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Arbuscular Mycorrhiza Inoculum to Support Sustainable Cropping Systems

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2004 Plant Manage m e nt Ne twork . Acce pte d for publication 14 January 2003. Publishe d 1 March 2004.

Arbuscular Mycorrhiza Inoculum to Support Sustainable Cropping Systems

Sym posium Hom e page

Yolande Dalp, Agriculture and Agri-Food Canada, Research Branch, 960 Carling Avenue Ottawa, Ontario, K1A 0C6; and Marcia Monreal, Agriculture and Agri-Food Canada, Research Branch, P.O. Box 1000A, Brandon, Manitoba, R7A 5Y3
C orre sponding author: Yolande Dalp . dalpe y@agr.gc.ca Dalp , Y., and Monre al, M. 2004. Arbuscular m ycorrhiza inoculum to support sustainable cropping syste m s. O nline . C rop Manage m e nt doi:10.1094/C M-20040301-09-R V.

Im pact State m e nt PDF ve rsion for printing

Abstract
Arbuscular mycorrhizae (AM) are symbiotic associations, formed between plants and soil fungi that play an essential role in plant growth, plant protection, and soil quality. The AM fungi expand their filaments in soil and plant roots. This filamentous network promote bi-directional nutrient movement where soil nutrients and water move to the plant and plant photosynthates flow to the fungal network. AM fungi are ubiquitous in the soil and can form symbiosis with most terrestrial plants including major crops, cereals, vegetables, and horticultural plants. In agriculture, several factors, such as host crop dependency to mycorrhizal colonization, tillage system, fertilizer application, and mycorrhizal fungi inoculums potential can affect plant response and plant benefits from mycorrhizae. Due to their obligate symbiotic status, AM fungi need to associate with plant for growth and proliferation. C onsequently, the cultivation of AM fungal strains and the maintenance of reference collections require methodologies and infrastructures quite different from those used with other microbial collections and inoculum production. Interest in AM fungi propagation for agriculture is increasing due to their role in the promotion of plant health, in soil nutrition improvement, and soil aggregate stability. The comprehensive life cycle of AM fungi and methods currently used for the propagation of inoculum and the maintenance of in vivo and in vitro source collections are described. Methods and regulations of large-scale production of commercial inoculum that provide users with products of high quality and efficiency are discussed.

Introduction Arbuscular my corrhizae (AM) are sy mbiotic associations formed between plants and soil fungi that benefit both partners. The phy tobiont correspond to approx imately 80% of plant species and the fungi are classified in the phy lum Glomeromy cota, including nine genera; Glomus , Paraglomus , Sclerocystis , Acaulospora , Entrophospora , Gigaspora , Scutellospora , Diversispora , Geosiphon, and Archaeospora (41 ). AM fungi (AMF) are ubiquitous in the soil with around 1 7 0 described species (46). The sy mbiosis is called arbuscular because the fungi form specialized tree-like structures (arbuscules = tree-like) inside root cells. Other structures produced by fungi are intra- and ex traradical spores (which are germinating structures useful for long-term preserv ation of species, propagation, and species identification purposes), intraradical hy phae, ex traradical hy phae, intracellular fungal storage structures called v esicles (which are lipid containing bodies) and, for some genera, aux iliary cells branching from ex traradical hy phae. Intraradical AM fungal my celium
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Arbuscular Mycorrhiza Inoculum to Support Sustainable Cropping Systems

form a network around and inside cortical cells of plant roots, ex traradical AM my celium can spread throughout the soil surrounding the root sy stem and increase the ability to ex plore soil areas, accessing water and nutrients for plant roots. Benefits to plants are improv ed water and nutrient uptake, enhanced P transport, and drought and disease resistance. Benefits to fungi are the supply of photosy nthates to the fungal network located in the cortical cells of the plant and the surrounding soil. All water, nutrients, and photosy nthates ex changes occur v ia the fungal filament network that bridged plant rhizosphere and plant roots. AM Interaction with Soil and Crops The increased capacity of plant roots for water and nutrients uptake from the soil when colonized by AMF is the main mechanisms proposed to ex plain the effect of AM in plant performance. This behav ior is particularly ev ident with soil nutrients that are more immobile such as phosphorus (P), zinc (Zn), and copper (Cu) . Improv ed phosphorus nutrition when colonized with AMF has been demonstrated for hundreds of cultiv ated plants. By ex tending past the P-depletion zone formed around the root sy stems, the fungal soil network is able to maintain P transport to plant for longer periods (1 9,21 ,27 ). Under high P soil conditions, AMF are almost of no use to the plants and the sy mbiosis is temporarily inhibited. As such, a reduction in P applications is recommended in order to stimulate and maintain sy mbiosis efficiency . Most agricultural crops such as flax , corn, sorghum, wheat, barley , potatoes, and sunflower can benefit from my corrhizal association. Some other crop plants do not form AM sy mbiosis; those belong to the Cruciferae, Brassicaceae, Chenopodiaceae, and Cary ophy llaceae families (3). Canola (Brassicaceae family ), an important crop in western Canada does not form AM. Efficiencies and limitations of registered my corrhizal inoculum, in terms of the cultiv ated crops, are clearly posted on sale products together with recommendation for use. Interaction of AM and Agricultural Practices Agricultural practices such as fertilizer applications, crop rotation, tillage, and liming affect field AM potential and root colonization lev els. For ex ample, high lev els of P fertilization hav e been found to slow down or inhibit my corrhizal efficiency in soy bean fields (1 2). Cropping of a soil with canola, a non-host plant species, delay ed my corrhiza dev elopment of maize and of flax (1 5; Monreal et al., unpublished data ). Higher soil infectiv ity was observ ed under reduced or no tillage practices (31 ) and liming increased my corrhizal colonization of barley roots and soil infectiv ity (1 7 ). Plant species differ in their fertilization requirements, and consequently their dependency on AMF v ary considerably from one crop to another (33). For ex ample, under field conditions, beans, corn, and leek hav e a much higher my corrhizal dependency than potato and wheat. This range of plant response to AMF has to be taken into account when managing a cropping sy stem or a crop rotation. Data on the potential of crop plants to benefit from my corrhizal sy mbiosis are av ailable at the my corrhizal producers lev el. Table 1 , taken from Plenchette et al. (33) and personal inv estigations, giv es ex amples of Relativ e Field My corrhizal Dependency (RFMD for some plants. Equation 1 giv es the formula for calculating RFMD.
Table 1. Relative Field Mycorrhizal Dependency (RFMD) for selected plants. Plant name RFMD* (%)
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Arbuscular Mycorrhiza Inoculum to Support Sustainable Cropping Systems

C abbage (Brassicaceae)* C arrot C hicory (witloof) Faba bean Garden beet (C henopodiaceae)* Garden pea Kentucky blue grass Kidney bean Leek Pepper Potato Tomato (according cultivars) Sweet corn Wheat (according cultivars) * Non-mycorrhizal plant.

0 99.2 82.4 93.5 0 96.7 72.4 94.7 95.7 66.1 41.9 59.2 - 78.0 72.7 44.5 - 56.8

RFMD =

DM of mycorrhizal plant - DM of non-mycorrhizal plant DM of mycorrhizal plant

100

[1]

Impact on Plant Protection and Microbial Interactions AM fungi are recognized as high potential agents in plant protection and pest management (34,43,48). In sev eral cases direct biocontrol potential has been demonstrated, especially for plant diseases caused by Phytophtora, Rhizoctonia , and Fusarium pathogens (1 ,49,52). Sev eral studies hav e confirmed sy nergism between AMF and biocontrol agents such as Burkholderia cepacia Palleroni & Holmes (37 ), Pseudomonas fluorescens Migula (1 1 ), Trichoderma harzianum Rifai (7 ), and V erticillium chlamydosporium Kamy schko ex Barron & Onions (35). These interactions suggest that AM might affect plant and soil microbial activ ity by stimulating the production of root ex udates, phy toalex ins, and phenolic compounds (30,32). A small increase of activ ity of plant defence genes, especially for the production of chitinases, glucanases, flav onoid biosy sthesis, and phy toalex ins, has been observ ed during my corrhizal growth; howev er these my corrhizal defence induction mechanisms remain transitory (1 8). AMF Impact on C Sequestration and Soil-aggregate Stability Ov er the y ears, a body of research has accumulated showing the effects of AMF biomass accumulated in the roots of colonized plants and in the surrounding soil. The AMF soil hy phae spread into the rhizosphere where they dev elop a network of microscopic filaments that make up to 80% of the total hy phae content in soil (24). For ex ample, in 4- to 5-week-old inoculated faba beans plants (V icia fava L.), my corrhizal fungi biomass v aried from 0.5 to 5% associated with low (1 6%) and high (62%) root colonization lev els, respectiv ely (25). Also, AMF spore biomass, measured in nine soil-field samples grown with cassav a for six months, was estimated between 89 to 93 lb/acre (44). Soil-aggregates stability is an important soil phy sical property that can be affected by AMF. Recently , a gly coprotein produced by AMF that promotes soil aggregation, glomalin, has been discov ered. Furthermore, higher than normal carbon diox ide concentrations help to promote soil aggregation by increasing the production of glomalin (38). These findings
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Arbuscular Mycorrhiza Inoculum to Support Sustainable Cropping Systems

could hav e important future implications in the use of my corrhizal fungi to promote the production of soil stable aggregates, improv e water infiltration, and soil C sequestration in agricultural sy stems. Propagation Cycle of Arbuscular Mycorrhizal Fungi The major biological characteristic of AMF is their obligate biotrophic nature. This means that each of their life cy cle steps requires the association with a liv ing plant. As with most of the filamentous fungi, AMF propagation can occur either by spores differentiation and germination (Figs. 1 a, 1 b) or by my celium ex tension through soil and roots (Figs. 1 c, 1 d). Spores are differentiated by budding intercalary or apically on hy phae. AMF species identification is based on spore characters, spore wall architecture, and the morphology of subtending hy phae. Some molecular tools to differentiate among AMF species and strains hav e been dev eloped. Howev er these new technologies remain to be tested for a v ariety of AM fungal strains and species (26,29,42,53). Sex ual reproduction has not y et been observ ed for these sy mbiotic fungi; therefore they are considered asex ual. Fungal filaments grow through soil particles and come in contact with y oung plant roots, the fungus threads its way through root surface, and then grow between and inside cortical cells (Figs. 1 e, 1 f, 1 g). The wide dispersal of the fungal network through its filaments giv es the plant-root my corrhizae access to a much larger v olume of soil than the root sy stem itself (Fig. 1 d). The establishment of my corrhizal networks in roots and soil constitute a soil-root fungal continuum, which is required for beneficial sy mbiotic ex changes between fungi and plant.

Fig. 1. Propagation cycle of AMF. a. Spores of (i) Gigaspora , (ii) Glomus, (iii) Entrophospora , and (iv) Acaulospora ; b. germinating spore; c. hyphal network and spores; d. hypha and spores around root; e. hyphal penetration inside root; f. intracellular arbuscules; g. intraradical vesicles; h. colonized plant.

Inoculum Propagation The main obstacle in the production of efficient and reliable AM fungal inoculum lies in their sy mbiotic behav iour, the fungi obligatory requiring a host plant for growth. Traditionally , my corrhizal fungi are propagated through pot-culture. Starting fungal inoculum, usually made of spores and
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colonized root segments, are incorporated to a growing substrate for seedling production (5). The fungi spread in the substrate and colonize root seedlings. Both colonized substrates and roots can then serv e as my corrhizal inoculum. Soilless similar culture sy stems such as aeroponic cultures enable the production of cleaner spores and facilitate uniform nutrition of colonized plants (20). The successful propagation of some AM fungal strains on root-organ culture allowed the cultiv ation of monox enic strains that can be used either directly as inoculum or as starting inoculum for large-scale production (1 3). (i) Pot-culture propagation. Unlike saprophy tic fungi, the largescale production of AMF inoculum, due to their obligate sy mbiotic status, requires control and optimization of both host growth and fungal dev elopment. The microscopic sizes of AMF, together with the complex identification processes also contribute to the pitfalls of inoculum propagation. The inoculum propagation process entails the following stages. Isolation of AMF pure culture strain. Pure culture strains can be obtained originally from a single spore that germinate and colonize roots of a host plant. AM fungal strains can also be generated from colonized root segments isolated directly from field plants. Monospecific cultures will then be obtained through subsequent pot-culture generation, using isolated spores or fine root segments as starting inoculum. A technical problem usually encountered with AMF is that spores can easily fall into dormancy and germination rates decrease dramatically (1 6). A coldtemperature treatment can be used to break dormancy (23,39). Research culture collection can prov ide users with reliable fungal cultures appropriate to start AM fungus propagation, accompanied with detailed information on species origin, spore morphology , and sometimes strain molecular biology and biochemistry . Choice of a host plant . The most important criteria required for the host plant is its high my corrhizal potential (i.e., its capacity to be colonized by the AMF strain and to promote its growth and sporulation), a tolerance to growth under growth chamber and greenhouse conditions, and an ex tensiv e root sy stem made of solid but non-lignified roots. Leek (Allium porrum L.), Sudan grass (Sorghum bicolor (L.) Moench), corn (Zea mays L.), and bahia grass (Paspalum notatum Flugge) are the most frequently used plant host for inoculum propagation (50). Optimum grow ing conditions . Pasteurized, steamed, or irradiated growing substrates are required in order to av oid culture contamination which could affect the quality of the inoculum. A well-aerated substrate is recommended, such as coarse tex ture sandy soil (1 4) mix ed with v ermiculite or perlite or Turface (8). Inadequate mineral nutrient composition may affect fungal dev elopment. Optimum P lev els v ary with the host plant and cultiv ated fungal strains and an ex cess of av ailable phosphorus can inhibit AMF propagation. Potassium, nitrogen, magnesium, and a selection of micro-element ratios may also affect inoculum dev elopment, especially when inert growing substrates are used and plant fertilization is performed artificially (9,45). Other edaphic factors such as pH, soil temperature (36), light intensity , relativ e humidity , and env ironment aeration must also be controlled to optimize AMF propagation. (ii) In v itro propagation on root-organ culture (Fig. 2). Rootorgan cultures consist of ex cised roots that proliferate under ax enic conditions on a sy nthetic nutrient media (Fig. 2d) supplemented with v itamins, minerals, and carbohy drates. Continuous cultures of v igorous root-organ cultures hav e been obtained through transformation of roots by the soil bacterium Agrobacterium rhizogenes Conn. (51 ). Since 1 988
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(4), sev eral dozen species and strains hav e been successfully propagated in v itro with v arious sy nthetic growth media and growth conditions (1 3), and tested with compartmentalized solid and liquid v essels. The monospecific strains av ailable can be used directly as starting material for largescale inoculum production, a sole Petri dish culture being enough to generate sev eral thousand of spores and meters of hy phae within 4 months.

Fig. 2. In vitro propagation. a. Isolated spores; b. germinating colonized root segment; c. carrot root in culture; d. AMF root-organ culture; e. closer view of an AMF root-organ culture.

In v itro bulk production of AMF inoculum is promising, offering clean, v iable, contamination-free fungi (Glomeromy cota in v itro collection, or GINCO) (Fig. 2e). The cost of in v itro inoculum may appear prohibitiv e compared to the cost of a greenhouse-propagated one, but its use as starting inoculum is a warranty of purity . Research Collections (In Vivo and In Vitro) Three major research collections (Table 2) manage inoculum maintenance and distribution. Their respectiv e activ ities, serv ices, and av ailability in AMF strains are posted on their respectiv e websites.
Table 2. Major research collections of AMF inoculum. Name and internet address Banque Europenne des Glomeromycota (BEG) www.kent.ac.uk/bio/beg Glomeromycota In Vitro C ollection (GINC O) res2.agr.gc.ca/ecorc/ginco-can/index_e.htm www.mbla.ucl.ac.be/ginco-bel International C ulture C ollection of Vesicular-Arbuscular Mycorrhizal Fungi (INVAM) invam.caf.wvu.edu Propagation mode Pot-culture Root-organ culture

Pot-culture

Their common purpose is mainly to prov ide research and industry scientists with pure and reliable material for starting inoculum production for both fundamental researches and applied technologies. Sev eral other laboratory and industry collections are distributed
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throughout the world to support either fundamental and applied researches or commercial activ ities. Long-term Preservation of AMF Inoculum Large-scale production of my corrhizal inoculum requires inv entory of product and the ability to prov ide clients with products of high and consistent quality . Although the detailed procedure for inoculum preserv ation is proprietary , methodologies for its preserv ation remain simple and inex pensiv e. Fungal v iability and my corrhizal efficiency can be maintained for sev eral months at room temperature (68 to 7 7 F) especially when semi-dry inocula are kept in their plastic containers or packaging. The major inconv enience of such a storage period is the occurrence of spore dormancy . Long-term storage (up to 1 to 2 y ears) may be conducted at 41 F cold temperature storage (Dalp et al., unpublished results ). This method is efficient for both in v iv o and in v itro propagated strains. As spore germination and my celium potential may be stimulated by cold treatment, strain v igour can usually be recov ered after long-term storage at cold temperature. Liquid inoculums should react similarly to the traditional dry ones. More sophisticated and ex pensiv e preserv ation techniques are performed by research culture collections. These include the maintenance of inoculum on liv ing plant-host grown on sterile growth substrate with regular check for mono-specificity of the cultiv ated strains, storage in liquid nitrogen tanks (1 0), and freeze-dry ing under v acuum. The last two techniques are the usual techniques used in repository culture collections (DAOM/CCFC; ATCC). Commercial Inoculum Production Small scale AMF inoculum production began in the 1 980s followed by large scale production in the 1 990s. At present, sev eral companies hav e officially registered and commercialized AMF inoculum. (i) Methodologies. The first generation of commercial inoculum appeared in the early 1 980s. Since then, basic methodologies used for in v iv o inoculum propagation hav e ev olv ed gradually . Pot-cultiv ation remains the preferred propagation technique, as it prov ides a conv enient and relativ ely economic method to produce my corrhizal inoculum on a large scale (40). Generally , my corrhizal fungi propagules, such as colonized roots, spores, and hy phae, are mix ed with a growing substrate, and the pots are seeded and incubated under controlled conditions (Fig. 3). The in v itro propagation on root-organ culture may not change drastically the traditional procedures but will certainly facilitate the quality control of strain purity and improv e the supply of massiv e amounts of spores as starting inoculum.

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Fig. 3. In vivo propagation. a. Seeding mycorrhizal substrates; b. mycorrhizal seedling production; c. growth chamber inoculum propagation; d. root growth and colonization; e. colonized seedlings; f. field inoculum propagation.

(ii) Obtaining m other inoculum for large-scale production. Both segments of colonized roots (0.08 to 0.1 6 inches long) containing hy pha and/or v esicles and fungal spores may be used as starting fungal propagules for the production of mother inoculum. Research collections can prov ide such material from either in v iv o or in v itro propagated fungi. Since root-organ culture technology has become av ailable, fungal propagules may be ex tracted from in v itro cultures grown at large scales on solid or semi-liquid growing media. (iii) Establishm ent of cultures. The establishment of my corrhizal cultures may proceed in different way s (Fig. 3): Mother inoculum added directly at seeding in large tray s or pots; Mother inoculum incorporated at seedling transplantation of 4to-6-week old plantlets; Mother inoculum added at transplantation of micro-propagated plantlets; Colonized seedlings produced in greenhouses and transplanted to the field. Composition of Commercial Inoculum The inoculum sold on the market are prov ided as granular substrates made from mix ed materials such as peat, compost, v ermiculite, perlite, sand, and/or ex panded clay in which segments of colonized roots, spores, and filamentous networks are distributed. Most of the time these roots, spores, and hy phal networks are not detectable because of their microscopic sizes. In terms of fungal content, the tendency is to introduce a mix of sev eral AMF in commercial inoculum. The most frequently used AMF species for commercial inoculum is ty pically Glomus intraradices Schenck & Smith. This species is well adapted to both in v iv o and in v itro propagation, can colonize a large v ariety of host plants, surv iv e to longterm storage, and is geographically distributed all ov er the world. These characteristics make the G. intraradices species an ex cellent candidate for commercial inoculum. Sev eral other AMF belonging mainly to Glomus species, but also to Gigaspora , Scutellospora , and Acaulospora genera, are gradually used for commercial inoculum production. These AMF are sometimes in a mix ture with growth-promoting bacteria and with ectomy corrhizal fungi, making a potentially better inoculum for plant
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Arbuscular Mycorrhiza Inoculum to Support Sustainable Cropping Systems

protection and production. Innovations and Future Developments One innov ativ e technique is the ready - and easy -to-use inoculum in which fungal propagules are ex tracted from growing media, concentrated, and mix ed with carriers such as peat, sand, v ermiculite, or ex panded clay . Products are av ailable in powdered form containing a specified number of activ e fungal propagules per v olume of inoculum. Liquid inoculum dedicated to horticultural use and isolated spores are also av ailable. Aeroponic inoculum production at large scale has been inv estigated by Souza et al. (47 ) but has not reached commercialization. Bioreactor assay s with liquid AMF root-organ culture propagation (22) may ev entually become suitable for commercialization for research needs. Howev er, as the fungi are produced in association with Agrobacterium -transformed roots, it is unlikely that its used can be allowed for field inoculation. Knowing the performance v ariability between AM fungal strains, the improv ement of commercial inoculum quality will almost certainly come from the selection of higher performance my corrhizal fungal strains better adapted to the plant host or crop to be colonized and to specific env ironmental growing conditions (2,28). Constraints and Regulations (i) Cost of inoculum v ersus fertilizers. Again, the obligate biotrophic nature of AMF which, unlike other fungi, implies the establishment of a plant propagation sy stem, either under greenhouse conditions or in v itro laboratory propagation. These techniques result in high inoculum production costs, which still remains a serious problem since they are not competitiv e with production costs of phosphorus fertilizer. Ev en if farmers understand the significance of sustainable agricultural sy stems, the reduction of phosphorus inputs by using AM fungal inocula alone cannot be justified ex cept, perhaps, in the case of high v alue crops. This could be the case of organic crop farmers, which can sell their products at premium price. (ii) Sanitary control. Another serious problem in commercializing inoculum comes from the need to control the biological composition of the product, especially from inv ading phy topathogenic microorganisms. At present, the inoculum produced using the pot-culture v ariants, either in greenhouses, growth chambers, or fields, is nev er completely free from ex ternal microorganisms. This is a problem ev en though the producers attempt to control pathogens with v arious agrochemicals. Farmers are usually aware of the risk of pathogens, so they av oid using inoculum containing host root residues. In most commercial inoculum, colonized root segments are chopped into 0.08-to-0.1 2-inch-long pieces so segments that remain are difficult to detect. When colonized roots are directly incorporated to carriers, their surface sterilization with a light solution of disinfecting product can be done without affecting the effectiv eness of an inoculum. When roots and rhizosphere material are used for inoculum preparation, handling with clean apparatus is adv ised. (iii) Efficiency of inoculum . In the field application of any microbial inoculum, it is essential to v erify that the inoculated microorganisms possess the characteristics and the potential described by the inoculum manufacturers. With AM inoculum, such ev aluations can be done using sev eral approaches such as morphological identification of AM spores to confirm the fungus identity , and by estimating the my corrhizal root colonization lev el of test plants (6). Tentativ e molecular techniques hav e been dev eloped for the detection of AMF inoculum strains and
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discrimination from indigenous AMF strains naturally occurring in soils. These techniques are not y et totally reliable due to the large genetic heterogeneity in AMF and, as such, these techniques are not routinely used for the detection of AMF. Similar situations are observ ed with the discrimination among strains when using internal transcribed spacer (ITS) sequences of ribosomal DNA genes (rDNA). Reliable molecular techniques to trace the inoculated strains using rep-PCR and specific primers dev elopments are under study (42). Such a technological breakthrough would greatly facilitate both fundamental and applied research on my corrhizae as well as improv e quality control of commercial inoculum. (iv ) Official registration for com m ercial products. The commercialization of my corrhizal inoculum is subjected to regional or national registration at agriculture departments and usually falls under the country s Fertilizer Act. In Canada, my corrhizal inoculum are considered to be supplements: products, other than fertilizers, manufactured, sold or represented for use in the improv ement of the phy sical condition of the soil or to aid plant growth or crop y ields. In the USA, registration of an AM inoculum may fall either in the fertilizer or the pesticide sectors, depending on the v ocation of the proposed my corrhizal product. Application for registration is required for such products and ex tensiv e information is attached to the registration request: (a) a list of ingredients and possible contaminants in the proposed inoculum; (b) the minimum concentration of each ingredient including the activ e my corrhizal fungi and the purpose of each of them; (c) official material safety data sheets; (d) the product label, showing the name and address of producer, the number of v iable fungal propagules or the sy mbiotic efficiency ex pressed as percentage of colonization ex pected by the inoculum, recommended plant host, soil conditions for effectiv eness, recommended application rate, storage conditions, and ex piration date; (e) manufacturing process; and (f) the testing protocol. Such quality control is important to ex clude poor quality microbial inocula from the market. Attached with prev ious information, statistically significant efficacy data from field tests done under different soil and climatic conditions are usually required in order to support the claims being made regarding the performance of the proposed inoculum. A detailed tax onomic description may also be requested together with strain history , geographic distribution, and ex isting literature on the my corrhizal potential of the fungal strain. In most countries, my corrhizal fungi are no longer considered detrimental for human and animal health. As such, no env ironmental infectiv ity or tox icity tests are required. The creation of an International Association of My corrhizal Inoculum Producers was discussed at the last International Conference on My corrhizae held August 1 0-1 5, 2003 in Montreal, in order to establish rules and regulations which would stimulate industrial production of high quality inoculum. Organic crop farmers since they hav e already started using AMF inoculum in larger-scale production, are potentially a new clientele base. Howev er, at this point, they are hav ing difficulties with inoculum application and with a clear measurement of beneficial effect in their crops (personal communication). For large-scale application of inoculum, future research focusing on achiev ing good contact between seed an inoculum is needed. Regardless of the method of inoculum application, new users should establish a portion of their crop without inoculum in order to assess the benefits obtained in the crop established with inoculum. Acknowledgments
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Arbuscular Mycorrhiza Inoculum to Support Sustainable Cropping Systems

The authors wish to thank Dr. Mary Leggett (Philom Bios Inc., Saskatoon, Saskatchewan, Canada) who organized the Inoculum Forum Conference; Mr. Clifford Hamilton for his technical assistance on image preparation; and S. Sguin, J. Cay ouette, and S. Redhead for comments on the manuscript. Literature Cited
1 . Abdel-Aziz, R. A., Radwan, S. M. A., Abdel-Kader, M. M., and Barakat, M. I. A. 1 9 9 7 . Biocontrol of faba bean root-rot using VA m y corrhizae and its effect on biological nitrogen fixation. Egy pt. J. Microbiol. 3 1 :2 7 3 -2 86 . 2 . Adholey a, A. 2 003 . Com m ercial production of AMF through industrial m ode and its large-scale application. 4 th Int. Conf. My corrhizae. Montreal Quebec Canada, August 1 0-1 5 2 003 , No. 2 4 0. 3 . Barker, S. J., Tagu, D., and Delp, G. 1 9 9 8. Regulation of root and fungal m orphogenesis in m y corrhizal sy m biosis. Plant Phy siol. 1 1 6 :1 2 01 -1 2 07 . 4 . Bcard, G., and Fortin, J. A. 1 9 88. Early ev ents of v esicular-arbuscular m y corrhiza form ation on Ri T-DNA transform ed roots. New Phy tol. 1 08:2 1 1 -2 1 8. 5. Brundrett, M., Bougher, N., Dell, B., Grov e, T., and Malajczuk, N. 1 9 9 6 . Working with m y corrhizas in forestry and agriculture. ACIAR (Austral. Cen. Int. Agric. Res.) Mono. 3 2 . 6 . Dalp, Y. 1 9 9 1 , Vesicular-arbuscular m y corrhizae. Pages 2 87 -3 01 in: Manual of Soil Sam pling and Methods of Analy sis. 3 rd Ed. Can. Soc. Soil Sci., Lewis Pub. of CRC Press. 7 . Datnoff, L. E., Nem ec, S., and Pernezny , K. 1 9 9 5. Biological control of fusarium crown and root rot of tom ato in Florida using Trichoderma harzianum and Glomus intraradices . Biol. Control. 5:4 2 7 -4 3 1 . 8. Dehne, H. W., and Backhaus, G. F. 1 9 86 . The use of v esicular-arbuscular m y corrhizal fungi and plant production. I. Inoculum production. J. Plant Dis. Prot. 9 3 :4 1 5-4 2 4 . 9 . Dixon, S., Sm ith, S. E., and Sm ith, F. A. 1 9 9 9 . Characterization of two arbuscular m y corrhizal fungi in sy m biosis with Allium porrum : Inflow and flux of phosphate across the sy m biotic interface. New Phy tol. 1 4 4 :1 7 3 -1 81 . 1 0. Douds, D. D., and Schenck, N. C. 1 9 9 0. Cry opreserv ation of spores of v esicular-arbuscular m y corrhizal fungi. New Phy tol. 1 1 5:6 6 7 -6 7 4 . 1 1 . Edwards, S. G., Young, J. P. W., and Fitter, A. H. 1 9 9 8. Interactions between Pseudomonas fluorescens biocontrol agents and Glomus mosseae, an arbuscular m y corrhizal fungus, within the rhizosphere. FEMS Microbiol. Lett. 1 6 6 :2 9 7 -3 03 . 1 2 . Ezawa, T., Yam am oto, K., and Yoshida, S. 2 000. Species com position and spore density of indigenous v esicular-arbuscular m y corrhizal fungi under different conditions of P-fertility as rev ealed by soy bean trap culture. Soil Sci. Plant Nutr. 4 6 :2 9 1 -2 9 7 . 1 3 . Fortin, J. A., Bcard, G., Declerck, S., Dalp, Y., St-Arnaud, M., Coughlan, A. P., and Pich, Y. 2 002 . Arbuscular m y corrhiza on root-organ cultures: A rev iew. Can. J. Bot. 80:1 -2 0. 1 4 . Gaur, A., and Adholey a, A. 2 000. Effects of the particle size of soil-less substrates upon AM fungus inoculum production. My corrhiza 1 0:4 3 -4 8. 1 5. Gav ito, M. E., and Miller, M. 1 9 9 8. Changes in m y corrhiza dev elopm ent in m aize induced by crop m anagem ent practices. Plant Soil 1 9 8:1 85-1 9 2 . 1 6 . Gem m a, J. N., and Koske, R. E. 1 9 88. Seasonal v ariation in spore abundance and dorm ancy of Gigaspora gigantea and in m y corrhizal inoculum potential of dune soil. My cologia 80:2 1 1 -2 1 6 . 1 7 . Ham el, C., Dalp, Y., Lapierre, C., Sim ard, R. R., and Sm ith, D. L. 1 9 9 6 . Endom y corrhizae in a newly cultiv ated acidic m eadow: Effects of three y ears of barley cropping, tillage, lim e, and phosphorus on root colonization and soil infectiv ity . Biol. Fertil. Soils 2 1 :1 6 0-1 6 5. 1 8. Harrison, M. J. 1 9 9 7 . The arbuscular m y corrhizal sy m biosis. Pages 1 -3 4 in: Plant-Microbe Interactions, Vol. 3 . G. Stancey , and N.T. Keen, eds. Chapm an and Hall, New York.
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Arbuscular Mycorrhiza Inoculum to Support Sustainable Cropping Systems

1 9 . Hodge, A. 2 000. Microbial ecology of the arbuscular m y corrhiza. FEMS Microbiol. Ecol. 3 2 :9 1 -9 6 . 2 0. Jarstfer, A. G, and Sy lv ia. D. M. 1 9 9 9 . Aeroponic culture of VAM fungi. Pages 4 2 7 -4 4 1 : My corrhiza: Structure, Function, Molecular Biology and Biotechnology . 2 nd edition. A. Varm a, and B. Hock, eds. Springer-Verlag, Berlin. 2 1 . Jeffries, P., and Barrea, J. M. 1 9 9 4 . Biogeochem ical cy cling and arbuscular m y corrhizas in the sustainability of plant-soil sy stem sp. Pages 1 01 -1 1 5: Im pact of Arbuscular My corrhizas on Sustainable Agriculture and Natural Ecosy stem s. S. Gianinazzi, and H. Schuepp, eds. Basel; Boston; Berlin; Birkhauser. 2 2 . Jolicoeur, M., William s, R. D., Chav arie, C., Fortin, J. A., and Archam bault, J. 1 9 9 9 . Production of Glomus intraradices propagules, an arbuscular m y corrhizal fungus, in an airlift bioreactor. Biotechnol. Bioeng. 6 3 :2 2 4 -2 3 2 . 2 3 . Juge, C., Sam son, J., Bastien, C., Vierheilig, H., Coughlan, A., and Pich, Y. 2 002 . Breaking dorm ancy in spores of the arbuscular m y corrhizal fungus Glomus intraradices : A critical cold-storage period. My corrhiza 1 2 :3 7 -4 2 . 2 4 . Kabir, Z., OHalloran, I. P., and Ham el, C. 1 9 9 7 . Ov erwinter surv iv al of arbuscular m y corrhizal hy phae is fav ored by attachm ent to roots but dim inished by disturbance. My corrhiza 7 :1 9 7 -2 00. 2 5. Kucey , R. M. N., and Paul, E. A. 1 9 82 . Biom ass of m y corrhizal fungi associated with bean root. Soil Biol. Biochem . 1 4 :4 1 3 -4 1 4 . 2 6 . Lanfranco, L., Wy ss, P., Marzachi, C., and Bonfante, P. 1 9 9 5. Generation of RAPD-PCR prim ers for the identification of isolates of Glomus mosseae, an arbuscular m y corrhizal fungus. Mol. Ecol. 4 :6 1 -6 8. 2 7 . Lange, N. R., and Vlek, P. L. G. 2 000. Mechanism of Calcium and Phosphate Release from Hy droxy -Apatite by My corrhizal fungi. Soil Sci. Soc. Am . J. 6 4 :9 4 9 -9 55. 2 8. Masanori, S., and Takuy a. M. 2 002 . Inoculation with arbuscular m y corrhizal fungi: The status quo in Japan and future prospects. Plant Soil 2 4 4 :2 7 3 -2 7 9 . 2 9 . Millner, P. D., Mulbry , W. W., and Rey nolds, S. L. 2 001 . Taxon-specific oligonucleotide prim ers for detection of Glomus etunicatum . My corrhiza 1 0:2 59 -2 6 5. 3 0. Morandi, D. 1 9 9 6 . Occurrence of phy toalexins and phenolic com pounds in endom y corrhizal interactions, and their potential role in biological control. Plant Soil 1 85:2 4 1 -2 51 . 3 1 . Mozafar, A., Anken, T. Ruh, R., and Frossard, E. 2 000. Tillage intensity , m y corrhizal and non m y corrhizal fungi, and nutrient concentrations in m aize, wheat, and canola. Agron. J. 9 2 :1 1 1 7 -1 1 2 4 . 3 2 . Norm an, J. R., and Hooker, J. E. 2 000. Sporulation of Phytophthora fragariae shows greater stim ulation by exudates of non-m y corrhizal than by m y corrhizal strawberry roots. My col. Res. 1 04 :1 06 9 -1 07 3 . 3 3 . Plenchette, C., Fortin, J. A., and Furlan, V. 1 9 83 . Growth responses of sev eral plant species to m y corrhizae in a soil of m oderate P-fertility . I. My corrhizal dependency under field conditions. Plant Soil 1 1 0:1 9 9 -2 09 . 3 4 . Quarles, W. 1 9 9 9 . Plant disease biocontrol and VAM fungi. IPM Pract. 2 1 :1 9. 3 5. Rao, M. S., Kerry , B. R., Gowen, S. R., Bourne, J. M., and Parv atha, R. P. 1 9 9 7 . Managem ent of Meloidogyne incognita in tom ato nurseries by integration of Glomus deserticola with Verticillium chlamydosporium . Pflanzenkrank. Pflanzenschutz 1 04 :4 1 9 -4 2 2 . 3 6 . Rao, A. V., and Tarafdar, J. C. 1 9 9 9 . Soil solarization for m ass scale production of arbuscular m y corrhizal fungal inoculum in Indian arid zone. Indian J. Agric. Sci. 6 9 :2 7 1 -2 7 4 . 3 7 . Rav nskov , S., Larsen, J., and Jakobsen, I. 2 002 . Phosphorus uptake of an arbuscular m y corrhizal fungus is not affected by the biocontrol bacterium Burkholderia cepacia. Soil Biol. Biochem . 3 4 :1 87 5-1 881 . 3 8. Rilling, M. C., Wright, S. F., Allen, M. F., and Field, C. B. 1 9 9 9 . Rise in carbon dioxide changes soil structure. Nature 4 00:6 2 8.
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Arbuscular Mycorrhiza Inoculum to Support Sustainable Cropping Systems

3 9 . Safir, G. R., Coley , S. C., Siqueira, J. O., and Carlson, P. S. 1 9 9 0. Im prov em ent and sy nchronization of VA m y corrhiza fungal spore germ ination by short-term cold storage. Soil Biol. Biochem . 2 2 :1 09 -1 1 1 . 4 0. Sahay , N. S., Sudha, A. S., Varm a, A., and Singh, A. 1 9 9 8. Trends in endom y corrhizal research. Indian J. Exp. Biol. 3 6 :1 06 9 -1 086 . 4 1 . Schuessler, A., Schwarzott, D., and Walker, C. 2 001 . A new fungal phy lum , the Glom erom y cota: Phy logeny and ev olution. My col. Res. 1 05:1 4 1 3 -1 4 2 1 . 4 2 . Sguin, S., Dsaulniers, N., Dalp, Y., and Lv esque, C. A. 2 003 . Dev elopm ent of AMF strains specific prim ers for detection in field-grown colonized roots. 4 th Int. Conf. My corrhizae. Montreal Quebec Canada, August 1 0-1 5 2 003 , No. 4 4 4 . 4 3 . Sharm a, S., and Dohroo, N. P. 1 9 9 6 . Vesicular-arbuscular m y corrhizae in plant health and disease m anagem ent. Int. J. Trop. Plant Dis. 1 4 :1 4 7 -1 55. 4 4 . Siev erding, E., Toro, S., and Mosquera, O. 1 9 89 . Biom ass production and nutrient concentrations in spores of VA m y corrhizal fungi. Soil Biol. Biochem . 2 1 :6 9 -7 2 . 4 5. Singh, C. S., and Jha, D. 1 9 9 4 . Mass inoculum production of v esicular arbuscular m y corrhizae (VAM): Effect of v arious bacteriological m edia and fertilizer solutions. Microbiol. Res. 1 4 9 :2 7 -2 9 . 4 6 . Sm ith, S. E., Read, D. J., and Harley , J. L. 1 9 9 7 . My corrhizal Sy m biosis. 2 nd Ed. Academ ic Press, London, Toronto. 4 7 . Souza, de, E. S., Burity , H. A., Espirito-Santo, A. C., and Silv a, da, M. L. R. B. 1 9 9 6 . Alternativ e for arbuscular m y corrhizal fungi inoculum production in aeroponic culture. Pesqu. Agropecu. Bras. 3 1 :1 53 -1 58. 4 8. St. Arnaud, M., Ham el, C., Caron, M., and Fortin, J. A. 1 9 9 5. Endom y corhizes VA et sensibilit des plantes aux m aladies: Sy nthse de la littrature et m canism es dinteraction potentiels. Pages 51 -87 in: La Sy m biose My corhizienne. tat des Connaissances. J. A. Fortin, Y. Pich, C. Charest, eds. Editions Orbis Frelisghburg Qubec. 4 9 . St. Arnaud, M., Ham el, C., Vim ard, B., Caron, M., and Fortin, J. A. 1 9 9 7 . Inhibition of Fusarium oxysporum f.sp. dianthi in the non-VAM species Dianthus caryophyllus by co-culture with Tagetes patula com panion plants colonized by Glomus intraradices . Can. J. Bot. 7 5:9 9 8-1 005. 50. Struble, J. E., and Skipper, H. D. 1 9 88. Vesicular-arbuscular m y corrhizal fungal spore production as influenced by plant species. Plant Soil 1 09 :1 1 9 4 1 1 96. 51 . Tepfer, D. 1 9 89 . Ri t-DNA from Agrobacterium rhizogenes : a source of genes hav ing applications in rhizosphere biology and plant dev elopm ent, ecology and ev olution. Pages 2 9 4 -3 4 2 : Plant-Microbe Interactions, Vol. 3 . T. Kosuge, and E. W. Nester, eds. McGraw-Hill Publishing, New York. 52 . Vigo, C., Norm an, J. R., and Hooker, J. E. 2 000. Biocontrol of the pathogen Phytophthora parasitica by arbuscular m y corrhizal fungi is a consequence of effects on infection loci. Plant Path. 4 9 :509 -51 4 . 53 . Yokoy am a, K., Tateishi, T., Marum oto, T., and Saito, M. 2 002 . A m olecular m arker diagnostic of a specific isolate of an arbuscular m y corrhizal fungus Gigaspora margarita. FEMS Microbiol. Let. 2 1 2 :1 7 1 -1 7 5.

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