, or by mail from the ISTA Secretariat. Safety Precautions Ensure you are familiar with hazard data and take appropriate safety precautions, especially during preparation of buffers, grinding, autoclaving, and weighing out of ingredients. It is assumed that this procedure is carried out in a microbiological laboratory by persons familiar with the principles of Good Laboratory Practice, Good Microbiological Practice, and aseptic technique. Dispose of all waste materials in an appropriate way (e.g. autoclaving or disinfection) and in accordance with local health, environmental and safety regulations. Treated Seed
239

This method has not been validated for the determination of Pea Early-Browning Virus (PEBV) or Pea Seed-borne Mosaic Virus (PSbMV) in treated seed. Seed treatments may affect the performance of this test. (Definition of treatment: any process, physical, biological or chemical, to which a seed lot is subjected, including seed coatings. See 7.2.3) Sample and sub-sample size The sample (total number of seeds tested) or sub-sample size to be tested depends on the desired tolerance standard (maximum acceptable percentage of seeds infested) and detection limit (theoretical minimum number of pathogen propagules per seed which can be detected). In any case, the subsample size should not exceed 100 seeds. Materials Reference material : PSbMV- and PEBV-in fes ted seeds or standard i zed reference mater ia l ( f l ou r of peas conta in ing PSbMV and/or PEBV). Microtit re plates: 96- wel l plates sui tab le for ELISA (CCP) . Antisera: Sui tab le for detect i on of PSbMV- and PEBVin fes ted seeds (e .g . PRI , Wageningen, The Nether lands) . Balance: capable of weighing to the nearest 0.01 g. pH meter: capable of being read to the nearest 0.1 pH uni t . Automatic pipettes: capable of pipet t i ng to the nearest 0.001 mL. Grinder: capable of gr ind ing peas to f i ne f l ou r (e .g . Retsch gr indomix GM 200) . Incubator: capable of operat ing at 4 2 C. Incubator: capable of operat ing at 37 2 C. ELISA plate reader. Tubes: 10 mL. Sample Preparation This can be done in advance of the assay.

240

1. It is vital to exclude any possibility of crosscontamination between seed samples, it is therefore essential to clean all surfaces, containers, hands, etc. both before and after handling each sample. 2. Count the number of seeds in a known weight. Estimate the thousand-seed weight (TSW) as: TSW = (weight of seeds/number of seeds) 1000
3.

Based on the est imated TSW, weigh out sub- samples of the requi red s ize in to new, clean bags or conta iners

Method Critical control points are indicated by CCP. 1. Coating of ELISA plates 1.1 Add appropriate (as defined by supplier) dilution of PSbMV- and PEBV-coating serum to coating buffer. Be sure that the antisera are not only suitable for diagnostics but also for the detection of viruses in seed extracts (CCP). 1.2 Coat one plate with 180 l of PSbMV-coating buffer per well. Coat another plate with 180 l of PEBV-coating buffer per well. 1.3 Cover ELISA plates with lid or wrap with plastic wrap to minimize evaporation. 1.4 Incubate plates overnight at 4 2 C. 2. Extraction of virus from the seed and incubation of extracts. 2.1 Count or weigh 100 seed subsamples. 2.2 Grind each subsample to fine flour in a grinder (CCP). 2.3 From each subsample weigh out 0.5 g of flour and transfer to a 10 mL tube. 2.4 Add 5 mL of extraction buffer to each tube. 2.5 Vortex each tube for 15 s. Allow extract to settle for at least 5 min. on the bench to facilitate pipetting. 2.6 Remove coating buffer from ELISA plates and immediately rinse plates three times thoroughly, using PBS/Tween 20 to remove residues. Alternatively, use a suitable washing device (CCP).
241

2.7 Immediately after rinsing, pipette 180 l of each seed extract into a well. Use 2 wells per subsample. 2.8 Add positive and negative controls to each ELISA plate. Use at least 2 dilutions for the positive controls: a low dilution, which gives a high extinction, and a high dilution, which gives an extinction just above the detection threshold (CCP). Negative controls must include a healthy seed extract. 2.9 Cover plates with lid or wrap with plastic wrap to minimize evaporation and incubate overnight at 4 2 C. 3. Incubation of conjugate 3.1 Prepare appropriate dilution of PSbMV- and PEBVconjugated antiserum using conjugate buffer as defined by the supplier. 3.2 Remove seed extracts from ELISA plates and rinse plates 3 times with washing buffer PBS/Tween 20 to remove residues of seed extract. Alternatively, use a suitable washing device (CCP). 3.3 Immediately after rinsing, add 180 l of diluted conjugate to each well of the ELISA plate. 3.4 Cover plates with lid or wrap with plastic wrap to minimize evaporation, and incubate for 3 h at 37 2 C. 4. Addition of substrate to ELISA plates 4.1 Prepare substrate solution (10 mg para- ni t rophenol phosphate in 20 mL of subst ra te buffer ) . 4.2 Remove conjugate from ELISA plates and rinse thoroughly, either 3 times by hand using washing buffer PBS/Tween 20, or, alternatively, using a reliable washing device (CCP). 4.3 Add 180 l of substrate solution to each well. 4.4 Incubate for 2 h at 20 2 C. 4.5 Measure extinction (A405) with ELISA plate reader (see General methods, point 2). General methods (common to many test procedures)

242

1. Grind ing of seeds Grind each subsample of 100 seeds to a fine flour. Be sure to use a grinder that can be cleaned thoroughly, since crosscontamination is likely during the grinding step. 2. Recording of ELISA ext inc t i on Record the results for all wells in the microtitre plate. Check first whether the positive and negative controls meet the expectations, since otherwise the results of the test are invalid and the test should be repeated. It is recommended to use a negative-positive threshold of 2.5 times the background of healthy samples. 3. Report ing resu l t s The result of a seed health test should indicate the scientific name of the pathogen detected and the test method used. When reported on an ISTA In ternat i ona l Seed Analys i s Cert i f i c a t , e resu l t s are entered under Other Determinat ions . In the case of a negative result (pathogen not detected in any of the sub-samples), the results should be reported in terms of the tolerance standard and detection limit. The tolerance standard depends on the total number of seeds tested, n, and is approximately 3/n (p = 0.95; Roberts et al., 1993). In the case of a positive result, the report should indicate the number of positive sub-samples out of the total number tested and the sample size or the maximum likelihood estimate of the proportion of infested seeds. Quality assurance Critical control points (identified in the methods by CCP) Sensitivity can be influenced by using different types of microtitre plates or materials. The quality of antisera from different sources is known to be variable. Therefore ensure that the antisera are suitable not only for diagnostics but also for the detection of viruses in seed extracts. Step 1.1.
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 A f i ne f l ou r

wi l l improve the extract i on ef f i cacy . Therefore , gr ind seeds for 20 s at 10 000 r .p .m. to get f i ne f l ou r . Note that some knives easi l y become blunt and therefo re gr ind less ef f i c i en t l y with t ime. Step 2.2 Coated microt i t r e plates wi l l lose act iv i t y rapid l y when they are le f t to dry on the bench for some t ime. Therefore l imi t t ime that empty microt i t r e plates s i t on bench as much as poss ib l e . Step 2.6. The use of appropr ia te posi t i ve and negat ive contro l s i s very important to val ida te the resu l t . Be sure that , apart f rom a high posi t i ve contro l , there i s always a low posi t i ve contro l in each plate . Step 2.8. High backgrounds in ELISA are often caused by poor washing of the microt i t r e plates between the di f f e ren t incubat ion steps . Washing can be done by hand using PBS/Tween 20 or with a washing device . The thorough washing of microt i t r e plates i s high ly cr i t i c a l in sever steps (2 .6 , 3.2 and 4.2) in the ELISA, part i cu l a r l y afte the incubat ion with the conjugated ant i se rum. Step 4.2. Coating buffer (pH 9.6) Na2CO3: 1.59 g/L . NaHCO3: 2.93 g/L . Extraction buffer (0.05 M, pH 7.4) NaCl: 8.0 g/L KH2PO4: 1.0 g/L . Na2HPO4 12H2O: 14.5 g/L . Tween 20: 1.5 mL. PVP (ELISA grade, e.g. molecular weight 10 000 Da): 20.0 g. Conjugate buffer (0.05 M, pH 7.4) NaCl: 8.0 g/L . KH2PO4: 1.0 g/L . Na2HPO4 12H2O: 14.5 g/L . Tween 20: 1.5 mL. BSA (ELISA grade, e.g. BSA fract ion 5): 5.0 g

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Substrate buffer (pH 9.6) Diethanolamine: 97 mL. HCl (32%): 15 mL. Washing buffer PBS/Tween 20 (0.05 M, pH 7.4) NaCl: 8.0g/L . KH2PO4: 1.0 g/L . Na2HPO4 12H2O: 14.5 g/L . Tween 20: 1.5 mL. Preparation of individual buffers 1. Weigh or measure out all ingredients into a suitable container. 2. Adjust volume to 1000 mL with distilled/de-ionized water and dissolve or mix ingredients as appropriate. 3. Check the pH with a pH meter. Storage of buffers Store buffers as mentioned above at 4 2 C for a week. References Clark, M.F. and Adams, A.N. (1977). Characteristics of the microplate method of enzyme linked immunosorbent assay for the detection of plant viruses. Journa l of General Viro l ogy 34 , 475483. Koenraadt, H.M.S. and Remeeus, P.M. (2007). Proposal for a new method for detect ing Pea Ear ly - Browning Virus and Pea Seed- borne Mosaic Virus with ELISA . ISTA Method Val ida t i on Report 5. Hamilton, R.I. and Nichols, C. (1978). Serological methods for detection of pea seed-borne mosaic virus in leaves and seeds of Pisum sat i vum . Phytopathology 68 , 539543.

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Maury, Y., Bossennec, J.M., Boudazin, G., Hampton, R.O., Pietersen, G. and Maguire, J. (1987). Factors influencing ELISA evaluation of transmission of pea seed-borne mosaic virus in infected pea seed: seed group size and seed decortication. Agronomie 7, 225230. Roberts, S.J., Phelps, K., Taylor, J.D. and Ridout, M.S. (1993). Design and interpretation of seed health assays. In Proceedings of the f i r s t ISTA Plant Disease Committee Symposium on Seed Health Test ing (ed. J .W. Sheppard) , Ottawa, Canada. Agr icu l t u re Canada, Ottawa, Canada, pp. 115125. Van Vuurde, J.W.L. and Maat, D.Z. (1985) Enzyme-linked immunosorbent assay (ELISA) and disperse-dye immuno assay (DIA): comparison of simultaneous and separate incubation of samples and conjugate for the routine detection of lettuce mosaic virus and pea early-browning virus in seeds. Nether lands Journa l of Plant Pathology 91 , 3 13.

Detection of Pseudomonas savastanoi phaseolicola on Phaseolus vulgaris [ISTA Validated Method]

pv.

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Crop: Phaseolus vulgar i(bean) s Pathogen: Pseudomonas savastano i pv. phaseol i co l a syn. Pseudomonas syringae pv. phaseolicola (Halo Blight) (Gardan et al., 1992) Prepared by: Kurowski, C.1 and Remeeus, P.M.21Harris Moran Seed Company, 9142 Mace Blvd, Davis, CA 95616, USA. E-mail: c.kurowski@harrismoran.com2Naktuinbouw, P.O. Box 40, 2370 AA Roelofarendsveen, The Netherlands. Email: p.remeeus@naktuinbouw.nl Sponsored by: ISF, ISHI-Vegetables Committee Revision history:Version 1.0, 1 January 2007. Background ISTA has published two working sheets (Nos. 65 and 66) for Pseudomonas savastanoi pv. phaseolicola (Psp; Van Vuurde and Van den Bovenkamp, 1987; Jansing and Rudolph, 1996). Not only do the extraction methods in these working sheets differ, they also differ from the stationary overnight soaking commonly used in the testing laboratories of the seed industry. In practice, this has proven to be adequate in terms of sensitivity. Therefore, stationary overnight soaking is part of the present version for the detection of Psp and was incorporated in the validation study (Kurowski and Remeeus, 2007). Both ISTA working sheets are based on dilution plating, although an immunofluorescence (IF) prescreening is part of working sheet No. 65. The present version (Kurowski and Remeeus, 2007) abandons this IF prescreening. Instead of plating in triplicate on modified sucrose peptone (MSP), as in working sheet No. 66, two plates of MSP and two plates of an additional medium, milk Tween (MT; Goszczynska and Serfontein, 1998), are used. MT can complement MSP, and has the advantage of being able to detect Xanthomonas
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axonopodis pv. phaseol i (Xap), which is not possible on MSP. Furthermore, MT allows Pseudomonas savastanoi pv. phaseolicola to be distinguished from colonies of Pseudomonas syringae pv. syringae. The value of MT for detection of Psp and Xap has been demonstrated in practice and in method validation studies for both pathogens (Kurowski and Remeeus, 2007; Sheppard and Remeeus, 2006). In the present version, the final identification of Psp is based on a pathogenicity assay on bean seedlings, as in working sheet No. 65; the phaseolotoxin assay of working sheet No. 66 is not widely used and does not identify all strains of the pathogen (Rico et al., 2003). A sub-culturing step, to further assist in identifying suspect colonies, has been added to the method. Validation studies Kurowski, C. and Remeeus, P.M. (2007) Copies are available: by E-mail from i s ta . o f f i c e@is ta ; . ch by mail from the ISTA Secretariat. Safety precautions Ensure you are familiar with hazard data and take appropriate safety precautions, especially during preparation of media, autoclaving, and weighing out of ingredients. It is assumed that this procedure is being carried out in a microbiological laboratory by persons familiar with the principles of Good Laboratory Practice, Good Microbiological Practice, and aseptic technique. Dispose of all waste materials in an appropriate way (e.g. autoclaving or disinfection) and in accordance with local health, environmental and safety regulations. Treated seed This method has not been validated for the determination of Pseudomonas savastanoi pv. phaseolicola (Psp) on treated
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seed. Seed treatments may affect the performance of the method. (Definition of treatment: any process, physical, biological or chemical, to which a seed lot is subjected, including seed coatings. See 7.2.3) Sample and sub-sample size The sample (total number of seeds tested) or sub-sample size to be tested depends on the desired tolerance standard (maximum acceptable percentage of seeds infested) and detection limit (theoretical minimum number of pathogen propagules per seed which can be detected). In any case, the subsample size should not exceed 1000 seeds. Materials Reference material : a known st ra in Pseudomonas of savastanoipv. phaseolicola or standardized reference material. Plates of MT medium: 9.0 cm Petri dishes (3 plates of each medium per sub-sample + controls). Plates of MSP medium: 9.0 cm Petri dishes (3 plates of each medium per sub-sample + controls). Plates of Kings B (KB) medium: for sub-culturing (at least 1 sectored plate per sub-sample). Polythene bags or containers: with sterile saline (0.85% NaCl) plus Tween 20 (0.02%; 0.2 mL/L) for soaking of seeds (volume [mL] required is equivalent to 2.5 TSW [g]). Dilut ion bottles: containing 4.5 mL of sterile saline (2 per sub-sample). Other volumes may be acceptable (see General methods). 70% ethanol or equivalent disinfect ing product: for disinfection of surfaces and equipment. Incubator: capable of operating at 28 2 C, 2025 C and 1820 C. Balance: capable of weighing to the nearest 0.001 g. pH meter: capable of being read to the nearest 0.01 pH unit.

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Automatic pipettes: check accuracy and prec i s i on regular l y . Bean seedlings: suscept ib l e to al l races of the pathogen for pathogenic i t y test (e .g . cv. Helda) . Cold room or refr igerator: operat ing at 5 4 C) . Sample preparation This can be done in advance of the assay. 1. It is vital to exclude any possibility of cross-contamination between seed samples. It is therefore essential to disinfect all surfaces, containers, hands, etc. both before and after handling each sample. This can be achieved by using swabbing or spraying equipment with gloves and 70% ethanol. 2. If the submitted sample is received in several packets, these should be combined by emptying into a new, clean polythene bag and mixing by hand to give a composite sample. 3. Count the number of seeds in a known weight. Estimate the thousand-seed weight (TSW) as: TSW = (weight of seeds/number of seeds) 1000 4. Based on the estimated TSW, weigh out subsamples of the required size into new, clean polythene bags or containers. Methods Critical control points are indicated by CCP. 1. Extraction 1.1 Suspend seeds in sterile saline plus Tween 20 (0.02% v/v) in a polythene bag or container. The volume of saline required in millilitres should be equivalent to 2.5 TSW (g). For example: if TSW = 300 g, the volume of saline required is 2.5 300 = 750 mL (Olivier and Remeeus, 2004). 1.2 Soak subsamples overnight (1618 h) at 5 4 C). 2. Dilution and plating

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2.1 Shake the polythene bag or container to obtain a homogenous extract before dilution. 2.2 Prepare a tenfold dilution series from the seed extract. Pipette 0.5 mL of the extract into 4.5 mL of sterile saline and vortex to mix (101 dilution). Pipette 0.5 mL of the 101 dilution into another 4.5 mL of sterile saline and vortex to mix (102 dilution) (see General methods). 2.3 Pipette 100 L of each dilution and the undiluted seed extract onto plates of each of the selective media (MT, MSP) and spread over the surface with a sterile bent glass rod (see General methods). 2.4 Incubate inverted plates at 28 2 C) and examine after 4-5 d (see section 5). 3. Positive control (culture or reference material) 3.1 Prepare a suspension of a known strain of Psp in sterile saline or reconstitute standardized reference material according to the suppliers instructions. 3.2 Dilute the suspension sufficiently to obtain dilutions containing approximately 102 to 104 cfu/mL. This may require up to seven tenfold dilutions from a turbid suspension. 3.3 Pipette 100 L of appropriate countable dilutions onto plates of each of the selective media (MT, MSP) and spread over the surface with a sterile bent glass rod. 3.4 Incubate plates with the sample plates. 4. Sterility check 4.1 Plate a dilution series from a sample of the extraction medium (i.e. saline + Tween 20), containing no seeds, and plate on each of the media as for samples. 5. Examination of the plates 5.1 Examine sterility check and recovery of positive controls on semi-selective medium (CCP). 5.2 Examine the sample plates for the presence of typical Psp colonies by comparison with the positive control plates. 5.3 After 4-5 d on MT, Psp colonies are creamy white, flat, circular, 4.55 mm in diameter (Fig. 1).

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5.4 After 4-5 d on MSP, Psp colonies are circular, raised globose, glistening and light yellow with a less dense centre. The medium around the colony turns light yellow after 3 d (Fig. 2). 5.5 The colony size and colour can differ within a sample. 5.6 Estimate the number of suspect and other colonies (see General methods). 6. Confirmation and identification of suspect colonies 6.1 Subculture suspect colonies to sectored plates of KB. To prevent cross-contamination of isolates, use a new sectored plate for each subsample. The precise numbers of colonies subcultured will depend on the number and variability of suspect colonies on the plate; if present, at least six colonies should be subcultured per subsample (CCP). 6.2 Subculture the positive control isolate to a sectored plate for comparison. 6.3 Incubate sectored plates for 2-4 d at 28 2 C). 6.4 Compare appearance of growth with positive control. On KB in general, Psp develops creamy or white circular and flat colonies (CCP)(Fig. 3). 6.5 Confirm the identity of isolates by pathogenicity on bean seedlings of known susceptibility (CCP). 6.6 Record results for each colony subcultured. 7. Pathogenicity assay (Fenwick and Guthrie, 1969; Van Vuurde and Van den Bovenkamp, 1989) 7.1 Incubate seeds of a bean cultivar known to be susceptible to all races of Psp (e.g. cv. Helda) in rolled germination paper for 3-4 d (crook-neck stage) at 1820 C in darkness. 7.2 Dip a sterile toothpick or needle in the bacterial culture on a 2-4 d KB culture (e.g. sectored plate). 7.3 Stab the needle through the cotyledon. Turn the toothpick or needle slightly while withdrawing to release bacteria. Re-infesting the toothpick or needle between inoculations is recommended. 7.4 Inoculate 2 seedlings per isolate. 7.5 Inoculate seedlings with the positive control isolate and a negative control with only a toothpick or needle (CCP).

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7.6 Inoculated seedlings are transferred to damp soil in a humidity chamber (7080% RH) for 45 d at 2025 C (light:dark 12:12). 7.7 Record symptoms after 45 d and again at 810 d if necessary. After 45 d but before deterioration of the cotyledons, the flat inner sides of the cotyledons are inspected for typical greasy spots at the point of inoculation (Fig. 4). Compare with positive control (CCP). General methods (common to many test procedures) 1. Preparation of tenfold dilution series Each dilution should be prepared by pipetting 0.5 mL (5%) from a well-mixed seed extract or previous dilution into a universal bottle (screw-capped) or similar container containing 4.5 mL (2%) of sterile diluent and then vortexing to mix prior to the next dilution step. A new sterile pipette tip should be used for each dilution step. Pipettes should be checked regularly for accuracy and precision and recalibrated as necessary. It is acceptable to prepare tenfold dilutions using other volumes, provided that the laboratory can demonstrate that the required accuracy and precision can be achieved. 2. Plating of dilutions This should be done as soon as possible after dilutions have been prepared, and certainly within 30 min. Working from the highest (most dilute) dilution to the undiluted extract, 0.1 mL is pipetted onto the centre of a surface-dry, labelled agar plate. The liquid should then be spread evenly over the entire surface of the medium with a bent glass rod. If care is taken to work from the highest to the lowest dilution (or undiluted extract) a single pipette tip and a single bent glass rod can be used for each sample. Ensure that all liquid has been absorbed by the agar before inverting and incubating plates. If necessary, allow plates to dry under a sterile

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airflow in a microbiological safety cabinet or laminar-flow hood. 3. Recording of dilution plates Record the results for all dilution plates. The most accurate estimate of bacterial numbers should be obtained from spread plates with total number between 30 and 300 colonies. However this may be further complicated depending on the relative numbers of suspect pathogen and other colonies. In order to minimize effort, start recording with the highest dilution (most dilute) and count the number of suspect and the number of other colonies. If the total number of colonies on a plate greatly exceeds 300 there is little value in trying to make a precise count if a more reliable count has already been obtained from a more dilute plate, in which case it is sufficient to record the number of colonies as m (many) if they are still separate or c (confluent) if they have run together. 4. Sectored plates Using a laboratory marker pen draw lines on the base of a standard 9 cm plate (Petri dish) to divide it into six equal sectors. Subculture single colonies from dilution plates and make a single zigzagged streak within a single sector on the plate. Take care to leave sufficient space between each isolate to ensure the growth does not coalesce. Thus six suspect colonies can be sub-cultured to each sectored plate. Separate plates should be used for each sample/sub-sample. If the purity of sub-cultured isolates is doubtful, they should be further streaked out on whole plates. 5. Reporting results The result of a seed health test should indicate the scientific name of the pathogen detected and the test method used. When reported on an ISTA In ternat i ona l Seed Analys i s Cert i f i c a t , e resu l t s are entered under Other Determinat ions .

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In the case of a negative result (pathogen not detected in any sub-samples), the results should be reported in terms of the tolerance standard and detection limit. The tolerance standard depends on the total number of seeds tested, n, and is approximately 3/n (P=0.95) (see Roberts et al., 1993); the detection limit per sub-sample is equal to the detection limit per mL multiplied by the volume of extract. In the case of a positive result, the report should indicate the mean number of pathogen propagules (cfu) per seed and either the number of positive sub-samples out of the total number tested and the sample size or the maximum likelihood estimate of the proportion of infested seeds. Quality assurance General A record should be kept of the date and results of pipette calibration checks. It is essential that operators have received appropriate training and use automatic pipettes correctly. Critical control points (Identified in the methods by CCP) Dilution plates prepared from positive control isolate(s) or reference material should give single colonies with typical morphology (Step 5.1). The numbers of colonies on dilution plates prepared from the positive control isolate(s) or reference material should be similar on both media (Step 5.1). Numbers of bacteria on dilution plates should be consistent with the dilution (i.e. should decrease approximately tenfold with each dilution) (Step 5.1). There should be no growth on dilution plates prepared as a sterility check (Step 5.1). Due to the potential for non-pathogenic isolates to be present in seed lots together with pathogenic isolates, it is essential to subculture (Step 6.1), if present, at least the minimum number of suspect colonies specified (six per sub255

sample). Compare the appearance of growth of sub-cultured isolates with that of positive controls (Step 6.4). and test all Pseudomonas-like sub-cultured isolates for pathogenicity (Step 6.5). Positive control isolates should be included in every pathogenicity test (Step 7.5). The positive control isolate should give typical symptoms in pathogenicity test (Step 7.7). The activity units per gram of some antibiotics may vary between batches. It may be necessary to adjust the weight or volume added to ensure that the final number of units per liter of medium is consistent (MT and MSP media). Preparation of sterile saline (van Vuurde et al., 1989) Sodium chloride (NaCl): 8.5 g/L . Disti l l ed/de- ionized water: 1000 mL. Preparation 1. Weigh out all ingredients into a suitable container. 2. Add 1000 mL of distilled/de-ionized water. 3. Dissolve and dispense into final containers. 4. Autoclave at 121 C and 15 p.s.i. for 15 min. 5. For extraction of seeds, add 0.2 mL of sterile Tween 20 per litre after autoclaving. Storage Provided containers are tightly closed, sterile saline may be stored for several months before use. Preparation of MSP agar medium (Mohan and Schaad, 1987) Sucrose: Proteose K2HPO 4: MgSO4 20.0 g. peptone No. 3: 5.0 g. 0.5 g. 7H2O: 0.25 g.
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Agar: 20.0 g. Disti l l ed/de- ionized water: 1000 mL. Cycloheximide*: 200.0 mg. Cephalexin*: 80.0 mg. Vancomycin*: 10.0 mg. Bromothymol blue (15 mg per mL 95% EtOH)*: mg. * Added after autoclaving Preparation

15.0

1. Weigh out all ingredients except antibiotics and bromothymol blue into a suitable container. 2. Add 1000 mL of distilled/de-ionized water. 3. Steam to dissolve. 4. Autoclave at 121 C and 15 p.s.i. for 15 min. 5. Prepare antibiotic and bromothymol blue solutions. 6. Allow medium to cool to approximately 50 C before adding antibiotic and bromothymol blue solutions. 7. Mix the molten medium gently to avoid air bubbles and pour plates (22 mL per 9.0 cm plate). 8. Leave plates to dry in a laminar flow bench or similar before use. Antibiotics (amounts for guidance only; CCP) a) Dissolve 2 g cycloheximide in 10 mL 70% ethanol. Add 1 mL/L. b) Dissolve 800 mg cephalexin in 10 mL 70% ethanol. Add 1 mL/L. c) Dissolve 100 mg vancomycin in 10 mL 70% ethanol. Add 1 mL/L. d) Dissolve 150 mg bromothymol blue in 10 mL 95 % ethanol. Add 1 mL/L. Filter sterilize when antibiotics are dissolved in water rather than 70% ethanol. Note: Nystat i n can be used as an al te rnat i ve for cyclohex imide to contro l fungi . Disso lve 400 mg nystat i n in 10 mL 70% ethanol ; add 1 mL to cool medium.
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Storage Store prepared plates inverted in polythene bags at 48 C and use within 4 weeks of preparation to ensure activity of antibiotics. Preparation of MT agar medium (adapted from Goszczynska and Serfontein, 1998) A Proteose peptone No. 3: 10.0 g/L . CaCl 2: 0.25 g/L . Tyrosine: 0.5 g/L . Agar: 15.0 g/L . Disti l l ed/de- ionized water. 500 mL. B Skim milk powder (Oxoid, Sigma; CCP): 10.0 g/L . Disti l l ed/de- ionized water: 500 mL. C Tween 80: 10.0 mL. D Nystatin*: 40 mg (1 mL). Cephalexin*: 80 mg (1 mL). Vancomycin*: 10 mg (1 mL). * Added after autoclaving Preparation 1. Weigh out all ingredients in section A into a suitable container. 2. Add 500 mL of distilled/de-ionized water. 3. Dissolve ingredients. 4. In a separate container, dissolve skim milk powder in 500 mL distilled water. 5. Separately prepare 10 mL Tween 80. 6. Sterilize preparations from section A, skim milk solution (section B) and Tween 80 (section C) separately at 121 C and 15 p.s.i. for 15 min.

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7. After sterilization of all components, aseptically add sterilized skim milk preparation and sterilized Tween 80 to sterilized ingredients in section A. 8. Prepare antibiotic solutions (section D). 9. Allow medium to cool to approximately 50 C and add antibiotics. 10. Mix gently to avoid air bubbles, and pour 22 mL of mixture onto each 9.0 cm plate. 11. Leave plates to dry on a laminar flow bench or similar before use. Antibiotics (amounts for guidance only, CCP) a) Dissolve 400 mg nystatin in 10 mL 70% ethanol. b) Dissolve 800 mg cephalexin in 10 mL 70% ethanol. c) Dissolve 100 mg vancomycin in 10 mL 70% ethanol. Filter sterilize when antibiotics are dissolved in water rather than 70% ethanol. Preparation of Kings B (KB) agar medium (King et al., 1954) Proteose peptone No. 3: 20.0 g/L . K2HPO 4: 1.5 g/L . MgSO4 7H2O: 1.5 g/L . Glycerol: 15.0 mL. Agar: 15.0 g/L . Distilled/de-ionized water: 1000 mL. Preparation 1. Weigh out all ingredients into a suitable oversize container (i.e. 250 mL of medium in a 500 mL bottle or flask) to allow swirling of medium just before pouring. 2. Add 1000 mL of distilled/de-ionized water. 3. Steam to dissolve. 4. Autoclave at 121 C and 15 p.s.i. for 15 min. 5. Allow medium to cool to approximately 50 C. 6. Pour plates (22 mL per 9.0 cm plate).

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7. Leave plates to dry in a laminar flow bench or similar before use. Storage Store prepared plates inverted in polythene bags at 48 C. Prepared plates can be stored for several months provided they do not dry out. References Fenwick, H.S. and Guthrie, J.W. (1969). An improved Pseudomonas phaseol i co l a pathogenic i t y test . Phytopathology, 59 , 11 (Abstract). Gardan, L., Bollet, C., Abu Ghorrah, M., Grimont, F. and Grimont, P.A.D. (1992). DNA relatedness among pathovar strains of Pseudomonas syringae subsp. savastanoi Janse (1982) and proposal of Pseudomonas savastanoi sp. nov. International Journal of Systematic Bacteriology, 42 , 606 612. Goszczynska, T. and Serfontein, J.J. (1998). Milk-Tween agar, a semiselective medium for isolation and differentiation of Pseudomonas syringae pv. syringae, Pseudomonas syringae pv. phaseolicola and Xanthomonas axonopodis pv. phaseoli. Journal of Microbiological Methods, 32 , 6572. Jansing, H. and Rudolph, K. ISTA Handbook on Seed Health Testing. Working Sheet No. 66 (Pseudomonas syringae pv. phaseolicola). King, E.O., Ward, M.K. and Raney, D.E. (1954). Two simple media for the demonstration of pyocyanin and fluorescin. Journal of Laboratory and Clinical Medicine, 44 , 301-307. Kurowski, C. and Remeeus, P.M. (2007). Proposal for a new method for detecting Pseudomonas savastanoi pv. phaseolicola on bean seeds. ISTA Method Validation reports 4, 122.

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Mohan, S.K. and Schaad, N.W. (1987). An improved agar plating assay for detecting Pseudomonas syr ingaepv. syringae and P. s. pv. phaseolicola in contaminated bean seed. Phytopathology 77 , 13901395. Olivier, V. and Remeeus, P.M. (2004). Additional experiment to select the extraction and dilution buffer for the detection of Xanthomonas axonopodis pv. phaseoli in bean seeds. ISHI report, Naktuinbouw, Research report 0305XAP. Rico, A., Lpez, R., Asencio, C., Aizpn, M., Asensio-S.Manzanera, C. and Murillo., J. (2003). Nontoxigenic strains of P. syringae pv. phaseolicola are a main cause of halo blight of beans in Spain and escape current detection methods. Phytopathology 93, 15531559. Roberts, S.J., Phelps, K., Taylor, J.D. and Ridout, M.S. (1993). Design and interpretation of seed health assays. In Proceedings of the First ISTA Plant Disease Committee Symposium on Seed Health Testing (ed. J.W. Sheppard), pp. 115125, Agriculture Canada, Ottawa, Canada. Sheppard, J.W. and Remeeus, P.M. (2006). Proposal for a new method for detecting Xanthomonas axonopodis pv. phaseoli on bean seeds. ISTA Method Validation reports 3, pp. 123. Van Vuurde, J.W.L. and Van den Bovenkamp, G.W. (1987). ISTA Handbook on Seed Health Testing, Working Sheet No. 65 (Pseudomonas syringae pv. phaseolicola). Van Vuurde, J.W.L. and Van den Bovenkamp, G.W. (1989) Detection of Pseudomonas syringae pv. phaseolicola in bean. In Detection of Bacteria in Seed and Other Planting Material (eds. A.W. Saettler, N.W. Schaad and D.A. Roth). The American Phytopathological Society Press, pp. 30-40.

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Fig- 1: Pseudomonas savastanoi pv. phaseol i co l a colonies on MSP plates after 4 d are circular, raised globose, glistening and light yellow, and the medium around the colony turns light yellow.

Fig- 2: Pseudomonas savastanoi pv. phaseol i co l a colonies on MT plates after 4 d are creamy white, flat, circular and 4.55 mm in diameter.

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Fig- 3: Iso la t i on Pseudomonas of savastanoi pv. phaseolicola by sectoring on KB medium showing white, creamy and flat colonies.

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C
Fig- 3: Germinated bean seedl ings A . The method of inoculation. B Typical Pseudomo-nas savastanoi pv. phaseolicola symptoms in a pathogenicity test, indicated as a typical greasy spot (C) at the point of inoculation.

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Detection of Xanthomonas axonopodis pv. phaseoli and Xanthomonas axonopodis pv. phaseoli var. fuscans on Phaseolus vulgaris [ISTA Validated Method] Crop: Phaseolus vulgar i(bean) s Pathogen: Xanthomonas axonopodis pv. phaseoli (common blight) and Xanthomonas axonopodis pv. phaseoli var. fuscans Prepared by: Sheppard, J.1, Kurowski, C.2 and Remeeus, P.M.3 1Canadian Food Inspection Agency. P.O. Box 960 Carling Avenue, Ottawa, Ontario, Canada 2Harris Moran Seed Company, 9142 Mace Blvd, Davis, CA 95616, US. E-mail: c.kurowski@harrismoran.com 3Naktuinbouw, P.O. Box 40, 2370 AA Roelofarendsveen, The Netherlands. E-mail: p.remeeus@naktuinbouw.nl Revision History: Version 1.0, 22 February 2006. Version 2.0, 18 August 2006. Background This method is derived from the validation studies carried out by ISTA in 2003, in collaboration with the International Seed Health Initiative for Vegetables (ISHI-Veg) (Sheppard and Remeeus, 2005). For routine testing of bean seed a combination of two complementary semi-selective media, MT and XCP1, is recommended with a pathogenicity test to confi rm suspect isolates.

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The two media, XCP1 and MT, have been chosen for their ease of use and selectivity for X. axonopodis pv phaseol i. In addition both media can be used to detect both X. axonopodis pv phaseoli and X. axonopodis pv phaseoli var fuscans. Although initially the morphology of fuscans and non-fuscans strains of X. axonopodis pv phaseoli appear to be similar on the media after a longer incubation the fuscans colonies are distinguished by a distinct brown pigmentation. A further advantage of MT medium is that it can also be used for identifying other bacterial seed-borne pathogens of beans, e.g. Pseudomonas savastanoi pv. phaseolicola and Pseudomonas syringae pv. syringae. Validation Studies Sheppard, J.W. and Remeeus, P.M. (2005) Copies are available: by E-mail from ista.offi ce@ista.ch; by mail from the ISTA Secretariat, Zrichstrasse 50, 8303 Bassersdorf, Switzerland. Detection limit 15 cfu/ml (theoretical, P=0.95). Safety Precautions Ensure you are familiar with hazard data and take appropriate safety precautions, especially during preparation of media, autoclaving, and weighing out of ingredients. It is assumed that this procedure is being carried out in a microbiological laboratory by persons familiar with the principles of Good Laboratory Practice, Good Microbiological Practice, and aseptic technique. Dispose of all waste materials in an appropriate way (e.g. autoclave, disinfect) and in accordance with local health, environmental and safety regulations. Treated Seed

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Seed treatments may affect the performance of this test. It must only be performed on untreated seed. Sample and sub-sample size The sample (total number of seeds tested) and sub-sample size to be tested depends on the desired tolerance standard (maximum acceptable percentage of seeds infested) and detection limit (theoretical minimum number of pathogen propagules per seed which can be detected); in any case the maximum sub-sample size should be 1000 seeds.

Materials Reference material - A known strain for both fuscans and non fuscans types of X. axonopodis pv. phaseoli or standardised reference material. Plates of MT medium - 9.0 cm Petri dishes (3 plates of each medium per sub-sample + controls) Plates of XCP1 medium - 9.0 cm Petri dishes (3 plates of each medium per sub-sample + controls) Plates of YDC - for sub-culture (at least 1 per sub-sample). Polythene bags or containers - of sterile saline (0.85% NaCl) plus Tween 20 (0.02% - 0.2 ml per litre) for soaking seeds (volume (ml) required is equivalent to 2.5 x TSW (g)). Dilution bottles - containing 4.5 ml of sterile saline (2 per sub-sample). Other volumes may be acceptable, see General Methods. 70% ethanol - for disinfection of surfaces, equipment Incubator: operating at 28C 2C

267

Balance: capable of weighing to the nearest 0.001 g Automatic pipettes - check accuracy and precision regularly Bean seedlings - susceptible to all races of the pathogen for pathogenicity test (e.g. Michelet, Contender) Sample Preparation 1. This can be done in advance of the assay. 2. It is vital to exclude any possibility of cross-contamination between seed samples. It is therefore essential to disinfect all surfaces, containers, hands, etc. both before and after handling each sample. This can achieved by swabbing/spraying equipment and gloved hands with 70% ethanol. 3. If the submitted sample is received in several packets, these should be combined by emptying into a new, clean polythene bag and mixing by hand to give a composite sample. 4. Count the number of seeds in a known weight. Estimate the Thousand Seed Weight (TSW) as:

5. Based on the estimated TSW, weigh out sub-samples of the required size into new, clean polythene bags or containers. Method [Critical control points are indicated by CCP ] 1. Extrac t i on 1.1. Suspend seeds in sterile saline plus Tween 20 (0.02% v/v) in a polythene bag or container. The volume of saline required in ml should be equivalent to 2.5 x TSW (g) e.g.

268

TSW = 300 g, therefore volume of saline required is 2.5 x 300 = 750 ml (Olivier and Remeeus, 2004). 1.2. Soak sub samples overnight (16-18 h) at 5C ( 4C). 2. Di lu t i on and plat i ng 2.1. Shake containers or polythene bags to obtain a homogenous extract before dilution. 2.2. Prepare a tenfold dilution series from the seed extract. Pipette 0.5 ml of the extract into 4.5 ml of sterile saline and vortex to mix (101 dilution). Pipette 0.5 ml of the 101 dilution into another 4.5 ml of sterile saline and vortex to mix (102 dilution) (see General Methods). 2.3. Pipette 100 l of each dilution and the un-diluted seed extract onto plates of each of the selective media (MT and XCP1) and spread over the surface with a sterile bent glass rod (see General Methods). 2.4. Incubate inverted plates at 28C 2C and examine after 4-5 d. 3. Posi t i ve contro l (cu l tu re or reference mater ia l ) 3.1. Prepare a suspension of a known strain of X. axonopodis pv. phaseoli, fuscans and non-fuscans, in sterile saline or reconstitute standardised reference material according to the suppliers instructions. 3.2. Dilute suspension suffi ciently to obtain dilutions containing approximately 102 to 104 cfu/ml. This may require up to seven ten-fold dilutions from a turbid suspension. 3.3. Pipette 100 l of appropriate countable dilutions onto plates of each of the selective media (MT and XCP1) and spread over the surface with a sterile bent glass rod. 3.4. Incubate plates with the sample plates. 4. Sterility check 4.1. Prepare a dilution series from a sample of the extraction medium (i.e., saline plus Tween 20), containing no seeds, and plate on each of the media as for samples. 5. Examination of the plates

269

5.1. Examine sterility check and recovery of positive control on semi-selective medium (CCP ) . 5.2. Examine the sample plates for the presence of typical X. axonopodis pv. Phaseoli colonies by comparison with the positive control plates. 5.3. After 4-5 d on MT, X. axonopodis pv phaseoli colonies are yellow distinguished by two zones of hydrolysis; a large clear zone of casein hydrolysis and a smaller milky zone of Tween 80 lysis (Fig. 1a and b). The fuscans of X. axonopodis pv phaseoli colonies produce a brown diffusible pigment. If not visible after 4 d inclubate for an additional day. Often the fuscans type colonies show Tween 80 lysis. 5.4. After 4-5 d on XCP1, X. axonopodis pv phaseoli colonies are yellow, glistening and surrounded by a clear zone of starch hydrolysis (Fig. 2b). The fuscans of X. axonopodis pv phaseoli colonies produce a brown diffusible pigment after 5 d of incubation (Fig 2a). Often the fuscans type colonies show Tween 80 lysis. 5.5. The colony size and colour can differ within a sample. 5.6. Estimate the number of suspect and other colonies (see General Methods). 6. Confi rmation/identifi cation of suspect colonies 6.1. Sub-culture suspect colonies to sectored plates of YDC. To avoid the potential for cross-contamination of isolates, use a new sectored plate for each sub-sample. The precise numbers of colonies sub-cultured will depend on the number and variability of suspect colonies on the plate: if present, at least six colonies should be sub-cultured per sub-sample (CCP ). 6.2. Sub-culture the positive control isolate to a sectored plate for comparison (Fig. 3). 6.3. Incubate sectored plates for 24-48 h at 28C 2C. 6.4. Compare appearance of growth with positive control. On YDC X. axonopodis pv phaseoli colonies are yellow and mucoid in appearance (Fig. 3) (CCP ). 6.5. Confi rm the identity of isolates by pathogenicity on bean seedlings of known susceptibility (CCP ). 6.6. Record results for each colony sub-cultured.

270

7. Pathogenic i t y (Saet t l e r , 1971) 7.1. Grow seedlings of a bean cultivar known to be susceptible to all races of X. axonopodis pv. phaseol i (e.g. Michelet or Contender at 2030C in small pots or modules until the fi rst leaf is just visible (usually about 10 days after sowing). 7.2. Water plants 1-3 h before inoculation to provide better conditions for infection. 7.3. Dip a sterile toothpick or needle into the bacterial culture of a 2-4 d old YDC culture (i.e. sectored plate). 7.4. Inoculate the seedling by stabbing the toothpick or needle through the primary node at an angle of about 45. Stop stabbing as the needle emerges from the opposite side of the node. Turn the toothpick or needle slightly while drawing to release bacteria. Support the seedling with one hand during the process. 7.5. The number of plants which should be inoculated will depend on the variability of the cultivar and experience of the operator, but 1-3 plants per isolate should usually be suffi cient. It is better to inoculate more isolates with 1 plant per isolate than one isolate with 3 plants. 7.6. Inoculate seedlings with the two positive control isolates fucans and non-fucans (following 7.3-7.5) (CCP ) and a negative control with only a sterile toothpick or needle (CCP ). 7.7. Grow on plants at 20-25C. 7.8. Record symptoms after, 8-10 d and again at 14 d. Compare with positive control (CCP ). Typical X. axonopodis pv. phaseolsymptoms i include dark green watersoaked lesions at the point of entry of the toothpick or needle. Lesions can become red-brown, elongate extending into the stem causing slight to severe stem cracking (Fig. 4). Symptons for fuscans and non-funscans are the same. Should the time from inoculation be extended wilting and fl agging of the top foliage followed by necrosis can occur 1418 d after inoculation.

271

Fig. 1a

Fig. 1b

Fig- 1: X. axonopodis pv. phaseol i colonies on MT plates after 4 d indicated by a large clear zone of casein hydrolysis (a) and a smaller milky zone of Tween 80 lysis (b).

Fig. 2a

Fig. 2b

272

Fig- 2: X. axonopodis pv. phaseol i colonies, fuscans (a) and nonfuscans (b), on XCP1 plates showing a clear zone of starch hydrolysis and fuscans on XCP1 showing a milky zone, after 4 d

Fig. 3a

Fig. 3b

Fig- 3: X. axonopodis pv. phaseol i colonies, fuscans (a) and nonfuscans (b), on YDC plates after 2 d are brown and yellow in appearance.

273

Fig. 4a

Fig. 4b

Fig- 4: Phaseolus vulgar i stem s 10 days afte r inocu la t i on with typica l les i on of X. axonopodis pv. phaseoli (a), and a negative control (b).

General Methods (common to many test procedures) 1. Preparation of ten-fold dilution series Each dilution should be prepared by pipetting 0.5 ml ( 5%) from a well-mixed seed extract or previous dilution into a universal bottle (screw-capped) or similar container containing 4.5 ml ( 2%) of sterile diluent and then vortexing to mix prior to the next dilution step. A new sterile pipette tip should be used for each dilution step. Pipettes should be checked regularly for accuracy and precision and re-calibrated as necessary. It is acceptable to prepare ten-fold dilutions using other volumes provided that the laboratory can demonstrate that the required accuracy and precision can be achieved. 2. Plating of dilutions. This should be done as soon as possible after dilutions have been prepared and certainly within 30 min. Working from the
274

highest (most dilute) dilution to the undiluted extract, 0.1 ml is pipetted onto the centre of a surface-dry, labeled agar plate. The liquid should then be spread evenly over the entire surface of the medium with a bent glass rod. If care is taken to work from the highest to the lowest dilution (or undiluted extract) a single pipette tip and a single bent glass rod can be used for each sample. Ensure that all liquid has been absorbed by the agar before inverting and incubating plates. If necessary allow plates to dry under a sterile air-flow in a microbiological safety cabinet or laminar flow hood. 3. Recording of dilution plates Record the results for all dilution plates. The most accurate estimate of bacterial numbers should be obtained from spread plates with total number between 30 and 300 colonies. However this may be further complicated depending on the relative numbers of suspect pathogen and other colonies. In order to minimize effort, start recording with the highest dilution (most dilute) and count the number of suspect and the number of other colonies. If the total number of colonies on a plate greatly exceeds 300 there is little value in trying to make a precise count if a more reliable count has already been obtained from a more dilute plate, in which case it is sufficient to record the number of colonies as m (many) if they are still separate or c (confluent) if they have run together. 4. Sectored Plates Using a laboratory marker pen draw lines on the base of a standard 9 cm plate (Petri dish) to divide it into six equal sectors. Sub-culture single colonies from dilution plates and make a single zigzagged streak within a single sector on the plate. Take care to leave sufficient space between each isolate to ensure the growth does not coalesce. Thus six suspect colonies can be sub-cultured to each sectored plate. Separate plates should be used for each sample/sub-sample. If the purity of sub-cultured isolates is doubtful, they should be further streaked out on whole plates.

275

5. Reporting Results The result of a seed health test should indicate the scientific name of the pathogen and the test method used. When reported on an ISTA Certifi cate, results are entered under Other Determinat ions . In the case of a negative result (pathogen not detected in any sub-samples), the results should be reported in terms of the tolerance standard and detection limit.

The tolerance standard depends on the total number of seeds tested, n, and is approximately 3/n (P=0.95) (see Roberts et al . , 1993); the detect i on l imi t per sub- sample i s equal to the detect i on l imi t per ml mult ip l i ed by the volume of extrac t . In the case of a positive result, the report should indicate the mean number of pathogen propagules (cfu) per seed and either the number of positive sub-samples out of the total number tested and the sample size or the maximum likelihood estimate of the proportion of infested seeds (Most Probable Number). Quality Assurance General A record should be kept of the date and results of pipette calibration checks. It is essential that operators have received appropriate training and use automatic pipettes correctly. Critical Control Points [Identified by CCP in the methods] Dilution plates prepared from positive control isolate(s) or reference material, should give single colonies with typical morphology (Step 5.1 and 6.4). The numbers of colonies on dilution plates prepared from the positive control isolate(s) or reference material should be

276

similar on both media (Step 5.1). Note that recovery of the fuscans type X. axonopodis pv. phaseoli is in general lower on MT than on XCP1. Numbers of bacteria on dilution plates should be consistent with the dilution (i.e. should decrease approximately ten-fold with each dilution) (Step 5.1). There should be no growth on dilution plates prepared as a sterility check (Step 5.1). Due to the potential for non-pathogenic isolates to be present in seed lots together with pathogenic isolates, it is essential to sub-culture (Step 6.1), if present, at least the minimum number of suspect colonies specifi ed (six per subsample) and to test all Xanthomonaslike sub-cultured isolates for pathogenicity (Step 6.5). Positive control isolates should be included in every pathogenicity test (Step 7.6). The positive control isolate should give typical symptoms in a pathogenicity test (Step 7.8). The quality of milk powder is vital to develop the hydrolysis of starch in MT medium. Two milk sources that work well are Oxoid and Sigma brands. The activity (g) of some antibiotics may vary between batches. It may be necessary to adjust the weight or volume added to ensure that the fi nal number of units per liter of medium is consistent (MT and XCP1 media). Preparation of steri le sal ine (van Vuurde et al., 1989) Compound Sodium chloride (NaCl) g/l 8.5
277

Distilled/de-ionized water Preparation

1 000 ml

1. Weigh out all ingredients into a suitable container. 2. Add 1 000 ml of distilled/de-ionized water. 3. Dissolve and dispense into final containers. 4. Autoclave at 121C, 15 psi for 15 min. 5. For extraction of seeds, add 0.2 ml of sterile Tween 20 per 1 000 ml. Storage Provided containers are tightly closed, may be stored for several months before use. Preparation of MT (Milk Tween) agar medium (adapted from Goszczynska and Serfontein, 1998) Compound g/l A Proteose peptone no. 3 10.0 CaCl2 0.25 Tyrosine 0.5 Agar 15.0 Distilled/de-ionized water 500 ml B Skim milk powder (Oxoid, Sigma) CCP 10.0 Distilled/de-ionised water 500 ml C Tween 80 10.0 ml D Nystatina 40 mg (1 ml) Cephalexinb 80 mg (1 ml) Vancomycinc 10 mg (1 ml) a, b, c Added after autoclaving Preparation

278

1. Weigh out all ingredients in section A into a suitable container. 2. Add 500 ml of distilled/de-ionised water. 3. Dissolve ingredients. 4. In a separate container, dissolve skim milk powder in 500 ml distilled water. 5. Separately prepare 10 ml Tween 80. 6. Sterilize preparations from section A, skim milk solution (section B) and Tween 80 (section C) separately at 121C, 15 psi for 15 min. 7. After sterilization, of all components aseptically add sterilized skim milk preparation and sterilized Tween 80 to sterilized ingredients in section A. 9. Prepare antibiotic solutions (section D). 10. Allow medium to cool to approximately 50C and add antibiotics. 11. Mix gently to avoid air bubbles and pour plates 22 ml per 9.0 cm plate. 12. Leave plates to dry in laminar flow bench or similar before use. Ant ib i o t i c s (amounts for guidance CCP only ), a. Dissolve 400 mg nystatin in 10 ml 70% ethanol. b. Dissolve 800 mg cephalexin in 10 ml 70% ethanol. c. Dissolve 100 mg vancomycin in 10 ml 70% ethanol. (Filter sterilize when antibiotics are dissolved in water rather than 70% ethanol.) Note Cycloheximide can be used as an alternative for nystatin to control fungi. Dissolve 500 mg of Cycloheximide in 10 ml 70% ethanol; add 1 ml to cool medium. Storage

279

Store prepared plates inverted in polythene bags at 4-8C and use within two weeks of preparation to ensure activity of antibiotics.

Preparation of XCP1 agar medium (Adapted from McGuire et al , . 1986) Compound g/l

A KBr 10.0 CaCl2 0.25 Soluble Potato Starch 10.0 Peptone 10.0 Agar 15.0 Crystal violet (1% aqueous) 0.15 ml Distilled water/de-ionised water 1 000 ml B Tween 80 10.0 ml C Nystatina 40 mg (1 ml) Cephalexinb 80 mg (1 ml) Fluorouracilc 10 mg (1 ml) Tobramycind a, b, c, d Added after autoclaving Preparation 1. Weigh out all ingredients in section A into a suitable container.
280

2. Add 1 000 ml of distilled water/de-ionized water. 3. Dissolve ingredients. 4. Add crystal violet. 5. Sterilize 121C, 15 psi for 15 min. 6. Sterilize 10 ml Tween 80 separately (section B) at 121C, 15 psi for 15 min. 7. Aseptically add Tween 80 to ingredients in section A. 8. Prepare antibiotic solutions. 9. Allow medium to cool to approximately 50C and add antibiotics. 10. Mix gently to avoid air bubbles and pour plates (22 ml per 9.0 cm plate). 11. Allow plates to dry in laminar fl ow bench or similar before use. Antibiotics (amounts for guidance only, CCP ) a. Dissolve 400 mg nystatin in 10 ml 70% ethanol. b. Dissolve 100 mg cephalexin in 10 ml 70% ethanol. c. Dissolve 30 mg fl uorouracil in 100 ml 70% ethanol. d. Dissolve 16 mg tobramycin in 100 ml 70% ethanol. (Filter sterilise when antibiotics are dissolved in water rather than 70% ethanol.) Note Cycloheximide can be used as an alternative for nystatin to control fungi. Dissolve 500 mg of Cycloheximide in 10 ml 70% ethanol, add 1 ml to cool medium. Storage Store prepared plates inverted in polythene bags at 4-8C and use within two weeks of preparation to ensure activity of antibiotics. Depending on the source of starch, pre-storage in the refrigerator for several days before use may result in more easily visible zones of starch hydrolysis.

281

Preparation of Yeast Dextrose Chalk (YDC) agar medium (Wilson et al . , 1967) Compound Bacto Agar Yeast Extract CaCO3 (light powder) D-Glucose (Dextrose) Distilled/de-ionized water 1 000 ml Preparation 1. Weigh out all ingredients into a suitable oversize container (i.e. 250 ml of medium in a 500 ml bottle/fl ask) to allow swirling of medium just before pouring. 2. Add 1 000 ml of distilled/de-ionized water. 3. Steam to dissolve. 4. Autoclave at 121C, 15 psi for 15 min. 5. Allow medium to cool to approximately 50C. 6. Swirl to ensure even distribution of CaCO3 and avoid air bubbles, and pour plates (22 ml per 9.0 cm plate). 7. Leave plates to dry in a laminar fl ow bench or similar before use. Storage Store prepared plates inverted in polythene bags at 8-20C. Prepared plates can be stored for several months provided they do not dry out. References Goszczynska, T. and Serfontein, J.J. (1998). Milk-Tween agar, a semiselective medium for i so l a t i on and di f f e ren t i t aPseudomonas t i on of syr ingae pv. syringae, Pseudomonas
282

g/l 15.0 10.0 20.0 20.0 1 000 ml

syr ingaepv. phaseol i co l a and Pseudomonas savastanoi pv. phaseolicola. Journal of Microbiological Methods 32 , 65-72. McGuire, R.G., Jones, J.B. and Sasser, M. (1986). Tween media for semi selective isolation of Xanthomonas campestris pv. vesicatoria from soil and plant material. Plant Disease 70 , 887-891. Roberts, S.J., Phelps, K., Taylor, J.D. and Ridout, M.S. (1993). Design and interpretation of seed health assays. In: Shepaard, J.W., (Ed.) Proceedings of the first ISTA Plant Disease Committee Symposium on Seed Health Testing, Ottawa, Canada. Pp 115-125. Agriculture Canada, Ottawa, Canada. Olivier, V. and Remeeus, P.M. (2004). Additional experiment to select the extraction and dilution buffer for the detection of Xanthomonas axonopodis pv. phaseoli in bean seeds. ISHI report, Naktuinbouw, Research report 0305XAP. Saettler, A.W. (1971). Seedling injection as an aid to identifying bean blight bacteria. Plant Disease Reporter 55 , 703-706. Sheppard, J.W. (1998). ISTA/ISHI Comparative test Xanthomonas campestris pv. phaseoli. View and explanation preliminary results from 3 labs. CFIA, Research report XAP 1998. Sheppard, J.W. and Remeeus, P.M. (2005). Proposal for a new method for detecting Xanthomonas axonopodis pv. phaseoli on bean seeds. ISTA Method Validation Reports 3. Van Vuurde, J.W.L. and van den Bovenkamp, G.W. (1989). Detection of Bacteria. In: Seed. A.W. Saettler, N.W. Schaad and D.A. Roth, editors.

283

Wilson, E.E., Zeitoun, F.M. and Fredrickson, D.L. (1967). Bacterial phloem canker, a new disease in Persian walnut trees. Phytopathology 57 , 618- 621.

Vegetable Crops of Family Apiaceae

(Dill, Celery, Carrot, Coriander, Parsnip, Parsley)
16. Seed unit: seed uni t

i s non- dehiscent in tac t schizocarp or mericarp ( f ru i t bodies) .

284

17. Submitted Sample Weight:

Anethum graveolens (Di l l ) = 40 g Apium graveolens(Celery, Celeric)= 25 g Cor iandrum sat i vum(Coriander) = 400 g Daucus carota(Carrot) = 30 g Past inaca sat i va (Parsnip) = 100 g Petrose l i num cr i spum (Parsley) = 40 g
18. Working Sample Weight:

Anethum graveolens (Dill) = 4g Apium graveolens(Celery, Celeric)= 1g Cor iandrum sat i vum(Coriander) = 40 g Daucus carota(Carrot) = 3g Past inaca sat i va (Parsnip) = 10 g Petrose l i num cr i spum (Parsley) = 4g 19. Purity Analysis: a. Pure Seed: In tac t seed uni t schizocarps s, and mericarps with or without perianth and regardless of whether they contain a true seed) shall be considered pure seed unless it is readily apparent that no true seed is present. (The term "readily apparent" shall be interpreted to mean that the purity analyst should not use a diaphanoscope, stereoscopic microscope, hand lens, pressing or other special equipment or means to detect whether true seeds are present.) b. Other Crop Seeds: Seeds of other species notified as crop. c. Weed Seeds: weeds. Seeds of other species notified as

285

d. Inert Matter:Seed piecesOne-hal f the or ig ina l s i ze or less , seed uni ts wherein it is readily apparent that no true seed is present, non-living material, nematode galls and fungus bodies.

20. Germination methods:

Number Temperature Substrata of seeds (C) BB; TS; RT 20-30 First Count (days) 7 Final Count (days) 21 Additional Additional Directions Directions General Fresh/ Dormant Requirements Seeds Light; water only -

Kind of Seed Anethum graveolens Dill Apium graveolens Celery and Celeriac Coriandrum sativum Coriander Daucus carota Carrot Pastinaca sativa Parsnip Alternate method for Parsnip Petroselinum crispum Parsley

TB

15-25; 20

10

21

Prechill

BB; RT 400 BB; RT; BP BB; TS; RT

15 20-30 20-30

7 5 7

14 14 28

Light moisture

Prechill; may retest at 10-30C Plant on top of sand, lightly cover seeds with sand, cover test with wet blotters. May prechill 4-5 days; may retest at 10-30C. May retest at 10-30C

20-30; 15-25

10

28

Standard moisture

TS; TB

15-25

10

28

Abbreviations: BB - Between blot te r s , PP - Pleated paper, RT - Rolled towels, S Sand, TB Top of blot te rs , TS Top of sand

Seedling Evaluation: Seedlings are considered normal if they possess those essential structures that are indicative of their ability to produce a plant under favorable conditions.

286

Abnormal Seedling Description Cotyledons

Less than hal f of the or ig ina l attached. Less than hal f of the or ig ina l necros i s or decay.

coty ledon t i s sue remain in coty ledon t i s sue f ree of

Epicotyl

Miss ing (may be assumed to be present i f coty ledons are in tac t ) .

the

Hypocotyl

Malformed: such as markedly shortened, cur led or th i ckened. Deep open cracks extending in to the conduct ing t i s sue . Watery.

Root

None. In carrot, celeriac and parsnip: miss ing or stubby pr imary root (even i f secondary roots are present ) . Seedl ings of carro Daucus t ( carota ) , celer i aApium c ( graveolens var. rapaceum) and parsnip (Pastinaca sativa) must possess a well-developed primary root.

287

SEED HEALTH TESTING IN UMBEL CROPS

1.

The main seed-borne Carrot pathogens with common names of the diseases they cause Pathogen
Alte rnar ia dauci (Kuhn) Groves & Skolko Alte rnar ia radic ina Meier , Drechs l . and Eddy syn. Stemphyl ium radic inum (Meier , Drechs l . and Eddy) Neergaard Cercospora carotae (Pass.) Kazn and Siem Gibbere l l a avenacea Cook syn. Fusanum avenaceum (Fr.) Sacc. Phoma rostmpii Sacc. Xanthomonas carotae (Kendrick) Dowson Viruses

Sr. No .
1 2

Common Names
Carrot leaf blight Black root rot , seedl ing

3 4 5 6 7

Cercospora bl ight of carrot ,leaf spot Brown root rot Phoma root rot Bacter ia l bl ight , root scab Carrot motley dwarf (a complex of three viruses, including carrot red leaf) Carrot red leaf virus

2.

The main seed-borne Parsn ip pathogens with common names of the diseases they cause Pathogen Common Names

Sr. No.
1 2 3

Alternar ia dauci (Ki ihn) Groves and Skolko syn. Black mould A. porr i (El l . ) Cif fe r r i daud f . sp . (Ki i hn) Neergaard Alternar ia radic ina Meier, Drechsl . and Black mould Eddy syn. Stemphyl ium radidnum Meier, Drechsl . and Eddy Erysiphe heraclei DC. ex St Amans Powdery

288

4 5

I te r son i l i a Virus

past inacea Channon

mildew Canker Strawberry Latent ringspot Virus

3.

The main seed-borne Pars ley pathogens with common names of the diseases they cause Pathogen Common Names

Sr. No .
1 2 3 4 5 6 7 4.

Alternar ia dauci (Ki ihn) Groves and Skolko syn. Black mould A. porr i (El l . ) Cif fe r r i daud f . sp . (Ki i hn) Neergaard Alternar ia radic ina Meier, Drechsl . and Black mould Eddy syn. Stemphyl ium radidnum Meier, Drechsl . and Eddy Erys iphe herac le DC. i ex St Amans Powdery mildew I te rson i l i a past inacea Channon Canker Phoma anethi (Pers . ex Fr . ) Sacc. syns. Leaf and Marssonina kirchner Hegyi i and Passalora stem spot kirchneri (Hegyi) Petr Rhizoctonia solani Kiihn Root & basal stem rot Septoria petroselini Desm. Leaf spot

Sr. No.
1 2 3 4 5 6 7

The main seed-borne Celery and Celer iac pathogens with common names of the diseases they cause Pathogen Common Names
Alternar ia dauci (Ki i hn) Groves and Skolko syn. A. porr i (El l . ) Cif fe r r i f . sp . daud (Ki i hn) Alternar ia radic ina Meier, Drechsl . and Eddy syn. Stemphyl ium radidnum Meier, Botrytis cinerea Pers. ex Pers. Cercospora apii Fresen Gibberella avenacea Cook syn. Fusanum avenaceum (Fr.) Sacc. Phoma apiicola Kleb. Septoria apiicola Speg. syn. S. apiiBlack mould, Root rot Black mould, Root rot Grey mould Ear ly bl ight , leaf spot Brown root rot Celery root rot , black- neck, scab, seedl ing canker Late bl ight , small 289

graveolent i s Dorogin 8 9 10 11 Vert i c i l l i um albo- atrum Reinke and Berth Erwin ia carotovora (Jones) Bergey et al. syn. Pectobacterium carotovorum (Jones) Waldee Pseudomonas apii Jagger Virus

leaf spot , large leaf spot Wilt Soft rot , crater spot Bacter ia l bl ight Strawberry latent r ingspot virus

Blotter method for the detection of Alternaria dauci on Daucus carota (car ro t ) [ ISTA Val ida ted Method]

Prepared by: Sheppard, J .W. , Cockere l l , V. and Roberts , S. J . ISTA- PDC Method Val ida t i on Sub- committee Sponsored by: ISTA-PDC Method Validation Sub-committee Revision History:Version 1.0, 01 January 2003. Background This method was originally published in the ISTA Handbook of Seed Health Test ing in November 1964 as S.3. No. 4 and revised in 1987 (Gambogi, 1987). It was incorporated into the Annexe to Chapter 7: Seed Health Test ing Methods as method 7-001 (Sheppard and Cockerell, 2002). It has been renumbered (7-001a) and slightly modified following studies conducted by the International Seed Health Initiative Vegetables in 1999 and 2001 (Van Bilsen, 2003). The studies compared blotter and malt agar methods and concluded that the two were equivalent. Note that seeds can be simultaneously tested for the presence of Alternaria radicina using the same method (see method 7-002a). Validation studies Van Bilsen (2003) Copies are available: by e-mail from ista.offi ce@ista.ch; or by mail from the ISTA Secretariat.

290

Safety Precautions Ensure you are fami l i a r with hazard data and take appropr ia te safety precaut ions . I t i s assumed that th i s procedure i s being carr i ed out in a microbio log i ca l laborato by persons fami l i a r with the pr inc ip l es of Good Laboratory Pract i ce , Good Microb io l og i ca l Pract i ce , and asept i c technique. Dispose of al l waste mater ia l s in an appropr ia te way (e .g . autoc lave, dis in fec t ) and in accordance with loca l heal th , envi ronmental and safety regula t i ons . Treated seed Seed treatments may af fec t the perfo rmance of th i s test . should only be perfo rmed on untreated seed. Materials It

Reference mater ia l - The use of reference cul tu res or other appropr ia te mater ia l i s recommended. Substra te - Blot te r s or f i l te r papers , 9.0 cm, ci r cu l a r (e .g Whatman No 1 or equiva lent ) , f ree f rom micro- organisms and inh ib i t o r s (3 per plate) . Pla tes - 9.0 cm ster i l e Petr i dishes (one per ten seeds) . Incubator - Operat ing at 20 2C, equipped with t imercontro l l ed near- ul t rav i o l e t l i gh t s (NUV, peak at 360 nm, e.g color number 08, Phi l i p s ; BLB, Sylvan ia ) . Freezer - Operat ing at 20 2C. Sample Preparation

1. I t i s vi ta l to exclude any poss ib i l i t y of cross - contamina between seed samples . This can be achieved by swabbing/spray ing equipment and gloved hands with 70% ethanol . 2. The test i s carr i ed out on a working sample as descr ibed in Sect ion 7.4.1 of the In ternat i ona l Rules for Seed Test ing .

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Method [Cr i t i c a l

contro l points are ind i ca CCP ted ] by

1. Place three 9.0 cm filter papers in each plate and soak with sterile distilled/de-ionized water. Drain away excess water. 2. Aseptically place 10 seeds, evenly spaced (CCP), on the surface of the filter paper in each plate. 3. Incubate for 3 d at 20 2C in the dark. 4. Transfer plates to freezer and maintain at 20 2C for 24 h. 5. After freezing, incubate for 6 d at 20 2C with alternating 12 h periods of darkness and NUV light (ISTA,1984; Tempe, 1968). Plates should be approx. 25 cm below the lights and should not be stacked. 6. Examine seeds under a stereoscopic microscope at x30 for fungal growth and up to x80 magnification for identification of conidia. Conidiophores are simple or slightly branched (Fig. 1), arising singly or in small groups from the surface of the seed or on aerial mycelium. Conidia are usually solitary, obclavate, up to 450 m long (including beak), pale olivaceous brown at first, becoming brown with age, with a long pale beak up to 3 times the length of the body (Ellis, 1971). Groups of sunken conidia are sometimes visible by the emerging clusters of their bright long beaks (Fig. 1, bottom left). Record the number of infected seeds in each plate (CCP). General Methods 1. Checking Tolerances Tolerances provide a means of assessing whether or not the variation in result within or between tests is sufficiently wide as to raise doubts about the accuracy of the results. A tolerance table, which can be applied to most direct seed health tests, can be found in Table 5.1 of Annexe 16 of the International Rules for Seed Testing or in Table G1 of the

292

Handbook of Tolerances and Measures of Prec i s i on for Seed Test ing(Miles, 1963) 2. Report ing Resul t s The result of a seed health test should indicate the scientific name of the pathogen, and the test method used. When reported on an ISTA Certificate, results are entered under Other Determinat ions . In the case of a negative result (pathogen not detected), the results should be reported in terms of the tolerance standard (e.g. infection level less than 1% with 95% probability). The tolerance standard depends on the total number of seeds tested, n, and is approximately 3/n (P=0.95) (see Roberts et al . ,1993). In the case of a positive result the report should indicate percentage of infected seeds. Quality Assurance Specif ic Training This test should only be performed by persons who have been trained in fungal identification or under the direct supervision of someone who has. Crit ical Control Points [Identified by CCP in the methods] Spreading hyphae may lead to contamination of other seeds. Seeds must therefore be spaced at least 20 mm from each other with not more than 10 seeds per 9.0 cm Petri dish (Step 2). Samples may be difficult to examine due to the growth of contaminants, especially Alternar i a tenuis , and/or A. radicina. Experience and great care is required for the detection of all occurrences (ISTA 1984) (Step 6). Supplementary examination may be required 13-14 d after plating of seeds to allow hampered or deeper inoculum to

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exceed contaminants by i t s f ruc t i f i (Step 6) . References

cat ion (Hewett , 1964)

El l i s , M.B. (1971) Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, Surrey, England. 609 pp. Gambogi, P. (1987) International Seed Testing Association, Handbook on Seed Health Testing, Working Sheet No 4 (2nd Ed.): Alternaria dauci on Daucus carota. International Seed Testing Association, Zurich, Switzerland. Hewett, P.D. (1964) Testing carrot seed infected with Alternaria porri f. sp. dauci. Proceedings of the International Seed Testing Association, 29 , 463-471. International Seed Testing Association (1984) Report of the 18th International Seminar on Seed Pathology, 9-16 July, 1984, Washington State University, Puyallup, U.S.A. Miles, S.R. (1963) Proceedings of the International Seed Testing Association, 28 (3), 644. Roberts, S.J., Phelps, K., Taylor, J.D. and Ridout, M.S. (1993) Design and interpretation of seed health assays. In: Sheppard, J.W., (Ed.) Proceedings of the First ISTA Plant Disease Committee Symposium on Seed Health Testing, Ottawa, Canada. pp. 115-125. Agriculture Canada, Ottawa, Canada. Sheppard, J.W. and Cockerell, V. (2002) Detection of Alternaria dauci on Daucus carota (Carrot). International Rules for Seed Testing. Annexe to Chapter 7: Seed Health Testing Methods. 7-001.

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Tempe, J . de (1968) : The quant i t a t i v e ef fec t of l i gh t and moisture on carro t seed in fec t i ons in blot te r . medium Proceedings of the International Seed Testing Association, 33, 547-553. Van Bilsen, J.G.P.M. (2003) Report of a comparative test on Alternaria dauci and Alternaria radicina on carrot seed. ISTA Method Validation Reports (submitted).

Fig- 1: Top: single conidia of Alternaria dauci and chains of conidia of the saprophyte A. tenuis on seed surface (left) and simple or slightly branched conidiophores with conidia of A. dauci developing from creeping hyphae on the blotter (right), x80 magnifi cation. Bottom: conidia of A. dauci on simple or slightly branched conidiophores borne on a single rootlet initial at x80 magnifi cation (left); conidium and simple conidiophores at x350 magnifi cation (right).

295

Fig-2: Top: conidiophores and conidia of Alternaria radicina and chains of conidia of the saprophyte A. tenuis on a rootlet initial x80 (left); spreading hyphae and fructifications of the pathogen on the blotter, x80 (centre); abundant growth and fructification of the pathogen on a rootlet initial, x50 (right). Bottom: conidia of Alternaria radicina, x350.

296

Detection of Xanthomonas hortorum pv. carotae (bacter i a l lea f bl ight on Daucus ) carota (carrot) [ISTA Validated Method] Prepared by: Asma, M. Bejo Zaden B.V., P.O. Box 50, 1749 ZH Warmenhuizen, The Netherlands. E-mail: m.asma@bejo.n l Sponsored by: ISHI-Veg Revision History:Version 1.0, 1 July 2005. Background The most commonly used method in seed health testing laboratories for the detection of Xanthomonas hortorum pv. carotae is based on a seed wash dilution-plating assay. This method involves washing seeds in buffer and plating serial dilutions of the extract on a semiselective medium. Various semi-selective media are currently used as described or adapted from the following papers: Cubeta and Kuan, (1986); Williford and Schaad, (1984); Kim et al., (1982); and McGuire et al., (1982). These media have been tested by ISHI-Veg and ISHI-Veg/ISTA in a number of comparative studies (Asma, 1999, Asma, 2000a and Asma, 2000b). In addition to comparing selective media the latter comparative study (Asma, 2000b) concluded that the confirmation method chosen had an effect on test results, with ELISA and IF giving false positive confirmations due to poor specificity of antisera. The 2000 study (Asma, 2000b) also looked at the effect of antibiotics and agar source on the performance of the test. Further work by Asma et al., (2002) has shown PCR to be a reliable and quick confirmation method when compared to pathogenicity tests. This method is derived from the previous comparative tests and the validation studies carried out by ISHI-Veg in 2003

297

(Asma, 2005). For routine testing of carrot seed a combination of two semi-selective media, MKM/MD5A or MKM/mTBM is recommended. If nystatin at a concentration of 35 mg/L is not enough to completely inhibit fungal growth, cycloheximide should be used. Either a pathogenicity test or a PCR test is used to confirm suspect isolates. Validation Studies:Asma, M. (2005) Copies are available: by E-mail from i s ta . o f f i c e@is ta ;. ch by mail f rom the ISTA Secretar i a t , Zr ichs t rasse 50, 8303 Bassersdor f , Switzer l and Safety Precautions Ensure you are familiar with hazard data and take appropriate safety precautions, especially during preparation of media, autoclaving, and weighing out of ingredients and handling of ethidium bromide. It is assumed that this procedure is being carried out in a microbiological laboratory by persons familiar with the principles of Good Laboratory Practice, Good Microbiological Practice, and aseptic technique. Dispose of all waste materials in an appropriate way (e.g. autoclave, disinfect) and in accordance with local health, environmental and safety regulations. Treated Seed Chemical seed treatments may affect the performance of this test. It should only be performed on untreated seed. Sample and sub-sample size The sample (total number of seeds tested) and sub-sample size to be tested depends on the desired tolerance standard (maximum acceptable percentage of seeds infested) and detection limit (theoretical minimum number of pathogen propagules per seed which can be detected); in any case the

298

maximum sub-sample size should be 10,000 seeds. A full discussion of these aspects can be found in Geng et al . (1987) , Roberts et al. (1993) and Roberts (1999). Materials Reference material - known strain of Xanthomonas hortorum pv. carotae (X. hortorum pv. carotae) or standardised reference material Plates of MKM medium - 9.0 cm Petri dishes (8 plates of MKM medium per sub-sample + controls) Plates of MD5A medium or mTBM medium - 9.0 cm Petri dishes (8 plates of MD5A or 8 plates of mTBM medium per sub-sample + controls) Plates of YDC - for sub-culture (at least 1 plate per subsample) Conical fl asks - of sterile saline (0.85% NaCl) plus Tween 20 (0.02% - 20l per 100 ml) for soaking of seeds (10 ml per 1000 seeds) Dilution bottles - containing 4.5 ml of sterile saline (2 per sub-sample); other volumes may be acceptable, see General Methods 70% ethanol - for disinfection of surfaces, equipment Incubator - operating at 28C Balance- capable of weighing to the nearest 0.001 g pH meter - capable of being read to the nearest 0.01 pH unit Automatic pipettes - capable of pipetting to the nearest 0.001 ml; check accuracy and precision regularly Carrot seedlings - susceptible to the pathogen for pathogenicity test e.g. cv. Napoli PCR Primers (Meng et al., 2004) - 3Sforw 5 CAT.TCC.AAg.AAg.CAg.CCA 3 3Srev 5 TCg.CTC.TTA.ACA.CCg.TCA 3 Universal Primers (adapted from Eden et al., 1991) - 1052F 5 gCA.Tgg.TTg.TCg.TCA.gCT.CgT 3 Bac R 5 TAC.ggC.TAC.CTT.gTT.ACg.ACT.T 3 Thermal cycler - ramping time 3C/s Agarose electrophoresis equipment Orbital shaker Sterile pipette tips

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Sterile bent glass rods Sample Preparation 1. This can be done in advance of the assay. 2. It is vital to exclude any possibility of cross-contamination between seed samples. It is therefore essential to disinfect all surfaces, containers, hands, etc. both before and after handling each sample. This can achieved by swabbing/spraying equipment and gloved hands with 70% ethanol. 3. If the submitted sample is received in several packets, these should be combined by emptying into a new, clean polythene bag and mixing by hand to give a composite sample. 4. Count the number of seeds in a known weight. Estimate the Thousand Seed Weight (TSW) as:

5. Based on the TSW, weigh out sub-samples of the required size into new, clean polythene bags. Method [Critical control points are indicated by CCP ] 1. Extrac t i on 1.1. Suspend seeds in sterile saline plus Tween 20 (0.02% v/v) in a conical fl ask. The volume of saline should be adjusted according to the number of seeds used (10 ml per 1,000 seeds). 1.2. Soak sub samples overnight (16-18 h) at 4-7C. 1.3. Shake for 5 min at room temperature (20-25C) on an orbital shaker set at 200 rpm. 2. Di lu t i on and plat i ng 2.1. Shake the fl asks to mix just before dilution.
300

2.2. Prepare a tenfold dilution series from the seed extract. Pipette 0.5 ml of the extract into 4.5 ml of sterile saline and vortex to mix (101 dilution). Pipette 0.5 ml of the 101 dilution into another 4.5 ml of sterile saline and vortex to mix (102 dilution). Pipette 0.5 ml of the 102 dilution into another 4.5 ml of sterile saline and vortex to mix (103 dilution) (see General Methods). 2.3. Pipette 100 l of each dilution and the un-diluted seed extract onto two plates of each of the two chosen semiselective media either MKM medium with MD5A medium or MKM medium with mTBM medium and spread over the surface with a sterile bent glass rod (see General Methods). 2.4. Incubate plates with positive control plates (Section 3) at 28C and examine after 4-8 d. 3. Posi t i ve contro l (cu l tu re or reference mater ia l ) 3.1. Prepare a suspension of a known strain of X. hortorum pv. carotae, e.g. NCPPB 425, in sterile saline or reconstitute standardised reference material according to the suppliers instructions. If a lyophilised culture is used, culture at least once on a non-selective medium prior to use to check the viability and morphology. 3.2. Dilute suffi ciently to obtain dilutions containing approximately 102 to 104 cfu/ml. This may require up to seven ten-fold dilutions from a turbid suspension. 3.3. Pipette 100 l of appropriate dilutions onto plates of both semi-selective media (MKM/MD5A or MKM/mTBM) and spread over the surface with a sterile bent glass rod. 3.4. Incubate plates with the sample plates (Section 2). 4. Sterility check 4.1. Prepare a dilution series from a sample of the extraction medium (i.e., saline plus Tween 20), containing no seeds, and plate on both semiselective media as for samples.

301

5. Examination of the plates 5.1. Examine sterility check and positive control plates (CCP ) . 5.2. Examine the sample plates for the presence of typical X. hortorum pv. Carotae colonies by comparison with the positive control plates. 5.3. On MKM after 4-6 d, X. hortorum pv. carotae colonies appear light yellow-cream, light brown to peach yellow, glistening, round and 2-4 mm in diameter (Fig. 1). 5.4. On MD5A after 7-8 d, X. hortorum pv. carotae colonies appear straw yellow, glistening, round smooth, convex with entire margins, and 2-3 mm in diameter (Fig. 2). 5.5. On mTBM after 7-8 d, X. hortorum pv. carotae colonies appear white or yellow or white-yellow, glistening, round, smooth, convex with entire margins, 1-2 mm in diameter and surrounded by a large clear zone of casein hydrolysis (Fig. 3). Casein hydrolysis on mTBM is not always present. 5.6. The colony size and color can differ within a sample. 5.7. Record the number of suspect and other colonies (see General Methods). 6. Confirmation/identification of suspect colonies 6.1. Sub-culture suspect colonies to sectored plates of YDC. To avoid the potential for cross-contamination of isolates, use a new sectored plate for each sub-sample. The precise numbers of colonies sub-cultured will depend on the number and variability of suspect colonies on the plate: if present, at least six colonies should be sub-cultured per sub-sample. 6.2. Sub-culture the positive control isolate to a sectored plate for comparison. 6.3. Incubate sectored plates for 48-72 h at 28C. 6.4. Compare appearance of growth with positive control. On YDC X. hortorum pv. carotae colonies are pale yellow and mucoid (Fig. 4).

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6.5. Confi rm the identity of isolates by pathogenicity on carrot seedlings of known susceptibility or by polymerase chain reaction (PCR). 6.6. Record results for each colony sub-cultured. 7. Pathogenic i t y CCP ( ) 7.1. Grow seedlings of a carrot cultivar known to be susceptible to X. hortorum pv. carotae (e .g . cv. Napol i ) in smal l pots or modules unt i l at leas t 3- 4 true lea f stage (approx imate ly 3- 4 weeks after sowing) . 7.2. Prepare a suspension in sterile tap water from each suspect bacterial culture on YDC medium and dilute to a concentration containing approximately 2x106 cfu/ml. The same procedure should be used for the positive control isolate. 7.3. Inoculate plants by spraying until runoff. Use one small pot with 3-4 plants per isolate. Include the positive control and a negative control. It is important not to rub the leaves after spraying, since this will cause false positive results (CCP ) . 7.4. Incubate inoculated plants at 27-28C enclosed in plastic bags (to provide conditions near 100% RH). After 48 h, remove the bags during daytime and replace at night. 7.5. Record symptoms after 7-10 d incubation. Typical X. hortorum pv. Carotae symptoms fi rst appear as small irregular yellowish water-soaked areas with a tiny light brown spot in the centre on inoculated leaves. Later, affected areas enlarge, become brown, and are often surrounded by a yellow halo (Fig. 5). Compare with positive control (CCP ). 8. Polymerase Chain Reaction (PCR) 8.1. Make a slightly turbid cell suspension (OD600nm approximately 0.05) in 1.0 ml sterile distilled water from the suspect cultures on YDC medium and the positive control. In addition a non-suspect isolate should be used as a negative control. The suspensions can be stored at -20C until identification.

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8.2. Use the following X. hortorum pv. carotae specific primers (Meng et al., 2004). 3Sforw 5 CAT.TCC.AAg.AAg.CAg.CCA 3 3Srev 5 TCg.CTC.TTA.ACA.CCg.TCA 3 8.3. Universal bacterial Primers should be used to validate the PCR reaction. These primers will give a product size of 441 bp. (adapted from Eden et al., 1991) compared to the 355 bp product from the X. hortorum pv. carotae specific primers. 1052F 5 gCA.Tgg.TTg.TCg.TCA.gCT.CgT 3 Bac R 5 TAC.ggC.TAC.CTT.gTT.ACg.ACT.T 3 8.4. Prepare the reaction mixture (page 7-020-17). Carry out PCR reactions in 0.2 ml thin walled PCR tubes in a fi nal volume of 10 l (8 l reaction mixture + 2 l bacterial suspension). 8.5. PCR profi le: An initial 5 min incubation at 95C followed by 35 cycles of 15 s at 94C, 15 s at 58C and 30 s at 72C. A fi nal 5 min incubation at 72C and 20 min at 20C 8.6. Fractionate 10 l of the PCR products by gel electrophoresis during 1.5 h at 150V on a 1.5% agarose gel in 0.5x Tris Borate EDTA (TBE buffer) stained with ethidium bromide. Include a 100 bp ladder. 8.7. Analyse the amplifi cation products for a X. hortorum pv. carotae specific product of 355 bp and a universal bacterial product of 441 bp with an ultraviolet trans-illuminator. Two bands (specifi c and universal) = positive identifi cation; one band (universal) = negative identifi cation; no bands = bacterial template absent, repeat reaction. General Methods (common to many test procedures) 1. Preparation of ten-fold dilution series Each dilution should be prepared by pipetting 0.5 ml ( 5%) from a well-mixed seed extract or previous dilution into a universal bottle (screw-capped) or similar containing 4.5 ml ( 2%) of sterile diluent and then vortexing to mix prior to the next dilution step. A new sterile pipette tip should be used for each dilution step. Pipettes should be checked regularly for accuracy and precision and re-calibrated as

304

necessary. It is acceptable to prepare ten-fold dilutions using other volumes provided that the laboratory can demonstrate that the required accuracy and precision can be achieved. 2. Plating of dilutions. This should be done as soon as possible after dilutions have been prepared and certainly within 30 min. Working from the highest (most dilute) dilution to the undiluted extract, 0.1 ml is pipetted onto the centre of a surface-dry, labeled agar plate. The liquid should then be spread evenly over the entire surface of the medium with a bent glass rod. If care is taken to work from the highest to the lowest dilution (or undiluted extract) a single pipette tip and a single bent glass rod can be used for each sample. Ensure that all liquid has been absorbed by the agar before inverting and incubating plates. If necessary allow plates to dry under a sterile air-flow in a microbiological safety cabinet or laminar flow hood. 3. Recording of dilution plates Record the results for all dilution plates. The most accurate estimate of bacterial numbers should be obtained from spread plates with total number between 30 and 300 colonies. However this may be further complicated depending on the relative numbers of suspect pathogen and other colonies. In order to minimize effort, start recording with the highest dilution (most dilute) and count the number of suspect and the number of other colonies. If the total number of colonies on a plate greatly exceeds 300 there is little value in trying to make a precise count if a more reliable count has already been obtained from a more dilute plate, in which case it is sufficient to record the number of colonies as m (many) if they are still separate or c (confluent) if they have run together. 4. Sectored Plates Using a laboratory marker pen draw lines on the base of a standard 9 cm plate (Petri dish) to divide it into six equal sectors. Sub-culture single colonies from dilution plates and

305

make a single zigzagged streak within a single sector on the plate. Take care to leave sufficient space between each isolate to ensure the growth does not coalesce. Thus six suspect colonies can be sub-cultured to each sectored plate. Separate plates should be used for each sample/sub-sample. If the purity of sub-cultured isolates is doubtful, they should be further streaked out on whole plates. 5. Reporting Results The result of a seed health test should indicate the scientific name of the pathogen and the test method used. When reported on an ISTA Certificate, results are entered under Other Determinations. In the case of a negative result (pathogen not detected in any sub-samples), the results should be reported in terms of the tolerance standard and detection limit. The tolerance standard depends on the total number of seeds tested, n, and is approximately 3/n (P=0.95) (see Roberts et al . , 1993); the detect i on l imi t per sub- sample i s equal to the detect i on l imi t per ml mult ip l i ed by the volume of extract . In the case of a positive result, the report should indicate the mean number of pathogen propagules (cfu) per seed and either the number of positive sub-samples out of the total number tested and the sample size or the maximum likelihood estimate of the proportion of infested seeds. Quality Assurance General A record should be kept of the date and results of pipette calibration checks. It is essential that operators have received appropriate training and use automatic pipettes correctly. Critical Control Points [Identified by CCP in the methods]

306

Dilution plates prepared from positive control isolate(s) or reference material, should give single colonies with typical morphology (Step 5.1). The numbers of colonies on dilution plates prepared from the positive control isolate(s) or reference material should be similar on both media (Step 5.1). Numbers of bacteria on dilution plates should be consistent with the dilution (i.e. should decrease approx. ten-fold with each dilution) (Step 5.1). There should be no growth on dilution plates prepared as a sterility check (Step 5.1). Most cultivars of bunching carrots are susceptible to X. hortorum . pv. carotae (Step 7.1) Positive control isolates should be included in every pathogenicity test (Step 7.3). The positive control isolate should give typical symptoms in a pathogenicity test (Step 7.5). The activity (units/mg) of some antibiotics may vary between batches. It may be necessary to adjust the weight or volume added to ensure that the final number of units per liter of medium is consistent (MKM, MD5A).

307

Fig- 1: Xanthomonas hortorum pv. Carotae colonies on MKM plates after 6 d indicated by light yellow-cream, light brown to peach yellow, glistening, round colonies and 2-4 mm in diameter.

Fig- 2: Xanthomonas hortorum pv. Carotae colonies on MD5A plates after 7 d indicated by straw yellow, glistening, round smooth, convex colonies with entire margins, and 2-3 mm in diameter.

Fig- 3: Xanthomonas hortorum pv. Carotae colonies on mTBM plates after 7 d indicated by white or yellow or white-yellow, glistening, round, smooth, convex colonies with entire margins, 1-2 mm in diameter and surrounded by a zone of casein hydrolysis. 308

Fig- 4: Typica l pale yel low and mucoid growth Xanthomonas of hortorum pv. carotae isolates on a sectored plate of YDC after 72 h at 28C.

309

Fig- 5: Typica lXanthomonas hortorum pv. carotae symptoms in a pathogenicity test indicated by small brown irregular areas surrounded by a yellow halo.

310

Universal band Specific band

Fig- 6: Agarose gel showing Xanthomonas hortorum pv. carotae specific products of 355 bp and universal bacterial products of 441 bp. Two bands (specific and universal) = positive identification; one band (universal) = negative identification.

Preparation of sterile saline Compound Sodium chloride (NaCl) Distilled/de-ionized water Preparation 1. Weigh out all ingredients into a suitable container.
311

g/l 8.5 1000 ml

g/500 ml 4.25 500 ml

2. Add 1000 ml (or 500 ml) of distilled/de-ionised water. 3. [For extraction of seeds, add 200 l of sterile Tween 20 per 1000 ml]. 4. Dissolve and dispense into fi nal containers. 5. Autoclave at 121C, 115 psi for 15 min. Storage Provided containers are tightly closed, may be stored for several months before use. Preparation of MKM agar medium Note. This medium is a modification of the KM-1 medium (Kim et al . , 1982) f rom which i t di f f e r s in ant ib i o t i c s composi t i on and concentra t i on . The amounts of phosphate salts have also been adjusted to achieve the correct pH without further adjustment. Compound NH4Cl K2HPO4 KH2PO4 Lactose monohydrate D(+) trehalose dihydrate Yeast extract 2-Thiobarbituric acid Bacto agar Tobramycin sulphatea Cephalexin monohydrateb Bacitracinc Nystatind g/l 1.0 1.2 1.2 10.0 4.0 0.5 0.2 17.0 0.00 2 0.01 0 0.05 0 0.03 5 g/500 ml 0.5 0.6 0.6 5.0 2.0 0.25 0.1 8.5 0.001 0.005 0.025 0.018

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a, b, c, d Added after autoclaving Preparation 1. Weigh out all ingredients except antibiotics into a suitable container. 2. Add 1000 ml (or 500 ml) of distilled/de-ionized water. 3. Dissolve and check pH which should be 6.6. 4. Autoclave at 121C, 115 psi for 15 min. 5. Prepare antibiotic solutions. 6. Allow medium to cool to approximately 50C and add antibiotic solutions. 7. Mix thoroughly but gently by inversion/swirling to avoid air bubbles and pour plates (22 ml per 9.0 cm plate). 8. Leave plates to dry in a laminar fl ow bench or similar before use. Antibiotics and other additions (amounts for guidance only, CCP ) a. Dissolve 20 mg tobramycin sulphate (Sigma T-1783 or Duchefa T-0153) in 10 ml 70% ethanol. Add 1 ml/l (0.5 ml/500 ml). b. Dissolve 200 mg cephalexin monohydrate (Sigma C-4895) in 10 ml 70% ethanol. Add 0.5 ml/l (0.25 ml/500 ml). c. Dissolve 500 mg bacitracin (Sigma B-0125 66K units/g or Duchefa B-0106, 70 K units/g) in 10 ml 70% ethanol. Add 1.0 ml/l (0.5 ml/500 ml). d. Dissolve 100 mg nystatin (Sigma N-3503, Duchefa N0138) in 10 ml 70% ethanol. Add 3.5 ml/l (1.75 ml/500 ml). Use 100 mg/l cycloheximide, instead of nystatin, when fungal growth on the selective media is not completely inhibited by 35 mg/l nystatin. Storage Store prepared plates inverted in polythene bags at 4C and use within two weeks of preparation to ensure activity of antibiotics.

313

Preparation of MD5A agar medium (Cubeta and Kuan, 1986) Compound g/l g/500 ml

MgSO4.7H2O 0.3 0.15 NaH2PO4 1.0 0.5 NH4Cl 1.0 0.5 K2HPO4 3.0 1.5 Bacto agar 17.0 8.5 a Cellobiose 10.0 5.0 b L-glutamic acid 0.005 0.0025 c L-methionine 0.001 0.0005 d Cephalexin monohydrate 0.01 0.005 e Bacitracin 0.01 0.005 f Nystatin 0.035 0.018 a, b, c, d, e, f Added after autoclaving Preparation 1. Weigh out all ingredients except antibiotics, L-glutamic acid, L-methionine and cellobiose into a suitable container. 2. Add 900 ml (or 450 ml) of distilled/de-ionised water. 3. Dissolve and check pH which should be 6.4, adjust if necessary. 4. Autoclave at 121C, 115 psi for 15 min. 5. Prepare antibiotic, L-glutamic acid, L-methionine and cellobiose solutions. 6. Allow medium to cool to approximately 50C before adding solutions. 7. Mix thoroughly but gently by inversion/swirling to avoid air bubbles and pour plates (22 ml per 9.0 cm plate). 8. Leave plates to dry in a laminar fl ow bench or similar before use.

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Antibiotics and other additions (amounts for guidance only, CCP ) a. Dissolve 10 g cellobiose in 100 ml of distilled/de-ionised water and fi ltersterilize. b. Dissolve 50 mg L-glutamic acid in 10 ml distilled/deionised water and fi ltersterilize. Add 1.0 ml/l (0.5 ml/500 ml). c. Dissolve 10 mg L-methionine in 10 ml distilled/de-ionised water and fi ltersterilize. Add 1.0 ml/l (0.5 ml/500 ml). d. Dissolve 200 mg cephalexin monohydrate (Sigma C-4895) in 10 ml 70% ethanol. Add 0.5 ml/l (0.25 ml/500 ml). e. Dissolve 500 mg bacitracin (Sigma B-0125, 66K units/g or Duchefa B-0106, 70 K units/g) in 10 ml 70% ethanol. Add 0.2 ml/l (0.1 ml/500 ml). f. Dissolve 100 mg nystatin (Sigma N-3503, Duchefa N-0138) in 10 ml 70% ethanol. Add 3.5 ml/l (1.75 ml/500 ml). Use 100 mg/l cycloheximide, instead of nystatin, when fungal growth on the selective media is not completely inhibited by 35 mg/l nystatin.

Storage Store prepared plates inverted in polythene bags at 4C and use within two weeks of preparation to ensure activity of antibiotics. Preparation of mTBM agar medium Note. This medium is a modification of Tween Medium B (McGuire et al . , 1986) f rom which it differs in adding 10.0 g/l skim milk powder and leaving out 0.4 mg/l tobramycine and 0.25 g/l CaCl2.

315

Compound

g/l

g/500 ml

H3BO3 0.3 0.15 KBr 10.0 5.0 Peptone 10.0 5.0 a Skim milk powder 10.0 5.0 Bacto agar 17.0 8.5 b Tween 80 10.0 ml 5.0 ml c Cephalexin monohydrate 0.065 0.033 d 5-Fluorouracil 0.012 0.006 e Nystatin 0.035 0.018 a, b, c, d, e Added after autoclaving Preparation 1. Weigh out all ingredients except skim milk powder, antibiotics and Tween 80 into a suitable container. 2. Add 900 ml (or 450 ml) of distilled/de-ionised water. 3. Dissolve and check pH which should be 7.4, adjust if necessary. 4. Autoclave at 121C, 115 psi for 15 min. 5. Prepare antibiotic and skim milk powder solutions. 6. Allow medium to cool to approximately 50C before adding skim milk powder, antibiotic solutions and Tween 80. 7. Mix thoroughly but gently by inversion/swirling to avoid air bubbles and pour plates (22 ml per 9.0 cm plate). 8. Leave plates to dry in a laminar flow bench or similar before use. Antibiotics and other additions (amounts for guidance only, CCP ) a. Autoclave solution (10.0 g/100 ml) separate. The quality of skim milk powder greatly affects the capabilities of mTBM. Milk sources that work well are BBL, Oxoid or Sigma. c. Dissolve 200 mg cephalexin monohydrate (Sigma C-4895) in 10 ml 70% ethanol. Add 3.25 ml/l (1.625 ml/500 ml).

316

d. Dissolve 100 mg 5-fl uorouracil (Sigma F-6627, Duchefa F0123) in 10 ml 70% ethanol. Add 1.2 ml/l (0.6 ml/500 ml). e. Dissolve 100 mg nystatin (Sigma N-3503, Duchefa N0138) in 10 ml 70% ethanol. Add 3.5 ml/l (1.75 ml/500 ml). Use 100 mg/l cycloheximide, instead of nystatin, when fungal growth on the selective media is not completely inhibited by 35 mg/l nystatin. Storage Store prepared plates inverted in polythene bags at 4C and use within two weeks of preparation to ensure activity of antibiotics. Preparation of Yeast Extract Dextrose Chalk (YDC) medium (Schaad, 1988) Compound Bacto Agar Yeast Extract CaCO3 (light powder) D-Glucose (Dextrose) Distilled/de-ionized water Preparation 1. Weigh out all ingredients into a suitable oversize container (i.e. 250 ml of medium in a 500 ml bottle/fl ask) to allow swirling of medium just before pouring. 2. Add 1000 ml (or 500 ml) of distilled/de-ionised water. 3. Autoclave at 121C, 115 psi for 15 min. 4. Allow medium to cool to approx. 50C . 5. Swirl to ensure even distribution of CaCO3 and avoid air bubbles, and pour plates (22 ml per 9.0 cm plate). g/l 17.0 10.0 20.0 20.0 1000 ml g/500 ml 8.5 5.0 10.0 10.0 500 ml

317

6. Leave plates to dry in a laminar fl ow bench or similar before use. Storage Store prepared plates inverted in polythene bags at room temperature. Prepared plates can be stored for several months provided they do not dry out. Preparation of Reaction Mixture for PCR Compound Sterile MilliQ 10x Buffer MgCl2 (50 mM) dNTPs (10 mM tota l , 2.5 mM
each)

Final concentration

1x 1.5 mM 200 M each 0.50 M 0.50 M 0.10 M 0.10 M 0.04 U/l

Volume (l) in 10 l 3.42 1.00 0.30 0.80 1.00 1.00 0.20 0.20 0.08 2.00

Primer 3Sforw (5 pmol/l) Primer 3Srev (5 pmol/l) Primer 1052F (5 pmol/l) Primer BacR (5 pmol/l) Taq Polymerase (5U/l) Bacterial suspension 10x Buffer Tris-HCL(pH 9.0) = 750 mM (NH4)2SO4 = 200 mM Tween 20 = 0.1% (v/v)

Preparation of Tris Borate EDTA (TBE) Buffer 0.5x Compound Tris Boric acid g/l 5.39 2.75 g/500 ml 2.70 1.38
318

Na2EDTA.2H2 O

0.37

0.19

The pH is 8.3 and requires no adjustment.

Preparation of 1.5% agarose gel for electrophoresis Compound Tris Borate EDTA (TBE) 0.5x Agarose Ethidium bromidea
1 agarose gel (20x20 cm)

1 liter 1000 ml 15.0 g 50.0 l

160 ml 2.4 g 8.0 l

a Dissolve 100 mg ethidium bromide in 10 ml distilled/de-ionized water. Add 50 l/l of this solution. Ethidium bromide is carcinogenic !

Preparation 1. To calculate the volume of the gel (in ml), multiply the area of the gel by the required thickness (0.4 cm). 2. Make sure that the gel tray is clean and dry before use. Seal the ends of the tray with tape. 3. Weigh out the desired amount of agarose and place in an Erlenmeyer fl ask with a measured amount of electrophoresis buffer, e.g. for a 100 ml gel add 1.5 g of agarose and 100 ml of 0.5x TBE buffer to a 200 ml fl ask. The larger flask insures against the agarose boiling over. 4. Dissolve the agarose in a boiling water bath or in a revolving-plate microwave oven. All the grains of agarose should be dissolved and the solution clear. 5. Allow the medium to cool to approx. 60C before adding ethidium bromidea stock solution, mix well and pour a gel with 0.4 cm thickness immediately. Wear suitable gloves when adding ethidium bromide. 6. Place the gel comb(s) in position in the gel tray 7. After the gel is completely set (approximately 30 minutes at room temperature) carefully remove the gel comb(s). 8. Remove the tape from the ends of the gel tray and put the tray in position in a agarose electrophoresis unit. For electrophoresis in a submarine mode, pour enough

319

electrophoresis buffer into the apparatus to cover the gel to a depth of at least 1 mm. 9. The same electrophoresis buffer used in the gel must also be used for the running buffer. References Asma, M. (1999). Test Report Comparative Test Xanthomonas campestr i s pv. carotae in carrot seed. ISHI Report , Bejo Zaden BV, Research Report P9411. Asma, M. (2000a). Report of the ISHI-ISTA comparative test Xanthomonas campestris pv. carotae in carrot seed. ISHI Report, Bejo Zaden BV, Research Report P9417. Asma, M. (2000b) Additioneel onderzoek Xanthomonas campestris pv. carotae. ISHI Report, Bejo Zaden BV, Research Report P9317-15 / P9416. Asma, M. (2005). Proposal for a new method for detecting Xanthomonas hortorum pv. carotae on carrot seeds. ISTA Method Validation Reports 2, 1-17. Asma, M., de Vogel, R., Woudt, B. and Krause, D. (2002). Evaluation of pathogenicity testing, rep-fi ngerprinting and PCR for the identifi cation of Xanthomonas campestris pv. carotae, ISHI Report Bejo Zaden BV, Research Report P9317-16. Cubeta, M.S. and Kuan, T.L. (1986). Comparison of MD5 and XCS media and development of MD5A medium for detection of Xanthomonas campestris pv. carotae in carrot seed, Phytopathology 76 , 1109. Eden, P.A., Schmidt, T.M., Blakemore, R.P. and Pace, N.R. (1991). Phylogenetic Analysis

320

of Aquaspirillum magnetotacticum using Polymerase Chain Reaction Amplifi ed 16S rRNA specifi c DNA. International Journal of Systematic Bacteriology 41 , 324- 325. Geng, S., Campbell, R.N., Carter, M. and Hills, M. (1987). Quality control programs for seed-borne pathogens. Plant Disease 67 , 236- 242. Kim, H.K., Sasser, M. and Sands, D.C. (1982). Selective medium for Xanthomonas campestr i s pv. translucens. Phytopathology 72 , 936. Kuan, T.L., Minsavage, G.V. and Gabrielson, R.L. (1985). Detection of Xanthomonas campestris pv. carotae in carrot seed. Plant Disease 69 , 758-760. Meng, X.Q., Umesh, K.C., Davis, R.M. and Gilbertson, R.L. (2004). Development of PCR based assays for detecting the carrot bacterial leaf blight pathogen Xanthomonas campestris pv. carotae from different substrates. Plant Disease 88 ,1226-1234. McGuire, R.G., Jones, J.B. and Sasser, M. (1986). Tween media for the semi-selective isolation of Xanthomonas campestris pv. vesicatoria from soil and plant material. Plant Disease 70 , 887-891. Roberts, S.J., Phelps, K., Taylor, J.D. and Ridout, M.S. (1993). Design and interpretation of seed health assays. In: Sheppard, J.W., (Ed.) Proceedings of the First ISTA Plant Disease Committee Symposium on Seed Health Testing, Ottawa, Canada. pp. 115-125. Agriculture Canada, Ottawa, Canada. Roberts, S.J. (1999). Thresholds, standards, tests, transmission and risks. In: Proceedings of 3rd ISTA Seed Health Symposium, Ames, Iowa, USA, 16-19 August 1999. pp. 20-24. ISTA, Zurich, Switzerland.

321

Schaad, N.W. (1988). Initial identifi cation of common genera. In: Schaad, N.W. (ed.) Laboratory Guide for Identifi cation of Plant Pathogenic Bacteria, pp. 1-15. APS Press, St. Paul, MN, USA. Wil l i f o r d , R.E. and Schaad, N.W. (1984) . Agar medium for selec t i ve i so la t i on Xanthomonas of campestr i s pv. carotae from carrot seeds. Phytopathology 74 , 1142.

Malt agar method for the detection of Alternaria dauci on Daucus carota (carrot) [ISTA Validated Method] Prepared by: Van Bilsen, J.1, Cockerell, V.2 and Roberts, S.J.2 1Bejo Zaden B.V., P.O. Box 50, 1749 ZH Warmenhuizen, The Netherlands. E-mail: J.vanBilsen@bejo.nl 2ISTA-PDC Method Validation Sub-committee Sponsored by: International Seed Health Initiative Vegetables (ISHI Veg) Revision History:Version 1.0, 01 January 2003. Background This method was originally published in the ISTA Handbook of Seed Health Test ing in November 1964 as S.3. No. 4 and revised in 1987 (Gambogi, 1987). It has been slightly modified following studies conducted by the International

322

Seed Health Initiative - Vegetables in 1999 and 2001 (Van Bilsen, 2003). The studies compared blotter and malt agar methods and concluded that the two were equivalent. The major modification is evaluation after 10 d incubation rather than 7 d. Note that seeds can be simultaneously tested for the presence of Al ternar i a rad ic i using na the same method (see method 7- 002b) . Validation studies: Van Bi l sen (2003) Copies are available: by e-mail from i s ta . o f f i c e@is ta .;ch or by mail f rom the ISTA Secretar i a t Safety Precautions Ensure you are familiar with hazard data and take appropriate safety precautions, especially during preparation of media, autoclaving, and weighing out of ingredients. It is assumed that this procedure is being carried out in a microbiological laboratory by persons familiar with the principles of Good Laboratory Practice, Good Microbiological Practice, and aseptic technique. Dispose of all waste materials in an appropriate way (e.g. autoclave, disinfect) and in accordance with local health, environmental and safety regulations. Treated seed Seed treatments may affect the performance of this test. It should only be performed on untreated seed. Materials Reference material - The use of reference cultures or other appropriate material is recommended. Malt Agar plates - See page 6. 9.0 cm plates (Petri dishes; one plate per ten seeds).

323

Incubator - Operating at 20 2C, equipped with timercontrolled near-ultraviolet lights (NUV, peak at 360 nm, e.g. color number 08, Philips; BLB Sylvania). Sample Preparation 1. It is vital to exclude any possibility of cross-contamination between seed samples. This can be achieved by swabbing/spraying equipment and gloved hands with 70% ethanol. 2. The test is carried out on a working sample as described in Section 7.4.1 of the International Rules for Seed Testing Method [Critical control points are indicated by CCP ] 1. Aseptically place a maximum of 10 seeds evenly spaced on the agar surface of each malt agar plate. 2. Incubate plates for 10 d at 20 2C, with alternating 12 h periods of darkness and NUV light. Plates should be approx. 25 cm below the lights and should not be stacked. 3. Sub-culture a reference culture to a malt agar plate at the same time seeds are plated and incubate with the test plates. 4. Examine plates visually, and under a stereoscopic microscope at x30 magnification, for fungal growth. Use a magnification of x50 x80 for identification of conidia. Colonies of Alternar i a dauci are brown or dark brown with ol i ve - grey aer ia l mycel ium and produce a brown di f f u s i b l e pigment in the medium. Conid iophores are s imple or s l i gh t l y branched (F ig . 1) , ar i s i ng s ing ly or in smal l groups f rom th sur face of the seed or on aer ia l mycel ium. Conid ia are usual l y sol i t a r y , obclavate , up to 450 m long ( inc l ud ing beak) , pale ol i vaceous brown at f i rs t , becoming brown with age, with a long pale beak up to 3 t imes the length of the body (E l l i s , 1971) . Groups of sunken conid ia are sometimes vis i b l e by the emerging clus te rs of the i r br ight long beaks

324

(Fig. 1, bottom left). Record the number of infected seeds in each plate (CCP ) . General Methods (common to many test procedures) 1. Checking Tolerances Tolerances provide a means of assessing whether or not the variation in result within or between tests is sufficiently wide as to raise doubts about the accuracy of the results. A tolerance table, which can be applied to most direct seed health tests, can be found in Table 5.1 of Annexe 16 of the International Rules for Seed Testing or in Table G1 of the Handbook of Tolerances and Measures of Prec i s i on for Seed Test ing(Mi l es , 1963) .

2. Report ing Resul t s The resu l t of a seed heal th test should ind i ca te the sc ient i f name of the pathogen and the test method used. When reported on an ISTA Cert i f i c a t e , resu l t s are entered under Other Determinat ions . In the case of a negative result (pathogen not detected), the results should be reported in terms of the tolerance standard (e.g. infection level less than 1% with 95% probability). The tolerance standard depends on the total number of seeds tested, n, and is approximately 3/n (P=0.95) (see Roberts et al . ,1993) . In the case of a positive result the report should indicate percentage of infected seeds. Quality Assurance Specific Training This test should only be performed by persons who have been trained in fungal identification or under their direct supervision. Critical Control Points [Identified by CCP in the methods]

325

Contaminants may greatly compete with the pathogen on the non-selective medium making detection laborious and difficult (Step 4). The malt agar source can influence the results. Whenever a new batch of malt agar is used a check on the quality should be made using a reference lot with a known infection level (Preparation of malt agar).

Fig- 1: Top: s ing le conid iaAlte of rnar ia dauci and chains of conid ia of the saprophyte A. tenuis on seed surface (left) and simple or slightly branched conidiophores with conidia of A. dauci developing from creeping hyphae (right), x80 magnification. Bottom: conidia of A. dauci on simple or slightly branched conidiophores borne on a single rootlet initial at x80 magnification (left); conidium and simple conidiophores at x350 magnification (right).

Preparation of Malt Agar Compound Malt Agar (CCP ) De-ionized/distilled Water g/l g/500ml

as specified by manufacturer 1000 ml 500 ml

326

Preparation 1. Weigh out ingredients into a suitable autoclavable container. 2. Add 1000 (or 500) ml of distilled water. 3. Steam to dissolve. 4. Autoclave at 15 PSI and 121C for 15 min. 5. Allow agar to cool to approx. 50C. 6. Pour 15-22 ml of molten agar into 9.0 cm Petri plates and allow solidifying at room temperature (20-25C) for 24 h before use. Storage Prepared plates may be stored at room temperature or at 4C for up to one month before use. References Ellis, M.B. (1971) Dematiaceous Hyphomycetes . Commonwealth Mycolog ica l Ins t i t u t e , Kew, Surrey , England. 609 pp. Gambogi, P. (1987) In te rnat i ona l Seed Test ing Assoc ia t i on , Handbook on Seed Health Test ing , Working Sheet No 4 (2nd Ed.): Al ternar i a dauci on Daucus carota. International Seed Testing Association, Zurich, Switzerland. Miles, S.R. (1963) Proceedings of the International Seed Testing Association, 28 (3), 644. Roberts, S.J., Phelps, K., Taylor, J.D. and Ridout, M.S. (1993) Design and interpretation of seed health assays. In: Sheppard, J.W., (Ed.) Proceedings of the First ISTA Plant Disease Committee Symposium on Seed Health Testing, Ottawa, Canada. pp. 115-125. Agriculture Canada, Ottawa, Canada.

327

Van Bilsen, J.G.P.M. (2003) Report of a comparative test on Al ternar i a dauci and Alternaria radicina on carrot seed. ISTA Method Validation Reports (submitted).

328

Malt agar method for the detection of Alternaria radicina on Daucus carota (car ro t ) [ISTA Validation Method] Pathogen: Alternar i a rad ic i (syn na . Stemphyl ium rad ic i num ) Prepared by: Van Bilsen, J.1, Cockerell, V.2 and Roberts, S.J.2 1Bejo Zaden B.V., P.O. Box 50, 1749 ZH Warmenhuizen, The Netherlands. E-mail: J . vanBi l s en@bejo .n l 2ISTA-PDC Method Validation Sub-committee Sponsored by: International Seed Health Initiative Vegetables (ISHI-Veg). Revision History:Version 1.0, 01 January 2003. Background This method was originally published in the ISTA Handbook of Seed Health Test ing in November 1964 as S.3. No. 5 and was revised by Gambogi (1987). It has been slightly modified following studies conducted using six seed-lots in 11 laboratories by the International Seed Health Initiative Vegetables in 1999 and 2001 (Van Bilsen, 2003). The studies compared blotter and malt agar methods and concluded that the two were equivalent. The major modification is evaluation after 10 d incubation rather than 7 d. Note that seeds can be simultaneously tested for the presence of Alternar i a dauci using the same method (see method 7-001b). Validation studies Van Bilsen (2003).

329

Copies are available: by e-mail from i s ta . o f f i c e@is ta .;ch or by mail f rom the ISTA Secretar i a t . Safety Precautions Ensure you are familiar with hazard data and take appropriate safety precautions, especially during preparation of media, autoclaving, and weighing out of ingredients. It is assumed that this procedure is being carried out in a microbiological laboratory by persons familiar with the principles of Good Laboratory Practice, Good Microbiological Practice, and aseptic technique. Dispose of all waste materials in an appropriate way (e.g. autoclave, disinfect) and in accordance with local health, environmental and safety regulations. Treated seed Seed treatments may affect the performance of this test. It should only be performed on untreated seed. Materials Reference material - The use of reference cultures or other appropriate material is recommended. Malt agar plates - See page 6. 9.0 cm plates (Petri dishes one plate per ten seeds). Incubator - Operating at 20 2C equipped with timercontrolled near-ultraviolet lights (NUV, peak at 360 nm, e.g. color number 08, Philips; BLB, Sylvania). Sample Preparation 1. It is vital to exclude any possibility of cross-contamination between seed samples. This can be achieved by swabbing/spraying equipment and gloved hands with 70% ethanol.

330

2. The test is carried out on a working sample as described in Section 7.4.1 of the International Rules for Seed Testing.

Method [Critical control points are indicated by CCP ] 1. Aseptically place a maximum of 10 seeds, evenly spaced, on the agar surface of each malt agar plate. 2. Incubate plates for 10 d at 20 2C, with alternating 12 h periods of darkness and NUV light. Plates should be approx. 25 cm below the lights and should not be stacked. 3. Sub-culture a reference culture to a malt agar plate at the same time the seeds are plated and incubate with the test plates. 4. Examine plates visually, and under a stereoscopic microscope at x30 magnification, for fungal growth. Use a magnification of x50 x80 for identification of conidia. Colonies of Alternar i a rad ic i are na i r r egu la r to ci r cu la r with luxur ious aer ia l mycel ium, dark ol i ve grey to grey ish - black f rom above, blu i sh - black f rom below (Meier et al.,, 1922). Conidiophores are simple or occasionally branched, arising usually singly from the surface of the seed, on the emerging radicle or on aerial mycelium (Fig. 1). Conidia are produced singly or in chains of 2, or rarely 3, ellipsoidal or barrel shaped, with little evidence of beak, up to 75 m long, olivaceous brown, (Ellis, 1971). Under the stereoscopic microscope, conidia appear blackish and glossy (Fig. 1). Record the number of infected seeds in each plate (CCP ). General Methods (common to many test procedures) 1. Checking Tolerances Tolerances provide a means of assessing whether or not the variation in result within or between tests is sufficiently wide

331

as to raise doubts about the accuracy of the results. A tolerance table, which can be applied to most direct seed health tests, can be found in Table 5.1 of Annexe 16 of the International Rules for Seed Testing or in Table G1 of the Handbook of Tolerances and Measures of Prec i s i on for Seed Test (Mi ing les , 1963). 2. Report ing Resul t s The result of a seed health test should indicate the scientific name of the pathogen and the test method used, including any pre-treatment. When reported on an ISTA Certificate, results are entered under Other Determinat ions . In the case of a negative result (pathogen not detected), the results should be reported in terms of the tolerance standard (e.g. infection level less than 1% with 95% probability). The tolerance standard depends on the total number of seeds tested, n, and is approximately 3/n (P=0.95) (see Roberts et al . ,1993) . In the case of a positive result the report should indicate percentage of infected seeds. Quality Assurance Specific Training This test should only be performed by persons who have been trained in fungal identification or under their direct supervision. Critical Control Points [Identified by CCP in the method] Contaminants may compete strongly with the pathogen on the malt agar medium, so that detection may be laborious and difficult (Step 4). The malt agar source can influence the results. Whenever a new batch of malt agar is used a check on the quality should be made using a reference lot with a known infection level (Preparation of malt agar).

332

Fig- 1: Top: conid iophores and conid ia Alte ofrnar i a radic i and na chains of conid ia of the saprophyte A. tenuis on a rootlet initial x80 (left); spreading hyphae and fructifications of the pathogen x80 (centre); abundant growth and fructification of the pathogen on a rootlet initial, x50 (right). Bottom: conidia of Alternaria radicina, x350.

Preparation of Malt Agar Compound Malt Agar (CCP ) De-ionized/distilled Water Preparation g/l g/500ml

as specified by manufacturer 1000 ml 500 ml

333

1. Weigh out ingredients into a suitable autoclavable container. 2. Add 1000 (or 500) ml of de-ionised/distilled water. 3. Steam to dissolve. 4. Autoclave at 15 PSI and 121C for 15 min. 5. Allow agar to cool to approx. 50C. 6. Pour 15-22 ml of molten agar into 9.0 cm plates (Petri dishes) and allow to solidify at room temperature (20-25C) for 24 h before use. Storage Prepared plates may be stored at room temperature or at 4C for up to one month before use. References Ellis, M.B. (1971) Dematiaceous Hyphomycetes . Commonwealth Mycolog ica l Ins t i t u t e , Kew, Surrey , England. 608 pp. Gambogi, P. (1987) ISTA Handbook on Seed Health Test ing , Working Sheet No. 5: Alternaria radicina on Daucus carota . International Seed Testing Association, Zurich, Switzerland. Meier, F.C., Drechsler, C., and Eddy, E.D. (1922) Black rot of carrots caused by Alternaria radicina n. sp. Phytopathology, 12, 157-166. Miles, S.R. (1963) Proceedings of the International Seed Testing Association, 28 (3), 644. Roberts, S.J., Phelps, K., Taylor, J.D. and Ridout, M.S. (1993) Design and interpretation of seed health assays. In: Sheppard, J.W., (Ed.) Proceedings of the First ISTA Plant Disease Committee Symposium on Seed Health Testing,

334

Ottawa, Canada.pp. 115- 125. Agr icu l t u re Canada, Ottawa, Canada. Van Bilsen, J.G.P.M. (2003) Report of the comparative test on Al ternar i a dauci and Al ternar i a rad ic i on na carro t seed. ISTA Method Val ida t i on Reports (submitted). NSHS METHOD 1 for Detection of Alternaria dauciin Carrot ( Daucus carota) METHOD 1.1 ISTA Freeze Blot te r test (Gambogi , 1987) METHOD 1.2 Blot te r / Pa thogenic i t y test (Sunseeds, Brooks, Oregon, USA) METHOD 1.1 PATHOGEN: Alternaria dauci ISTA Freeze Blotter test (Gambogi , 1987) 1. Carrot seeds from the working sample, without pretreatment, are placed equidistantly, at least 20 mm from each other, on moistened blotters. 2. Incubate seeds either for 10 days at 20C in cycles of 12 hours darkness and 12 hours light, preferably NUV, or for 3 days at 20C, then at -20C overnight, and 7 days at 20C in cycles of 12 hours darkness and 12 hours light, preferably NUV. The latter method is the most widely used for routine analyses. 3. Seeds (seedlings) are examined under a stereoscopic microscope at 30 x magnification for fungal growth and up to 80 x for identification, which is based on sporulation of the fungus. 4. Supplementary examinations after 13-14 days after plating of seeds may be required (Hewett, 1964).

335

5. The results are expressed as a percentage of the total number of incubated seeds. REFERENCES Gambogi P, 1987. Carrot. Leaf blight, damping off. Daucus carota, Alternaria dauci (Khn) Groves & Skolko. ISTA Handbook on Seed Health Testing. 2nd Edition, Working Sheet No 4, 1-5. Hewett PD, 1964. Testing carrot seeds infected with Alternaria porri f. sp. dauci. Proceedings of the International Seed Testing Association, 29(3):463-471.

METHOD 1.2 PATHOGEN: Alternaria dauci Blotter/Pathogenicity test (Sunseeds, Brooks, Oregon, USA) Sample size: 3000 seeds Blotter Incubation 1. Six 9 x 12 plastic boxes with transparent covers are surface sterilized with 95% denatured ethyl alcohol. 2. Germination blotters placed in the boxes and moistened with sterile deionized water. 3. Carrot seeds rare sprinkled over the blotters in each box. 4. The seeds are incubated at 22C for 2 days under a 12 hr NUV light/ 12 hr dark cycle. 5. The seeds are then placed in a freezer at -20C overnight. 6. The seeds are returned to the 22C environment and incubated for 10-14 days under a 12 hr NUV light/ 12 hr dark cycle.

336

Pathogen identification 1. The seeds are scanned under a stereoscopic microscope at (7X) to detect a soft brown rot covered with a fine mass of mycelium with large sword-shaped conidia protruding from the mass. 2. Conidia are examined under a compound microscope at 1000X, to confirm their identity as A. dauci . 3. If further confirmation of the identity of the fungi is considered necessary, conidia growing on seeds in the blotter test are transferred to Carrot Extract medium to produced inoculum. Pathogenicity test 1. Carrot seedlings are grown to the 4-5 leaf stage. 2. Seedlings are sprayed to run-off with a 1.0 x104 spore concentration of inoculum. 3. Seedlings are incubated in plastic bags for 48 hr at 2530C. 4. Seedlings are then placed in a greenhouse at 22-28C for at least 14 days. They are sprayed with water and covered with plastic every night. 5. Symptoms may appear after 14 days as small dark brown to black lesions with a yellow border that first appears on leaf margins. Lesions may increase in size to cover the entire leaflets, giving seedlings a scorched appearance. 6. Isolates should be recovered from diseased tissues and confirmed as A. dauci on cul tu re plates as in Step 2 of pathogen ident i f i c a t i o n . Medium preparation

337

1. Add 25 g of dried carrot leaves and 15 g of agar to 1 liter of water. 2. Autoclave the mixture.

Vegetable Crops of Family Chenopodiaceae

(Sp inach Beet , roo t and Swiss chard , French ) charde
1.

Seed unit: Seed uni t i s clus ter of f ru i t s , accord ing to botanica l def in i t i o n , having true seeds ins ide . Submitted Sample Weight: Beta vulgar i (Beetroot s ) = 40 g Beta vulgaris subsp.Mari t ima (Swiss chard) = 40 g Spinacea oleracea (Sp inach) = 40 g

2.

3.

Working Sample Weight: Beta vulgar i (Beetroot s ) = Beta vulgaris subsp.Mari t ima (Swiss chard) = Spinacea oleracea (Sp inach) = 4 g 4 g 4 g
338

4. Purity Analysis: a. Pure Seed: In tac t seed uni t s , with or without prot rud ing axis , and in the case of pieces of seed uni ts , any piece which i s la rger than one- hal f the or ig ina l s ize shal l be cons idered pure seed. For Beta spp . the clus te rs reta ined on a 200 mm x 300 mm rectangular s ieve with square- ended s lo t s 1.5 mm x 20 mm when shaken for one minute shal l be pure seed, dis regard ing whether clus ter s conta in t rue seed or not . However prot rud ing sta lk pieces that are longer than the width of the clus ter , are to be ent i re l y removed pr io r to s iev ing . b. Other Crop Seeds: Seeds of species other than in tended species , not i f i ed as crop. c. Weed Seeds: Seeds of species other than in tended species , not i f i ed as weed. d. Inert Matter:Seed piecesOne-hal f the or ig ina l s i ze or less , non- l i v i ng mater ia l , nematode gal l s and fungus bodies .

5. Germination:
Number Temperature Substrata of seeds (C) First Count (days) Final Count (days) Additional Directions General Requirements See sections 4.7.3 and 4.12.6.a. Light moisture Additional Directions Fresh/ Dormant Seeds Prechill

Kind of Seed

Beta vulgaris Field, sugar, and garden beet; mangel; swiss chard Spinacia oleracea Spinach

RB; BB; PP

20; 20-30

10

BB; RT

15; 10

21

Prechill

339

Abbreviations: BB - Between blot te r s , Rol led blot te rs .

RT - Rol led towels , RB

Seedling Evaluation: Seedlings are considered normal if they possess those essential structures that are indicative of their ability to produce a plant under favorable conditions. General Description Multiple-seeded units of New Zealand spinach and Beta spp., shall be regarded as having germinated if they produce one or more normal seedlings. Only one seedling per multiple unit is to be counted. Seedling type: Epigeal dicot. Food reserves: Leaf-like cotyledons and perisperm. Shoot system: The hypocotyl elongates carrying the cotyledons above the soil surface. The epicotyl usually does not show any development within the test period. Root system: A primary root; secondary roots may develop within the test period. Abnormal Seedling Description Cotyledons

Less than half of the original cotyledon tissue remaining attached. Less than half of the original cotyledon tissue free of necrosis or decay.

Epicotyl
340

Miss ing (may be assumed to be present i f are in tac t ) .

coty ledons

Hypocotyl

Deep open cracks extending in to the conduct ing t i s sue . Malformed: such as markedly shortened, cur led or th i ckened. Watery.

Root

None. Weak, stubby or miss ing pr imary root with weak secondary or advent i t i ous roots .

Seedling

One or more essent ia l st ruc tu res impai red as a resu l t of decay f rom pr imary in fec t i on ( fo r disco loured seedl i ngs of Beta spp. , see note 2) . Alb ino .

Notes
4. See sect ion 4.7.3 for

di rec t i ons to wash samples of Beta pr io r to plant ing . Chemical inh ib i t o r s in the clus te or seed coat may work together with excess water to rob the embryo of oxygen and thus prevent germinat ion . I t i s important , therefo re , to ensure the seeds or clus ters are dr ied before plant ing . 5. Toxic substances f rom the clus ter Beta s ofmay cause disco lour i ng of the hypocoty l and/or root . Seedl ings which are s l i gh t l y disco loured are to be class i f i e d as normal; however, i f there i s excess ive disco lo ra t i on , retes t in soi l or by washing in running water for 3 hour 6. Frequent counts must be made on multigerm beet since the growing seedlings will separate from the cluster making it difficult to identify its source. Any
341

cluster which produces at least one normal seedling is classified as normal; only one normal seedling per cluster is to be counted.

SEED HEALTH TESTING CHENOPODIACEAE

IN FAMILY

1.

The main seed-borne Beetroot and Swiss chard pathogens with common names of the diseases they cause Pathogen
Alte rnar ia al te rnata (Fr . ) Alte rnar ia tenuis Auct. Cercospora beticola Sacc Keiss le r ,

Sr. No.
1 2

Common Names
syn. Seedling rot, leaf spot Leaf spot

342

Col le to t r i c hum dematium (Pers . ex Fr . ) Grove f .

spinaciae(El l spinaciaeEl l . and Halst . ) and Halst .

Anthracnose

Arx, syn, C.

4 5 6 7

8 9 10 11

Erys iphe betae (Vai iha) Weltz ien , Powdery mildew syn. E. communis (Wal l r . ) Fr . betae f. (Vanha) Jacz. Fusar ium oxysporumSchlechtend. :F r .Fusarium yellows and f . sp. betae (D. Stewart) W. C. Snyder root rot & H. N. Hans. (Texas isolates) Peronospora far inosa (Fr.) Fr., syns. P. Downy mildew schacht i iFuckel , P. effusa (Grcv. ) Rabcnh Pleospora betae (Berl.) Nevodovsky, Blackleg, syns. P. damping- off , leaf betae Bjocrling, P. bjoer l i ng Byford, ii Phoma betac Frank Ramularia beticola Fautr. and Lamb Leaf spot Corynebacterium betae Keyworth, Howell and Dowson Pseudomonas syr ingaepv. aptata (Brown and Jamicson) Stevens Viruses Si lver ing of red beet Bacter ia l bl ight , spot , black streak, black spot Arabis mosaic virus , Raspberry r ingspot virus Tomato black Virus (beet r ingspot virus) Lychnis r ingspot virus Eelworm canker

12

Ditylenchus dipsaci (Kiihn) Filipjev

2. The main seed-borne spinach pathogens with common names of the diseases they cause Sr. No .
1 2 3 4 5 6

Pathogen
Cladosporium variabile (Cooke) de Vries, syn. Heterosporium variabile Colletotrichum dematium (Pers. ex Fr.) Grove f. spinadae (Ell. and Halst.) Arx, syn. C. spinaciae El l . and Halst Colletotrichum spinaciicola Chupp and Sherf Phyllosticta spinadae Zimm. Rhizoctonia solani Kiihn Verticillium dahliae Kleb

Common Names
Lea f spot Anthracnose Anthracnose Leaf spot Damping off Wilt

343

Vegetable Crops of Family Malvaceae

(Okra)
1. 2.

Seed unit: True seed accord ing to botanica l def in i t i o n . Submitted Sample Weight:

344

Abelmoschus esculentus (Okra)

3.

= 1,000 g

Working Sample Weight: Abelmoschus esculentus (Okra) = 140 g

4. Purity Analysis:

a. Pure Seed: Seed, with or without testa , and in the case of pieces of seed uni ts , any piece which i s la rger than one- hal f the or ig i na l s ize shal l be considered pure seed. b. Other Crop Seeds: Seeds of species other than in tended species , not i f i ed as crop. c. Weed Seeds: Seeds of species other than in tended species , not i f i ed as weed. d. Inert Matter:Seed piecesOne-hal f the or ig ina l s i ze or less , non- l i v i ng mater ia l , nematode gal l s and fungus bodies . 5. Germination:
Number Temperature Substrata of seeds (C) First Count (days) 7 Final Count (days) 14 Additional Directions General Requirements Additional Directions Fresh/ Dormant Seeds -

Kind of Seed

Abelmoschus esculentus Okra

400

20-30

Abbreviations: S - In sand.

General Description Seedling type: Epigea l dicot . Food reserves: Coty ledons, which are much convoluted in the seed; they expand and become th in , lea f - l i ke and photosynthet i c .
345

Shoot system: The hypocoty l elongates carry ing the coty ledons above the soi l sur face . The epicoty l usual l y does not show any development with in the test per iod . Root system: A pr imary root , with secondary roots usual l y develop ing with in the test per iod . Abnormal Seedling Description Cotyledons

Less than hal f of the or ig ina l attached. Less than hal f of the or ig ina l necros i s or decay.

coty ledon t i s sue remain in coty ledon t i s sue i s f ree

Epicotyl

Miss ing (may be assumed to be present i f are in tac t ) .

coty ledons

Hypocotyl

Deep open cracks or gra iny les i ons extending in to the conduct ing t i s sue . Malformed: such as markedly shortened, cur led or th i ckened.

Root

None. Weak, stubby or miss ing pr imary root with weak secondary or advent i t i ous roots .

Seedling

One or more essent ia l st ruc tu res impai red as a resu l t decay f rom pr imary in fec t i on . Alb ino .

of

SEED HEALTH TESTING IN FAMILY

MALVACEAE
346

1.

The main seed-borne Okra pathogens with common names of the diseases they cause Pathogen
Ascochyta abelmoschi Harter Choanephora cucurbi tamm(Berk , and Rav.) Thaxter Fusar ium solan(Mart i ) . ) Sacc. Glomerel l a cingula (Stonem.) ta Spauld and Schrenk. syn. Gloeospor iwn cingula tum Atk. Rhizocton ia solan Kiihn i Viruses

Sr. No .
1 2 3 4 5 6

Common Names
Ascochyta bl ight pod-spot Okra frui t Wilt

Okra leaf curl Mosaic

Note: Glomerella cingulata is plant a pathogenic fungus that causes disease on many different hosts inlcuding quince and apple bitter rot and anthracnose on many fruit and vegetable species. Glomerel la cingula ta is the sexual stage (teleomorph) while the asexual stage (anamorph) is called Colletotr ichum gloeosporioides .

347

Vegetable Crops of Family Asteraceae

(Le t tu ce )
1.

Seed unit: Achene conta in ing t rue seed accord ing to botanica l def in i t i o n . Submitted Sample Weight: Lactuca sat i va L. (Lettuce) = 30 g

2.

3.

Working Sample Weight: Lactuca sat i va L. (Lettuce) = 3g

4. Purity Analysis: a. Pure Seed: Achene, with or without beak, or with or with out pappus, unless it is obvious that no seed is present. Seed, with or without per icarp / t es ta , and in the case of pieces of seed uni ts , any piece which i s la rger than one- hal f the or ig ina l s ize shal l be cons idered pure seed. b. Other Crop Seeds: Seeds of species other than intended species, notified as crop. c. Weed Seeds: Seeds of species other than intended species, notified as weed. d. Inert Matter:Seed pieces One-hal f the or ig ina l s i ze or less , non- l i v i ng mater ia l , nematode gal l s and fungus bodies . 5. Germination:

348

Kind of Seed

Number of Temperature Substrata seeds (C)

First Count (days)

Final Count (days)

Additional Directions General Requirements Light

Additional Directions Fresh/ Dormant Seeds Prechill

Lactuca sativa Lettuce and celtuce

400

TB

20; 15

Abbreviations: TB Top of blot te rs .

General Description Seedling type: Epigea l dicot . Food reserves: Coty ledons which expand and become th in , lea f - l i ke and photosynthet i c . Some var ie t i e s develop elongated pet io l es at the base of the coty ledons .

Shoot system: The hypocoty l elongates and carr i es the coty ledons above the soi l sur face . The epicoty l usual l y does not show any development with in the test per iod . Root system: A long pr imary root . Abnormal Seedling Description Cotyledons

Less than hal f of the or ig ina l coty ledon t i s sue remain in attached. Less than hal f of the or ig ina l coty ledon t i s sue f ree of necros i s or decay (see notes 5 and 6) . With any degree of physio log i ca l necros i s (see notes 5 1 and 6) .

Epicotyl

Miss ing (may be assumed to be present i f are in tac t ) . Any degree of necros i s or decay.

coty ledons

Hypocotyl

349

Deep open cracks extending in to the conduct ing t i s sue . Severely twis ted or gra iny . Watery.

Root

None. Pr imary root t ip blunt , swol len and disco loured . Pr imary root with spl i t s or les i ons .

Seedling

Swol len coty ledons assoc ia ted with extremely short or vest ig i a l hypocoty l and root . One or more essent ia l st ruc tu res impai red as a resu l t of decay f rom pr imary in fec t i on . Alb ino .

Notes 7. Toxic materials in the substrate will cause short, blunt roots. 8. Seedlings grown on top of white filter paper will be shorter than those on blue blotters. 9. Remove attached seed coats for seedling evaluation. 10. Seedlings with slight dormancy or light sensitivity may be slow to germinate. 11. Physio log i ca l necros i s i s manifes ted on le t tuce coty ledons by sof tened grey, brown, black or reddish areas appear ing adjacent to the midr ib and la te ra l veins . This must not be confused with natura l pigmentat ion of some var ie t i e s , or with insect damage. Physio log i ca l necros i s i s often accompanied by shortened hypocoty l s and roots , with the seed coat f requent ly remain ing attached to the coty ledons. Seedl ings showing any degree of physio log i ca l necros i s should be class i f i e d as abnormal . In "Remarks" ind i ca te the percentage of necrot i c seedl ings ( th i s percentage should inc lude necrot i c seedl ings which are also 2 otherwise abnormal) .
350

12. Seedlings with extensive physiological necrosis on the cotyledons may be slower in growth than those without such affected areas. Hypocotyl and root length may be affected by other factors such as proximity to light, delayed germination or dormancy.
1

The requi rement to be f ree of any degree of physio log i ca l necros i s di f f e r s f rom the AOSA descr ip t i on , in which only seedl ings with more than 50% of the coty ledons being necrot i c are considered abnormal .
2

Note 5 as i t appears here di f f e r s f rom Note 5 appear ing in the AOSA Seedl ing Evaluat i on Handbook.

SEED HEALTH TESTING IN FAMILY

ASTERACEAE

1.

The main seed-borne Lettuce pathogens with common names of the diseases they cause Pathogen Common Names

Sr. No.
1

Marssonina panattoniana (Ber l . ) Magnus Ring spot, syn. Microdochium panatton ianum anthracnose (Ber l . ) Sutton et al in . Galea et al. Septor ia lactucae Pass syn. Aschochyta lactucaeRostrup Pseudomonas dchor i i (Swingle ) Stapp V i ru ses Leaf spot Leaf bl ight Arabis mosaic Lettuce mosaic virus , Tobacco r ing spot virus

2 3 4

351

Vegetable Crops of Family Liliaceae

(Asparagus, Onion, Leek, Chives)

1. 2.

Seed unit: True seed accord ing to botanica l def in i t i o n . Submitted Sample Weight: Asparagus of f i c i na L. lis (Asparagus) = 1000 g Al l i um cepa L. (Onion) = 80 g Al l i um porrum L. (Leek) = 70 g Al l i um schoenoprasum L. (Chives) = 30 g

3.

Working Sample Weight: Asparagus of f i c i na L. lis (Asparagus) = 100 g Al l i um cepa L. (Onion) = 8g Al l i um porrum L. (Leek) = 7g Al l i um schoenoprasum L. (Chives) = 3g

4. Purity Analysis: a. Pure Seed: Seed, with or without testa , and in the case of pieces of seed uni ts , any piece which i s la rger than one- hal f the or ig i na l s ize shal l be considered pure seed.

352

b. Other Crop Seeds: Seeds of species other than in tended species , not i f i ed as crop. c. Weed Seeds: Seeds of species other than in tended species , not i f i ed as weed. d. Inert Matter:Seed piecesOne-hal f the or ig ina l s i ze or less , non- l i v i ng mater ia l , nematode gal l s and fungus bodies .

5. Germination:
Number Temperature Substrata of seeds (C) First Count (days) 7 7 7 Final Count (days) 14 14 14 Additional Directions General Requirements Lightly covered (sand test) Lightly covered (sand test) Light moisture; Lightly covered (sand test) Presoak 24 hours Additional Directions Fresh/ Dormant Seeds

Kind of Seed

Allium cepa Onion Allium porrum Leek Allium schoenoprasum Chives Asparagus officinalis Asparagus

BB; S; RT BB; S; RT BB; S; RT

20 20 20

S; BB; RT

20-30

21

Abbreviations: BB Between blot te r s , Sand.

RT Rol led towels , S in

14.10 Liliaceae (Lily Family) I - Asparagus Asparagus of f i c i na , l i asparagus s General Description Seedling type: Hypogeal monocot.

353

Food reserves: Endosperm which i s hard, semi- t ransparent and non- starchy ; minor reserves in the coty ledon. The endosperm surrounds the ent i re embryo. Cotyledon: A s ing le cyl i nd r i ca l coty ledon; fo l l ow ing germinat ion al l but the basal end remains embedded in the endosperm to absorb nutr i en ts . Shoot system: The epicoty l elongates and carr i es the terminal bud and pr imary leaves above the soi l sur face . The epicoty l may bear severa l smal l scale leaves . A short hypocoty l i s barely dis t i ngu i shab le jo i n i ng the root to the basal end of the coty ledon, which emerges f rom the seed. Root system: A long s lender pr imary root .

Abnormal Seedling Description Cotyledon

Detached f rom seedl i ng .

Epicotyl

Miss ing . Terminal bud miss ing or damaged. Deep, open cracks . Malformed, such as markedly shortened, cur led , or th i ckened. Spind ly . Watery. (See also note 1)

Hypocotyl

Not evaluated.

Root

354

No pr imary root . Stubby pr imary root , with weak secondary roots (but see note 3 for ornamental asparagus) .

Seedling

One or more essent ia l st ruc tu res impai red as a resu l t decay f rom pr imary in fec t i on . Alb ino .

of

Notes 4. Several epicotyls may arise simultaneously and may be considered normal if at least one appears to be vigorous and has a terminal growing point. 5. Some seeds do not conta in an embryo. (Remainder of note 2 of AOSA Seedl ing Evaluat i on Handbook deleted . ) 6. Ornamental asparagus Asparagus ( setaceusand A. densiflorus) has a thickened primary root, in contrast to the long, slender root of the garden asparagus (A. officinalis). 14.11 Liliaceae (Lily Family) II - Onion, leek and chives Allium cepa, onion Allium porrum, leek Allium schoenoprasum, chives

General Description Seedling type: Epigeal monocot. Food reserves: Endosperm which is hard, semi-transparent and non-starchy; minor reserves in the cotyledon. Cotyledon: A single cylindrical cotyledon; following germination the tip remains embedded in the endosperm to absorb nutrients.

355

Shoot system: The coty ledon emerges with the seed coat and endosperm attached to the t ip . A sharp bend known as the "knee" forms; cont inued elongat ion of the coty ledon on each s ide of th i s knee pushes i t above the soi l sur face . The coty ledon t ip i s pul led f rom the soi l and st ra igh tens except for a s l i gh t kink which remains at the s i te of the knee. The f i r s t fo l i age lea f emerges through a s l i t near the base of th coty ledon, but th i s does not usual l y occur dur ing the test per iod . The hypocoty l i s a very short trans i t i ona l zone between the pr imary root and the coty ledon. Root system: A long s lender pr imary root with advent i t i ous roots develop ing f rom the hypocoty l . The pr imary root does not develop secondary roots . Abnormal Seedling Description Cotyledon

Short and th ick . Without a def in i t e bend or "knee." Spind ly or watery .

Epicotyl

Not observed dur ing the test per iod .

Hypocotyl

Not evaluated.

Root

No pr imary root . Short , weak or stubby pr imary root .

Seedling

One or more essent ia l st ruc tu res impai red as a resu l t decay f rom pr imary in fec t i on . Alb ino .
356

of

Notes 3. Excess moisture may cause a delay in germination causing some seed lots to appear dormant. 4. Blotter or towel tests of onion are commonly overcome with fungus. To reduce this problem on a retest, seeds should be spaced farther apart.

SEED HEALTH TESTING IN FAMILY

LiliACEAE

1.

The main seed-borne Al l ium spp.pathogens with common names of the diseases they cause Pathogen
Alte rnar ia porr (Eil l . ) Botryt i s alMunn lii Ci fe r r i

Sr. No .
1 2 3

Common Names
Purple blotch Damping- off , grey mould, neck rot Seedl ing damping- off , 357

Botryt i s byssoidea Walker

4 5 6 7 8 9 10 11 12 13

Cladospor ium al l i i - cepae (Ranojev ic ) M. B. El l i s syn. Heterosporium allii-cepae Ranojevic Colletotrichum circinans (Berk.) Vogl., syns. C, dematium (Pers. ex Fr.) Grove F. circinans (Berk. ) Arx Fusarium spp. Peronospora destructor (Berk.) Casp Pleospora herbarum (Pers. ex Fr.) Rabenh., syn. Stemphylium botryosum Wallr. Puccinia allii Rud., syn. Puccinia porri Wint. Sclerotium cepivorum Berk. Urocystis cepulae Frost Virus Ditylenchus dipsaci (Ktihn) Filipjev

neck rot

Smudge, damping-off Wilt Downy mildew Black stalk rot , Leaf mould Rust White rot Smut Onion yel low dwarf virus Bloat , eelworm rot

358