Phuong Phap Quan Li Mau Benh Thuc Vat

PHNG PHP QUN L MU BNH THC VT
Phng php qun l mu bnh thc vt
Mi ti liu ghi chp nu khng th c kim tra li th cng ch l nhng mnh giy vn m thi R.W.G. Dennis, theo British Ascomycetes (1968), J. Cramer, Lehre, Germany.
ii
LI CM N
Chng ti xin chuyn li cm n su sc ti TS. John Alcorn, ngi hiu nh cun sch ny. Xin cm n T chc h tr pht trin Quc t Australia (AusAID) cung cp ngun kinh ph qu bu cho vic ra i v xut bn cun sch. Xin ginh li cm n c bit cho ng Eli Szandala, Vn phng cc chuyn vin bo v thc vt cao cp, B Nng nghip, Thy sn v Lm nghip Australia iu hnh chng trnh xut bn cun sch ny. Cun sch ny m t nhiu phng php c tho lun bi cc thnh vin tham gia trong hi tho t chc ti Chiang Mai, Thi Lan ngy 20 thng 11 nm 2004. Chng ti xin cm n TS. Srisuk Poonpolgul (Bangkok, Thi Lan), b Srisurang Likhitekaraj (Bangkok, Thi Lan), TS. Pornpimon Athipunyakom (Bangkok, Thi Lan), ng Woothisak Butranu (Bangkok, Thi Lan), TS. Kartini Kramadibrata (Bogor, Indonesia), TS. Lee Su See (Kepong, Malaysia), TS. Hien Thuy Phan (H Ni, Vit Nam) v ng Eli Szandala (Canberra, Australia) v nhng ng gp qu bu cho s thnh cng ca cun sch ny. Cc tc gi chnh TS. Roger Shivas v TS. Dean Beasley, Bo tng bnh cy, S Nng nghip v Thy sn Queensland, Australia. Cc tc gi tham gia TS. John Thomas v TS. Andrew Geering, Khoa virus hc, S Nng nghip v Thy sn Queensland, Australia. TS. Ian Riley, Chuyn gia tuyn trng hc, Trng i hc Adelaide, Bang Nam Australia, Australia. Commonwealth, Australia 2005 Cun cm nang Phng php qun l mu bnh thc vt c m bo quyn tc gi. Tuy nhin, bn c th ti xung, in v sao chp cho vic s dng ring, khng v mc ch thng mi hoc s dng trong phm vi c quan ca mnh. Ngoi mc ch s dng c php trong phm vi quy nh ca Lut bn quyn nm 1968, tt c cc quyn tc gi khc u c m bo. ISBN 0-9751686-9-X
iii
BNG CH VIT TT
AMV ACIAR ASEAN ASEANET AusAID BO BRIP DAFF DAR DNA ELISA FAO GA GBIF GPS KBA ICTV IJSB IJSEM IMI IPPC ISPM LNYV MEA MTA NA NSW OCPPO PCR PDA PEMV PRA PRSV QDPIF RIRDC RNA SEM SPA SPS SCBV SWG TMV TSWV TWA UV VIDE WFCC WTO Virus khm l linh lng Trung tm nghin cu Nng nghip Quc t Australia Hip hi cc Quc gia ng Nam Hi thit lp v iu hnh mng li khu vc ng Nam T chc h tr pht trin Quc t Australia Bo tng Bogoriense Bo tng bnh cy Queensland B Nng nghip, Lm nghip v Thy sn Bo tng bnh cy New South Wales Deoxyribonucleic Acid K thut min dch lin kt men T chc lng thc ca Lin Hp Quc Glycerol Agar Phng tin thng tin a dng sinh hc ton cu H thng nh v ton cu Kings B Agar y ban Quc t v phn loi virus Tp ch Quc t v phn loi hc vi khun Tp ch Quc t v tin ha v phn loi vi sinh vt hc CABI Bioscience, UK Centre Cng c Quc t v bo v thc vt Tiu chun Quc t v kim dich ng thc vt Virus m vng hoi sinh rau dip Malt Extract Agar Vn bn tha thun chuyn giao mu Nutrient Agar New South Wales Vn phng chuyn vin bo v thc vt cao cp Phn ng trng hp chui Khoai ty - ng Agar Virus khm bin dng u nh gi nguy c dch hi Virus m vng u S Nng nghip v Thy sn Queensland T chc nghin cu v pht trin nng thn Ribonucleic Acid Knh hin vi in t qut Sucrose Peptone Agar Kim dch ng thc vt Virus hnh que hi ma Cn trng lng tiu chun Virus khm l thuc l Virus m ho c chua Mi trng agar - nc my Tia cc tm C s d liu trao i thng tin gim nh virus Hip hi lu gi mu vi sinh vt Th gii T chc thng mi Th gii
iv
LI TA
Cun cm nang v qun l mu bnh thc vt ny c B Nng nghip, Thy sn v Lm nghip Australia ti tr thc hin. Cun sch c ra i vi mc ch cung cp ti liu tham kho cho cc nc trong khu vc nhm xy dng danh mc dch hi t tiu bn cc mu bnh thu c, p ng nhu cu ca thng mi Quc t i vi hng ha nng nghip. S thnh lp t chc thng mi Th gii (WTO) nm 1995 l tin cho s ra i ca mt k nguyn mi trong t do thng mi. Hip nh WTO v vic p dng cc bin php kim dch ng thc vt (Hip nh SPS) thc y s pht trin ca thng mi ha nng sn, tuy vy mc tiu ny b lng qun mt s nc ang pht trin. nhiu nc trn th gii, b su tp su v bnh hi rt ngho nn dn n nhng hn ch trong vic m t tnh trng lnh mnh ca nn nng nghip cng nh lm nghip nc . cng l nguyn nhn khin nhiu t chc bo v thc vt Quc gia khng th thc hin cng tc phn tch nguy c dch hi mt cch y v tin cy. Tnh trng ny cn tip din s l mt bt li ln cho cc nc ang pht trin khu vc trong vic m phn m rng th trng cho hng nng sn. Theo iu khon 9 ca hip nh SPS, cc nc pht trin chp thun tr gip v chuyn mn cho cc nc ang pht trin thnh vin pht trin k nng c lin quan n hip nh SPS v p ng cc iu kin m Hip nh SPS ra. Australia ng gp trch nhim ca mnh bng nhiu cch khc nhau thng qua cc chng trnh tr gip song phng trong khu vc, vi ngun vn ti tr ca T chc h tr pht trin Quc t Australia (AusAID). Cc hot ng tr gip c tin hnh trn nhiu lnh vc, trong c vic xy dng danh mc cc dch hi trn c s mu tiu bn. Vn phng cc chuyn vin bo v thc vt cao cp thuc B Nng nghip, Lm nghip v Thy sn Australia m nhn cng vic ny, i tng nhn c s gip trong chng trnh l cc nc khu vc ng Nam . Chng trnh ch trng n vic xy dng nng lc ca cc nh khoa hc nm lnh vc c bn sau: iu tra dch hi thc vt Chn on Bo qun mu Ph trch v qun l b su tp mu Qun l d liu Cun cm nang ny tm tt li nhng phng php v k thut thng c s dng trong tt c cc vn v bnh cy, bao gm c thng tin v nhng bnh hi c tm quan trng v kinh t, cc phng php thu thp mu bnh trn ng rung, phng php nui cy trn mi trng nhn to, phng php phn lp, bo qun mu cng nh cc thng tin, cc mo nh trong vic qun l tiu bn mu bnh v mu vi sinh vt hi. p ng nhu cu s dng rng ri, cun cm nang ny c dch sang ting Vit, ting Thi v ting Bahasa. Lois Ransom Chuyn vin bo v thc vt cao cp B Nng nghip, Thy sn v Lm nghip Australia. 1
MC LC
LI CM N BNG CH VIT TT LI TA MC LC DANH MC HNH DANH MC BNG 1
1.1 1.2 1.3 1.4
III IV 1 2 5 5 6
6 8 8 9
M U
U
Nhng trch nhim mang tnh ton cu Tnh trng lu gi mu sinh hc cc nc ASEAN Tm quan trng ca vic lu gi h s tiu bn Xy dng v pht trin cng tc lu gi mu bnh cy cc nc ASEAN
2
2.1 2.2 2.3 2.4
LU GI MU BNH HI THC VT
Bo tng mu B su tp vi sinh vt Xy dng b mu bnh cy Danh mc dch hi
10
10 10 11 12
3
3.1 3.2 3.3
XY DNG B SU TP MU BNH
Ngun mu Thu thp mu bnh ngoi ng rung Thu thp v x l mu bnh i vi l, thn v qu i vi r v t i vi nm ln Cch ghi phiu iu tra mu bnh iu tra v ly mu
15
15 15 17 18 19 20 21 22
3.3.1 3.3.2 3.3.3 3.4 3.5
4
4.1 4.2 4.3
PHNG PHP KIM TRA MU BNH

Nhum mu nm v vi khun Knh hin vi quang hc Chp nh mu Cch qun l v t tn file nh
24
24 26 27 27
4.3.1
5 PHNG PHP PHN LP NM, VI KHUN V TUYN TRNG TRONG PHNG TH NGHIM 29 2
5.1 5.1.1 5.1.2 5.1.3 5.2 5.3 5.3.1 5.3.2 5.3.3 5.4
Phn lp nm Bung gi m Phng php pha long Pht trin v sn sinh bo t Phn lp vi khun Phn lp tuyn trng Mu t Mu cy bnh Bo qun v lm tiu bn Mi trng nui cy
30 31 32 32 34 35 36 38 39 40
6
6.1
CCH BO QUN V LU GI MU BNH

Tiu bn mu bnh Tiu bn kh Tiu bn chun Mu vi sinh vt kh Tiu bn lam ng gi v vn chuyn tiu bn Mu vi sinh vt phn lp Nui cy trn mi trng agar Bo qun bng du khong Bo qun trong nc v trng Bo qun ng kh Bo qun trong t Bo qun bng silica gel Bo qun trn giy thm Bo qun lnh su Bo qun bng nit lng 6.1.1 6.1.2 6.1.3 6.1.4 6.1.5
44
44 44 46 47 47 48 48 49 49 50 50 50 50 51 51 51
6.2 6.2.1 6.2.2 6.2.3 6.2.4 6.2.5 6.2.6 6.2.7 6.2.8 6.2.9
7
7.1
GIM NH VI SINH VT GY HI
Nm Nm gy bnh trn r Nm gy bnh trn thn Nm gy bnh trn l Nm gy bnh trn qu v ht Nm g st Nm than en 7.1.1 7.1.2 7.1.3 7.1.4 7.1.5 7.1.6
52
52 53 53 54 56 56 58
7.2 7.3 7.4 7.4.1 7.4.2 7.5 7.5.1 7.5.2 7.5.3 7.6 7.6.1 7.6.2 7.6.3 7.6.4
Vi khun Phytoplasma Virus v viroid Cc loi virus v thng tin lu gi Triu chng bnh virus thc vt Tuyn trng Nhng yu cu v gim nh Nhn dng tuyn trng k sinh thc vt Gim nh n loi Cc k thut phn loi Knh hin vi in t qut (Scanning electron microscopy - SEM) K thut ha sinh v phn t Huyt thanh (min dch) K thut nucleic axit
59 61 61 62 63 66 67 67 68 69 69 69 69 70
8
8.1
QUN L H S MU BNH
H thng c s d liu (Database)
71
71
9
9.1 9.2 9.3 9.4 9.5
QUN L B SU TP MU
U
73
73 73 74 75 76
iu kin phng mu Phng tr b cnh cng hi tiu bn mu Phng tr nhn hi mu vi sinh vt Cho mn mu Bo m an ton cho mu bnh
10
TI LIU THAM KHO CHN LC
78
DANH MC HNH
Hnh 1 Hnh 2 Hnh 3 Hnh 4 Hnh 5 Hnh 6 Hnh 7 Hnh 8 Hnh 9 Hnh 10 Hnh 11 Hnh 12 Hnh 13 Trang thit b cn thit cho vic xy dng mt phng tiu bn v mu vi sinh vt...11 Ngun thng tin gip cho vic xy dng danh mc dch hi xp theo th t tin cy 12 V d v danh mc dch hi trn cy u ...................................................................12 Thng tin cn thit cho mt h s dch hi, c xy dng da trn ISPM 8 ...........14 Dng c thng dng ly mu bnh .........................................................................17 L lm kh mu nghi ng nhim virus. .........................................................................19 V d mt nhn ly mu c s dng ti phng lu gi mu bnh BRIP ...............22 Mt ct nghing ca bung mu nm phn lp c nh sng en.........................33 Cch vch phn lp vi khun trn mt agar. ................................................................35 Khay Whitehead dng tch tuyn trng t t v cy ...........................................37 Dng phu Baermann tch tuyn trng t t v cy bnh...................................37 Tch tuyn trng t m bnh bng phng php phun sng ...................................39 V d mt nhn dn trn gi tiu bn s dng ti phng tiu bn BRIP....................45
Hnh 14 Cc gi tiu bn Bo tng mu bnh Ustilaginales Vnky (HUV) v Bo tng bnh cy ca QDPI&F (BRIP) ..........................................................................................................48 Hnh 15 Hnh 16 Hnh 17 Hnh 18 Hnh 19 Triu chng bnh virus (t tri qua phi): vng bin mu, m vng v khm l ..65 Hnh thi mt trc ca mt s nhm tuyn trng ......................................................68 Mun Catalogue ca KE EMu database ....................................................................71 a ch Internet v trang ch ca hai database phn loi tin cy..............................72 Vt b ca nhn trong cc git nc ng trn np a Petri ....................................75
DANH MC BNG
Bng 1 Mt s triu chng bnh thng gp .............................................................................16
M U
Tm quan trng ca vic lu gi tiu bn sinh vt, bao gm c cc mu thc vt b bnh v cc vi sinh vt gy bnh c ni n trong hng lot bi bo trn cc tp ch khoa hc. Trong mt bi c nhan Trch nhim tng h ca phn loi nm hc v bnh cy, Walker (1975) trch dn t nht 18 ti liu tham kho v chnh tc gi trong nhn mnh tm quan trng ca phn loi hc trong sinh hc ng dng cng nh tm quan trng ca cng tc phn loi nm, bao gm nm hc ni chung v bnh cy ni ring. Nhng bi bo ny c nh hng ti vic tr gip pht trin cc bo tng bnh hi v phn loi hc hay khng? Cu tr li c l l khng, ly dn chng t thc t Australia, hu ht cc bo tng mu bnh Australia u do cc c quan ca Chnh quyn cc bang ti tr. Nguyn nhn ti sao chnh ph li lng l trong vic u t vo cc bo tng (phng) tiu bn sinh hc trong khi chng l nn tng cho nhng nghin cu v phn loi cn ang l vn gy tranh ci. C l vn l ch nhng nh phn loi hc khng ch n nhu cu ca nhng ngi c kh nng s dng chngnhiu nh phn loi hc thng khng ngh n nhu cu v cng vic ca cc nh sinh hc ng dng trong c cc nh bnh cy, thm ch mt s cn c xu hng tch mnh ra khi nhng lnh vc nghin cu v thc hnh sinh hc rng ln hn (Walker, 1975). Cng vi s ra i ca T chc thng mi th gii nm 1995 v nhng iu lut ca t chc ny p dng cho hng ha nng nghip, tnh trng sc khe cy trng tr thnh mt vn chnh tr thng mi ln. Chu p lc t nhiu thnh phn khc nhau, cc Quc gia ang phi s dng cc iu khon ca Hip nh kim dch ng thc vt (Hip nh SPS) 1 ti a ha li th cnh tranh hay ni cch khc l thc y m ca nhng th trng trc y b ng li do nhng nghi ng v vn kim dch v loi tr vic nhp khu nhng hng ha c nguy c nh hng xu ti cc ngnh sn xut nng nghip trong nc. Da trn nhng c s khoa hc v nh gi nguy c dch hi, Hip nh SPS a ra iu kin bo v cc ngnh sn xut nng nghip trc s xm nhp ca dch hi 2 t ngoi vo, ng thi to iu kin cho s pht trin ca thng mi ha nng sn. Hip nh SPS cho php cc thnh vin qun l thng mi i vi hng ha nng nghip da trn tnh trng sc khe cy trng v an ton thc phm. Tuy nhin cc gii hn ra phi r rng v phi c bng chng k thut. 1.1 NHNG TRCH NHIM MANG TNH TON CU
Cng c Quc t v bo v thc vt (IPPC) v Hip nh SPS quy nh: cc nc xut khu phi c trch nhim cung cp cho cc nc nhp khu mt danh mc dch hi c kh nng i theo hng xut khu. C th nh sau: IPPC quy nh cc nc c hng ha xut khu phi cung cp cc thng s k thut v sinh hc hp l cn thit cho vic nh gi nguy c dch hi
Bin php kim dch ng thc vt (trong hip nh SPS) l cc tiu chun v quy nh ca mt Quc gia v cc vn nh: s ln tp vi sinh vt, c t, kim loi nng, tn d thuc bo v thc vt trong thc phm, sinh vt hi, c di. 2 nh ngha dch hi c s dng ch cc ng vt chn t, vi sinh vt v tuyn trng gy hi cho thc vt v sn phm thc vt.
to iu kin cho vic xc nh nhng thng tin c th v tnh trng dch hi ca mt sn phm chun b xut khu. 3 4 iu khon 6.3 trong Hip nh SPS ghi r Nu cc thnh vin c hng ha xut khu tuyn b rng nhng khu vc trong lnh th ca h l nhng khu vc khng c hoc t su bnh th s phi a ra cc bng chng cn thit chng minh mt cch khch quan cho cc thnh vin nhp khu rng khu vc khng c hoc t su bnh. thc hin c iu ny, cc thnh vin xut khu s phi to iu kin cho cc thnh vin nhp khu thanh tra, kim tra hay thc hin cc th tc lin quan nu h yu cu. Ph lc B, mc 3(b) trong Hip nh SPS ghi r mi thnh vin u phi bo m rng mi thc mc hp l m cc thnh vin i tc a ra u phi c tr li tha ng v phi cung cp cho i tc cc ti liu lin quan n: (b) cc bin php phng tr, gim st, x l sn xut v kim dch; kh nng khng thuc ca dch hi v cc th tc c p dng trong lnh th . p ng cc quy nh trn, c th thc hin cng tc nh gi nguy c dch hi v a ra cc iu l v kim dch thc vt vi mc ch ngn nga s xm nhp v ly lan ca vi sinh vt gy hi, cc nc cn phi duy tr cng tc lu tr h s dch hi. Theo Tiu chun Quc t v bin php kim dch ng thc vt (ISPM) s 8 5 , vic cung cp h s dch hi tin cy v vic xc nh tnh trng dch hi l khng th thiu trong hng lot cc hot ng c cp trong IPPC cng nh cc nguyn tc c cp trong ISPM s 1: Nguyn tc kim dch thc vt trong thng mi Quc t. ISPM 8 ghi r: Tt c cc nc u c th s dng cc thng tin v tnh trng dch hi cho cc mc ch sau: Cc mc ch nh gi nguy c dch hi; Lp k hoch cho cc chng trnh qun l dch hi trong phm vi mt quc gia, trong khu vc hay trong phm vi Quc t; Xy dng cc danh mc dch hi ca Quc gia; Xy dng v duy tr cc khu vc sch su bnh. pht huy ht quyn li trong t do thng mi ca WTO, cc nc cn phi tun th mi iu kin trong Hip nh SPS do IPPC v WTO ra. Nu nh dch v kim dch thc vt nhm mc ch nh gi nguy c ly lan dch hi trong hng ha thng mi th vic xy dng c s h tng tt, lm nn tng cho cng tc bo v thc vt l v cng cn thit.
Cng c Bo v thc vt Quc t, 1997: iu VII: Hp tc Quc t: 1. Cc t chc tham gia hp ng phi hp tc cht ch trong phm vi c th thc hin mc tiu ca Cng c, c bit l c) hp tc trong phm vi c th cung cp cc thng tin k thut v sinh hc cn thit cho vic nh gi nguy c dch hi. 4 ISPM 11, nh gi nguy c dch hi i vi dch hi kim dch, 1.2 Thng tin: Vic cung cp thng tin chnh thc lin quan n tnh trng dch hi l quy nh bt buc theo IPPC (iu VIII.1c) m cc i tc phi thc hin (iu VIII.2). 5 ISPM 8, Xc nh tnh trng dch hi trong mt khu vc, IPPC, FAO, Thng 11, 1998.
1.2
TNH TRNG LU GI MU SINH HC CC NC ASEAN
Nm 2001-2002, T chc h tr pht trin Quc t Australia (AusAID) ti tr cho cng vic bc u ca chng trnh vi mc ch nh gi li tnh trng lu gi sinh vt hi v mu tiu bn bnh cy cc nc trong khi ASEAN. Cng vic ny bt ngun t mt quyt nh c thng qua ti cuc hp ln th hai ca y ban iu hnh Hi thit lp v iu hnh mng li khu vc ASEAN (ASEANET 6 LOOP) t chc nm 2000. Cc i biu ti cuc hp tn thnh kin v vic xy dng bo co v cc bo tng (phng) lu gi sinh vt cc nc thnh vin. Tc gi ca cc bn bo co v tnh trng lu gi tiu bn dch hi cc nc ASEAN u nhn xt rng trong phm vi d ln hay nh, khng mt nc no trong khu vc c kh nng m t y v tnh trng sc khe nn nng nghip ca mnh. Vn l ch, trong phm vi ln hn, s lng tiu bn bnh cy trong cc phng mu bnh trong khu vc qu nh. S lng tiu bn cn trng trong cc phng lu gi tiu bn ln hn nhiu so vi s lng tiu bn bnh cy. Cc nh cn trng hc nhn chung cng thng tho vi cng vic bo qun tiu bn v qun l mu hn l cc nh bnh cy. 1.3 TM QUAN TRNG CA VIC LU GI H S TIU BN
Cc ngun thng tin v s tn ti ca mt sinh vt hi rt nhiu v sn c vi tin cy khc nhau. Tuy nhin, trong phm vi thng mi Quc t, h s lu tr da trn cc tiu bn c xc nhn trong cc phng mu t tiu chun l ngun thng tin ng tin cy nht v tnh trng sc khe thc vt ca mt nc. H s dch hi bao gm mu tiu bn v d liu i km tiu bn gm c: a im ly mu, ngy ly mu, ngi ly mu, k ch v c tnh ca vi sinh vt hi. Cc phng mu bnh hot ng tt bao gi cng c rt nhiu mu khc nhau ca cng mt loi t cc k ch khc nhau, t cc khu vc a l v sn xut nng nghip khc nhau. Cc mu tiu bn c th c kim tra li xc nh li kt qu gim nh hoc c c cc thng tin chnh xc hn v iu kin ly mu v v phn b ca bnh. Mt khc, cc bo co c xut bn (cng b) m khng c mu tiu bn bnh lm chng u khng c gi tr v cn l mt cn tr cho thng mi nng nghip. Cc bo co khng ng s tht s c th gy kh khn, tn thi gian v tin ca bc b yu cu ca i tc thng mi sau ny. Tiu bn v cc mu vt khc trong phng mu sinh hc l v kh c lc trong vic tr gip tip cn th trng v gii thch cho vic thc hin cc bin php ngn nga s xm nhp ca sinh vt hi t ngoi vo.
ASEANET l Hi thit lp v iu hnh mng li BioNET Quc t trong khu vc ng Nam , y l mt t chc hot ng da trn s hp tc trong khu vc tng cng s t lc trong phn loi hc sinh vt.
1.4
XY DNG V PHT TRIN CNG TC LU GI MU BNH CY CC NC ASEAN
Nu cc nh khoa hc v nhng ngi cng tc trong lnh vc bnh cy hiu c s cn thit phi gi mu bnh vo cc phng th nghim c ch nh th vic xy dng cc phng mu bnh cc nc ng Nam s d dng hn. Chnh ph Australia u tin tr gip t chc cc hi tho nh bao gm cc nh bo v thc vt gii thch tm quan trng ca vic lu gi mu sinh hc trong mi lin quan vi thng mi v vai tr ca cc nh bo v thc vt trong vic pht trin cng tc ny. Thm vo , cc chng trnh o to dnh cho nhng ngi lm cng tc bnh cy cng c t chc bo m rng h bit cch bo qun mu v vn chuyn mu ti cc phng lu gi c ch nh. Cun cm nang ny cha ng nhng thng tin cn thit gip cho tt c nhng ngi lm cng tc bnh cy thc hin trch nhim ca h trong vic xy dng v pht trin bo tng Quc gia v mu bnh cy v mu vi sinh vt hi, cng nh cung cp cc ti liu cn thit cho nhng ngi qun l bo tng.
2
2.1
LU GI MU BNH HI THC VT
BO TNG MU
Bo tng mu l ni lu gi cc mu tiu bn sinh hc cht, kh, c p hoc cc mu thc vt v nm c x l bo qun di hn, tt c cc mu tiu bn u phi c thng tin i km. a s bo tng mu sinh vt ch dnh mt phn nh lu tr tiu bn nm v bnh cy trong khi ch mt s t bo tng mu chuyn lu tr nm v bnh cy. Thc t cc bo tng bnh cy u c chc nng nh hai bo tng, l lu tr tiu bn thc vt b bnh v lu tr vi sinh vt gy bnh nh nm, vi khun, virus, viroid, tuyn trng, phytoplasma v vi sinh vt c dng vi khun k sinh ni bo. Bo tng nm v cc thng tin c lu tr trong bo tng l i tng s dng ca cc nh phn loi hc, cc nh nm hc, cc nh bnh cy, cc nh khoa hc v bo v thc vt, cc cn b kim dch, cc nh su tm sinh vt v nhng ngi hoch nh chnh sch trong rt nhiu lnh vc bao gm c vic duy tr an ninh sinh hc v a dng sinh hc. Tt c cc bo tng c chnh thc cng nhn trn th gii u c tn vit tt, v d Bo tng Bogoriense (BO) v bo tng CABI Bioscience, Trung tm UK (IMI). Mi tiu bn trong mt b su tm u c nh s th t, ng trc cc s th t l tn vit tt ca bo tng. Nu bit s truy nhp ca mt tiu bn ta c th tm ra ni lu gi tiu bn vi s tr gip ca cun Danh mc tra cu cc bo tng (Index Herbariorum). Cun sch cha ng thng tin v hn 3.000 bo tng trn th gii v hn 9.000 nhn vin lm cng tc lu gi trong cc bo tng ny. Danh mc tra cu ny c th c truy cp qua mng internet qua a ch [http://sciweb.nybg.org/science2/IndexHerbariorum.asp] v cc thng tin c th c tm kim t cc thng s nh: tn c quan, t chc, tn vit tt, tn nhn vin, chuyn ngnh nghin cu. Cun danh mc tra cu Index Herbariorum l kt qu ca mt d n hp tc gia Hip hi phn loi thc vt Quc t v Vn thc vt New York. 2.2 B SU TP VI SINH VT
B su tp vi sinh vt s lu gi cc mu nm v vi khun cn sng trng thi n nh cho n khi c s dng nhng ln sau. C nhiu cch bo qun mu vi sinh vt khc nhau t cch lin tc cy truyn cho ti nhng phng php lm gim hoc gin on qu trnh trao i cht. Hip hi lu gi mu vi sinh vt th gii (WFCC) [www.wfcc.info] ng mt vai tr v cng quan trng trong vic la chn, duy tr, mc chnh xc v s phn b ca cc mu vi sinh vt cng nh mu t bo sinh vt. Mc ch ca lin on l thc y v h tr vic xy dng cc b su tm mu vi sinh vt v cc dch v c lin quan, l cu ni thit lp mng thng tin gia cc trung tm lu gi v nhng ngi s dng bo m s tn ti lu di ca cc trung tm ny. WFCC xy dng mt h thng d liu mu vi sinh vt vi phm vi ton cu. H thng c s d liu ny c duy tr ti Vin di truyn Quc gia Nht Bn v lu tr thng tin ca gn nh 500 b su tp t 62 nc khc nhau. Cc thng tin c lu gi bao gm tn cc t chc, cch qun l, dch v v nhng dn liu khoa hc ca cc b su tp. H thng c s d liu ny hnh thnh mt ngun thng tin quan trng cho tt c 10
cc hot ng lin quan n vi sinh vt v cng ng vai tr trung tm cho cc hot ng v d liu ca cc thnh vin WFCC. 2.3 XY DNG B MU BNH CY
Cc tiu bn c lu gi trong bo tng mu v cc b su tp vi sinh vt l hon ton khc nhau. Bo tng mu lu gi cc tiu bn cht trong khi b su tp mu vi sinh vt lu gi cc vi sinh vt cn sng. Cc nh bnh cy cn phi lm vic vi c hai loi mu (mu cy cht v mu vi sinh vt cn sng). Thng thng mi bo tng bnh cy u c lu gi mt b su tp vi sinh vt. mt s ni, hai nhm khc nhau hot ng trong cng mt t chc hoc thm ch hai t chc khc nhau trong cng mt Quc gia m trch hai b su tp ring r. Gii php l tng nht l bt c vng no hay mt Quc gia no cng nn c mi quan h cht ch gia bo tng mu bnh v b su tp mu vi sinh vt gy bnh. Nhng trang thit b c bn cn thit cho mt phng mu tiu bn v mu vi sinh vt hi c a ra Hnh 1. Cc trang thit b vn phng c th c dng chung cho c hai v d nh chng trnh qun l d liu tiu bn.
Phng mu tiu bn Phng chng cn trng chuyn dng My iu ha khng kh (20-23C) My ht m (40-60%) Gi, t gi mu (kim loi) Chung bo chy Thit b qun l My tnh, my in, my qut nh Phn mm c s d liu Knh hin vi Knh lp soi ni Knh hin vi in t qut My nh k thut s (gn c vo knh hin vi) Gi mu, nhn, bn ch thch (giy khng c axit) Th vin in t, in thoi, hp th T cha Phng mu vi sinh vt Bung cy v trng Ni hp T lnh T (lnh su) Tup gi mu My lm lnh kh/T cha nit lng Gi
Hnh 1
Trang thit b cn thit cho vic xy dng mt phng tiu bn v mu vi sinh vt
11
2.4
DANH MC DCH HI
Danh mc dch hi l tp hp cc sinh vt gy hi trn mt k ch c th ti mt nc hoc mt khu vc nht nh. Ngoi nhng thng tin lin quan n tnh trng sc khe cy nng nghip, cy lm nghip, ngun gc (bn x hay du nhp), danh mc dch hi c tm quan trng c bit, nht l i vi cc nc ang tm kim th trng nc ngoi xut khu hng ha. Nhng danh mc dch hi ng tin cy nht l nhng danh mc c xc nh v c mu tiu bn i km, nhng danh mc dch hi c xy dng trn c s cc bo co, ti liu c xut bn m khng c tiu bn i km c coi l khng c tin cy cao. Mc tin cy cn ph thuc vo k nng ca ngi ly mu v ngi gim nh, phng php gim inh v danh ting ca tp ch ng ti. Nhng bi m tc gi l cc nh phn loi hc v c xut bn trn cc tp ch chuyn ngnh th gii chc chn s ng tin cy hn cc bi khng c ng trn cc tp ch chuyn ngnh (Hnh 2).
Ngun thng tin sp xp theo tin cy 1. 2. 3. 4. 5. B su tp dch hi thuc B Nng nghip, cc trng, vin nghin cu. Ti liu tham kho ch yu: cc tp ch khoa hc, cc bi bo cng b cc cng trnh nghin cu, sch, bo co kim dch, bo co t cc c quan v sc khe thc vt v kim dch thc vt. Ti liu tham kho th yu: trch yu bo v thc vt ca CABI. Ti liu lin quan khc: cm nang, ti liu hi tho, nh gi nguy c dch hi. Cc ngun thng tin: tho lun vi cc chuyn gia trong v ngoi nc, cc bi bo ph thng, thng tin qua mng (internet).
Hnh 2 Ngun thng tin gip cho vic xy dng danh mc dch hi xp theo th t tin cy
Cc thng tin c a vo danh mc dch hi ph thuc vo nhu cu ca ngi s dng. Nhng thng tin g l cn thit? Mc ch s dng thng tin? Hnh 3 l v d v mt danh mc dch hi bao gm tn y ca cc vi sinh vt gy bnh trn 1 k ch v tn thng gi ca bnh.
CARICACEAE Carica papaya L. (u ) Alternaria tenuis Nees m v qu. Ascochyta caricae Pat. m en. Botryosphaeria rhodina (Berk. & M.A. Curt.) Arx Thi qu. Colletotrichum acutatum J.H. Simmonds Thn th trn qu. Corynespora cassiicola (Berk. & M.A. Curt.) C.T. Wei m l. Glomerella cingulata (Stoneman) Spauld. & H. Schrenk m qu chn. Macrophomina phaseolina (Tassi.) Goid. Tht ngang thn. Phytophthora cinnamomi Rands Thi r. Sclerotium rolfsii Sacc. Cht rp cy con. Thanatephorus cucumeris (A.B. Frank) Donk Cht rp cy con. Verticillium dahliae Kleb. Cht ho. Hnh 3 V d v danh mc dch hi trn cy u
12
Danh mc dch hi c xy dng t cc cng tc: Quan st; Ly mu; Bo qun mu trong iu kin ph hp; iu tra c k hoch tt c cc nng sn v cc khu vc c lin quan; Hp tc vi cc t chc khc; Vic lu gi cc b su tp phi p ng cc tiu chun Quc t v kim dch thc vt (tiu chun 8) (ISPM 8). T xa n nay, vic xy dng danh mc dch hi thng gp nhiu kh khn do vic tip cn thng tin t cc phng tiu bn ri rc cc c quan, t chc khc nhau trong c: Cc B Nng, Lm nghip trc thuc Quc gia; Cc bo tng lch s t nhin; Cc vin nghin cu nng nghip chuyn ngnh; Cc nh khoa hc lm cng tc nghin cu v cc vin hn lm. Cc thng tin lu tr trong cc phng tiu bn bnh cy c tm quan trng c bit. V vy, vic xy dng h thng c s d liu in t ngi s dng c th truy cp d dng l vic lm ht sc cn thit. H thng cng ngh thng tin hin nay li sn c, to iu kin cho vic tm tiu bn v thng tin lu tr mt cch nhanh chng v thun li. Chi ph cho phn mm thp, tuy nhin chi ph cho vic np thng tin v d liu li tn km hn. Vic np c s d liu l vn kh ph cp trong giai on u ca vic xy dng h thng c s d liu, v vy cc c quan lm cng vic ny i hi phi lm vic lin tc v cp nht thng tin thng xuyn. Mt vn ng lu tm na l s lung v cht lng ca cc thng tin c bn. a s cc ni, cng vic gim nh, phn loi chim phn ln bao gm vic kim tra li cc kt qu gim nh v ghi chp trc y cng nh gim nh cc mu gi n m cha c phn loi. Cc nc cn phi thit lp mt h thng tiu chun d liu ti thiu cho cc h s dch hi p ng cc tiu chun Quc t do IPPC (ISPM 8) ra. ISPM 8 lit k cc thng tin c bn cn thit xy dng mt h s dch hi (Hnh 4).
H s dch hi bao gm nhng g? 1. 2. 3. 4. 5. 6. Tn khoa hc ca dch hi (chi, loi, di loi) Trng thi hoc giai on ca vng i Nhm phn loi Phng php phn loi (bao gm c tn ngi phn loi) Ngy ly mu (bao gm c tn ngi ly mu) Thng tin chi tit v ni ly mu a. a im (thnh ph & bang, huyn, tnh) b. Nc c. Thng tin v GPS (kinh & v ) 7. Tn khoa hc ca k ch (chi, loi, di loi)
13
8. Tnh trng b hi ca k ch 9. Tnh trng ph bin ca bnh 10. Ti liu tham kho
Hnh 4
Thng tin cn thit cho mt h s dch hi, c xy dng da trn ISPM 8
Hu ht cc nc khng c trung tm ln lu gi cc thng tin v tnh trng sc khe cy trng bi v cc phng lu gi mu v h s dch hi thng phn b ri rc rt nhiu cc c quan khc nhau. iu ny nh hng ti s nht qun ca thng tin lu gi v cht lng thng tin lu gi v mi ni c s u tin khc nhau i vi i tng lu gi. Cng vi s pht trin gn y v cng ngh thng tin, chng ta c th khc phc c tnh trng tn ti ny. Cng ngh lu tr c s d liu bng h thng my tnh to iu kin cho cc h thng qun l d liu cc a im xa nhau c th ni kt c vi nhau, v vy m tt c cc im u c th truy cp c h thng mng c s d liu. C th ni rng s pht trin ca cng ngh gip cho vic xy dng mt h thng c s d liu duy nht cp Quc gia hoc cp khu vc, thng qua h thng ny thng tin c th c cp nht thng xuyn cp c s. H s bnh hi c tip cn thng qua mt trang ch trn h thng internet v mt s thng tin ch nhng ngi lin quan mi c th truy cp c (thng qua m kha bo v). Thng qua h thng ny, ngi s dng c th cung cp cc thng tin lin quan n danh mc loi, phn b v k ch. Mt h thng c s d liu c kh nng thu thp v tng hp d liu v sc khe cy trng t rt nhiu t chc khc nhau vi iu kin l: Cc c quan lm cng tc lu gi mu sinh vt hi phi lu gi h s mu bnh trong cc h thng c s d liu in t kt ni vi mng internet.; C kh nng pht trin v iu chnh cc 'gateway' (cng) hoc 'broker' chuyn dng m cc phn mm cn kt ni vi cc ngun thng tin c s d liu khc nhau; Cc c quan phi sn lng chia s thng tin v cam kt duy tr phng lu gi ca h nh mt thnh vin ca h thng mng d liu (bao gm c vic p ng cc tiu chun Quc t).
14
3
3.1
XY DNG B SU TP MU BNH
NGUN MU
Ngun tiu bn ch yu trong cc phng mu bnh cy c cung cp bi cc nh bo v thc vt lm vic ti cc phng th nghim chn on bnh hi thuc cc vin v cc c quan nghin cu trc thuc B. Cc mu bnh, sau khi c thu thp t cc cuc iu tra ngoi ng, thng c a n cc phng th nghim gim nh. Ti y, cc nh khoa hc s xem xt c nn lu li nhng mu bnh ny hay khng. Bt c mu bnh no i din cho 1 vi sinh vt ln u tin c pht hin ti mt a im mi hay trn mt k ch mi u phi c gi n mt trong cc phng lu gi mu bnh c uy tn. a s phng lu gi mu bnh u c i ng nhn vin c th xc nh c cc nhm vi sinh vt hi khc nhau. Cn b v sinh vin ti cc trng i hc v cc c quan nghin cu cng l nhng ngi thng cung cp mu cho cc phng lu gi mu bnh cy. Hn na, tt c cc cng trnh nghin cu khi c xut bn trn cc tp ch, sch u phi lit k t nht tn mt phng lu gi mu bnh ni h gi mu n. Nhng mu tiu bn ny c s dng kim tra v nhn dng cc vi sinh vt hi thu thp t cc cuc iu tra v cc cng trnh nghin cu v sinh thi, dch hi, di truyn tin ha, hnh thi, phn t. Ngoi ra, s lng mu cng c tng thm khi c cy truyn v lm kh trao i gia cc phng lu gi mu. Vic tng, biu, mua bn mu cng l mt hnh thc lm tng s lng mu trong mt phng lu gi mu. S chuyn nhng s hu gia cc phng lu gi mu do khng kh nng duy tr cng l mt vic ng khch l bo v cc b su tp mu bnh c gi tr. 3.2 THU THP MU BNH NGOI NG RUNG
Hu ht cc mu tiu bn trong mt phng lu gi vi sinh vt hi u c thu thp t ng rung hoc mi trng ngoi t nhin. Cc cy bnh u c xc nh nh cc triu chng v du hiu c trng. Triu chng l nhng thay i bn ngoi ca cy hoc cc b phn ca cy b nhim bnh m mt thng c th nhn thy c (Bng 1). Triu chng xut hin do thc vt b suy gim kh nng quang hp, sinh sn, ht nc hoc trao i cht. Triu chng
Thn th Mui en Cht li Lot Cht rp cy con
M t
Vt hoi sinh mu en do Colletotrichum gy ra Cc tn nm k sinh en v dy c(Meliolales), thng xut hin trn b mt l cy nhit i. M bnh b cht, lan rng rt nhanh. Vt hoi sinh, thng lm xung trn thn g, cnh hoc r. Cy con b gy v thi im tip gip vi mt t trc hoc ngay
15
Triu chng
M t
sau khi ny mm do nm Pythium hoc Rhizoctonia gy ra.
Kh cnh Sng mai
Rng l, cht cnh thm ch cht ton b cy Phn mu hi trng xut hin trn b mt l v thn cy do s xut hin ca bc bo t v bo t ng ca cc loi thuc b Peronosporales. Hin tng m cy ch sinh trng nhanh v khng bnh thng, ko di t gn, c bit l trn l v hoa Chi b phn ly thnh tng b chi nh cong hoc xon. Hin tng sng ln hoc to thnh u khng bnh thng Chy nha t m k ch. Cc vt thng hoc m bnh xc nh. S bin i mu sc tng khong xanh m, nht trn l. y l triu chng ca rt nhiu bnh do virus gy ra. Hoa bin tnh thnh dng cu trc ging nh l. Hin tng cc si nm, cnh bo t phn sinh v bo t phn sinh xut hin trn b mt cy c dng bt mu trng, nm phn trng thuc b Erysiphales. Cc bc mng nc, khi v gii phng ra nm. Cc nt sng xut hin r do nhng loi tuyn trng nht nh (Meloidogyne) gy nn. M thc vt b mm v phn r do enzyme ca vi sinh vt hi sn sinh ra (c th cng, mm, kh, t, en hoc trng). Cc khi bo t do nm g st thuc b Uredinales sn sinh ra. Khu vc b bnh sn si, th rp, ging nh c mt lp v cng. M trng ging nh b gii nc nng. Triu chng bnh trn l, cc m gia vt bnh ri rng to thnh nhng l hng trn l cy. Cc khi bo t trn l, trn thn v hoa do nm than en (Ustilaginomycetes) gy ra. Cc m mu en trn l v thn ca nm hoi sinh b mt (thng l Capnodiales) sng nh cc cht tit ra t cn trng (thng l rp). Cc b phn ca cy b bin i thnh mu xanh, c bit l hoa. Hin tng cy mt tnh trng, cc b phn ca cy r xung. S phn ly mnh ca chi v mm rt gn nhau hoc ti cng mt im.
Bin dng l, hoa Bin dng chi Nt sng Chy gm Vt m Khm Ha l Phn trng
Mn si Nt sng Thi G st So Vt bng Thng l Than en Mc en Lc ha Ho Chi rng
Bng 1 Mt s triu chng bnh thng gp
16
Du hiu ca bnh l s c mt ca cc vi sinh vt di cc dng khc nhau, v d nh dng qu th m mt thng c th nhn thy c. Mt s du hiu thng thy ca bnh nh sau:

a cnh, cnh bo t phn sinh, qu cnh: cc cu trc nm nh dng qu th sn sinh ra bo t. m: dng qu th ca nm m (nm r hoc nm m); Th si nm: khi si nm; Dch tit: dch lng v dnh tit ra t vt thng hoc cc l t nhin (kh khng, b khng); Si nm dng r: cc si nm tp hp b li ging nh si dy (thng c mu ti). THU THP V X L MU BNH
3.3
Vic la chn mu bnh cho d vi mc ch gim nh hay nghin cu v phn loi hc u phi ht sc cn thn. Thi im thu mu cy bnh thch hp nht l giai on u hoc gia ca bnh, khi vi sinh vt hi vn ang trng thi hot ng. Nhng mu bnh b nhim qu nng thng khng s dng c v giai on ny vi sinh vt hi c th khng hot ng na, thay vo l cc vi sinh vt hoi sinh xm nhp vo cc cc m bnh cht hoi. V vy, phn lp vi sinh vt hi t cc mu bnh giai on ny thng rt kh. Vic la chn v tr ly mu trn cy bnh cng rt quan trng. Ngi thu thp mu bnh cn phi c kin thc c bn v triu chng bnh v nguyn nhn gy bnh bo m rng mu ly c c vi sinh vt hi. Trong mt s trng hp, triu chng c th xut hin v tr ny ca cy nhng vi sinh vt hi th c th c tm thy v tr khc. V d nh bnh ho: triu chng thng xut hin u tin trn l trong khi vi sinh vt gy bnh li k sinh trong h thng mch dn ca r v thn. Mt danh mc cc dng c cn thit cho iu tra thu thp mu bnh c trnh by hnh 5.
Dng c Ko ct cy Knh lp cm tay Ko GPS Bn Cp p mu Bay, xng nh Bt d Ca tay Ti liu tham kho Giy bo Ti giy Phong b Dao Nhn Ti nilon Bt ch Thng
Hnh 5 Dng c thng dng ly mu bnh
Mt s nguyn tc cn tun theo khi thu thp v x l mu bnh:
Nhn dng cy k ch. Nu cha xc nh c tn cy k ch th phi ly mu cy khe, c bit l hoa v qu. Ngi ly mu phi bo m rng mu cy khe ly v chnh l cy k ch. iu ny c bit quan trng khi ly mu bnh than en v mt s bnh ph hy hoa ca mt s loi trong h Ha 17
tho v nhng bnh ny thng pht trin trn nhng tp on k ch khc nhau.
S dng ti giy ly gi mu bnh. Khng bao gi s dng ti nilon gi mu ti v mu vn c th h hp, lm m ti to iu kin cho vi sinh vt hoi sinh xm nhp v pht trin nhanh, ph hy cc m thc vt. Ti nilon ch c th c s dng gi cc mu t trong thi gian ngn. ng, gi mu cn thn trnh va p v hi nc ngng t. B mt m s to iu kin cho vi sinh vt hoi sinh pht trin, khin cho mu bnh khng th s dng c. S dng bt ch vit nhn (mc s khng thch hp v c th b nhe khi m t). Xin cc giy php cn thit thu thp v vn chuyn mu bnh. mt s khu vc vic ly mu sinh vt c th b hn ch, v d nh cc vn Quc gia hoc cc khu vc do t nhn qun l. Vic vn chuyn mu t nc ny sang nc khc c th phi cn n giy php xut nhp khu v giy php kim dch. i vi l, thn v qu
3.3.1
Nn ly mu l c b mt kh ro, nu trong iu kin ma m, b mt l t th c th dng giy bo thm kh trc khi kp mu gia cc lp giy bo hoc cc loi giy thm nc khc (khng nn s dng giy n v khi t giy n c th tan r ra v kh tch chng ra khi mu l). Khi p v lm kh mu l, cn ch ri l ra sao cho khng trng ln nhau. Nu l dy v mng nc, cn thay giy bo hng ngy cho n khi l kh hn. Khi ly mu thn cy b bnh cn ly v tr bao gm c m khe v m bnh. Gi cn thn mi thn bnh vo mt t bo v chng rt d b xy st hoc gy khi c gi thnh mt b chung. Khi ly mu qu mng nc, tht qu nhiu cn chn nhng mu mi xut hin triu chng hoc triu chng ang giai on gia ca s pht trin. Cc vi sinh vt gy thi th yu v hoi sinh thng xm nhp qu giai on cui ca s pht trin bnh, gy cn tr cho vic gim nh vi sinh vt gy bnh. Gi mi qu bnh vo mt t giy bo ring r. Khng dng ti nilon gi mu qu. Bnh g st v nm than en Khi ly mu nm g st cn kim tra c 2 mt l tm bo t ng mu nu en v bo t h mu vng da cam. Nm than en thng ph hy cc b phn hoa ca cc loi trong h Ha tho. Xc nh ng tn k ch l iu kin cn thit gip cho vic gim nh nm than en, tuy nhin vic xc nh tn k ch i khi gp kh khn nu h hoa b ph hy. Cn ch gi mu bnh bng giy bo cn thn bo t nm g st v nm than en khng b ri ra ngoi. Bnh vi khun M bnh vi khun thng b phn r rt nhanh, v vy kh thu thp v vn chuyn mu bnh ti phng th nghim xa. t mu bnh vo ti giy v dng giy bo m 18
bc li trnh cho mu khi b kh. Nn gi mu ni mt m, trnh nh nng mt tri. i vi mu m l v tn li do vi khun, nn dng giy bo p li cho kh lu gi. Nhiu vi khun gy bnh c th sng st hng thng, thm ch hng nm trong cc mu kh nhit phng. Bnh virus Mu bnh cy nghi ng nhim virus sau khi thu thp nn bo qun tm thi trong cc l lm kh (Hnh 6). L lm kh c th c lm bng cch ly mt l nha, tinh th Clorua Canxi (CaCl2) khan vo n 1/3 l, dng bng ph ln ngn cch gia cc tinh th Clorua Canxi v mu bnh. Cch bo qun mu ny tt nht nhit 04oC, tuy nhin cng c th p dng nhit mi trng. Dng ko hoc li dao ct l. Nu l b bi bn hoc cn trng bm vo, c th dng nc hoc cn lau sch trc khi ct. Ct l thnh tng mu nh 3 - 5 mm, nn ly gn phn gia ca phin l, sau t 5 - 10 mu l vo mt l lm kh. Li ko hoc dao phi c kh trng bng cn hoc dung dch sodium hypochlorite (NaOCl) 10% gia cc mu khc nhau trnh b tp. Ngoi cch x l mu nh trn, c th gi mu bnh virus trong ti nilon cng vi mt t giy m v gi trong thng cho n khi a n phng th nghim. Bng cch ny, l cy vn ti, gi c sc trng cn thit.
np vn bng nha
mu l (3-5 mm) bng
Tinh th canxi clorua
Hnh 6
L lm kh mu nghi ng nhim virus.
3.3.2
i vi r v t
Thng thng cc m r hay cc cu trc vi sinh vt hi vng r thng rt mng manh. Khng nn nh cy v c th lm t r, khng ly c phn r hay vi sinh vt hi, gy kh khn cho vic gim nh. 19
Lc nh loi b phn t bm vo r, nu c th nn ra r nh nhng (trong trng hp nh kim tra tuyn trng th khng nn ra). Trong t c rt nhiu vi sinh vt hoi sinh xm nhp vo cc phn r cht hoc ang cht, cn tr vic phn lp vi sinh vt gy bnh. Khi loi b t khi r, khng dng bn chi hoc cc vt dng tng t v c th lm mt cc phn r quan trng cho vic gim nh. Bc r trong giy bo vn chuyn n phng th nghim. Nn ly mu t dng cho vic phn tch r hn v bnh sau ny. Tuy nhin, hn ch ch yu ca vic ly mu t l chng c th kh nng v cng knh trong trng hp iu tra ly mu nhiu im. Khi xem xt c nn ly mu t hay khng cng nn tnh n thi gian v khng gian cn thit cho vic x l mu t sau khi em v. Cc vi sinh vt hi trong t thng khng phn b ng u m c xu hng tp hp thnh tng nhm trong iu kin thch hp hoc xung quanh im xm nhim trn cy k ch. Cch tt nht l nn ly ngu nhin mt s mu t. Kinh nghim cho thy rng mu thu thp c cng nhiu th vic nh gi tng quan v bnh cng chnh xc. S lng mu t ly tng im iu tra c th khc nhau ph thuc vo iu kin thc t. Cch lm thng thng l ghi li s lng mu ly, sau trn ln vo nhau mt cch k lng ri t ly mu i din. Nu ly mu bnh tuyn trng, cn cn thn khi va p lm try xc tuyn trng nm trong t. Khng nn ly mu t qu t hoc qu kh. t ly mu nn su t nht 5-10cm so vi mt t v y l vng r cy nn vi sinh vt hi tp trung nhiu nht. Nu triu chng bnh tp trung thnh tng m trn khonh rung th nn ly 2 mu t ring r trn m rung b nhim nng v m rung khng th hin triu chng c th so snh. Mi mu t nn ly khong 250-300 g. Nu c th nn ly mu t c cha c r cy, r c th ln trong t hoc ring. i vi cy thn tho, ly khong 25-100 g r l ty thuc vo loi cy, v d i vi rau c th ly t r (khong 25 g) trong khi i vi cc loi cy c r to nh chui th nn ly nhiu hn (khong 100 g). i vi cy thn g c th phi o su ti 30 cm gn gc cy hoc o n khi nhn thy ng ranh gii gia m khe v m bnh. Mu t nn gi trong ti nilon v t nhn giy hoc nha vo bn trong ti (ghi nhn bng bt ch nu dng nhn giy). Nn mu ni mt m, trnh nh nng mt tri. Gi mu cn thn, gi mu phn tch hoc x l mu cng sm cng tt. Nu iu kin khng th gi mu i hoc x l mu ngay th nn bo qun mu trong t lnh ti 4 - 8 C trong vi ngy m khng s mu b hng. 3.3.3 i vi nm ln
Nm ln, c bit l cc loi thuc b Agaricales rt d tm khi iu tra nhng li l mt trong nhng nhm kh vn chuyn n phng th nghim nht. V vy, nn ch ly mt s mu cc giai on sinh trng khc nhau ca nm. Khng nn ly mu nm bng cch nh ln, trnh lm gy thn nm khi ly mu. Dng dng c o 20
nm khng lm hng phn gc. Nn dng giy bo bc tng c th ring r v t cn thn trong cc hp ng sao cho nm khng b nt. Mu nm ln cn phi c lm kh cng nhanh cng tt. Ty tng trng hp c th c th p dng mt trong cc cch sau: Sy trong t sy c qut thng gi (45C, qua m); Dng my sy qu bng in; Dng cc ngun nng lng khc nh: than ci, bp ga, n du, di nh nng mt tri. Vic ly du vt bo t, c bit l mu ca chng, c th tr gip rt nhiu cho vic gim nh, nht l i vi cc loi nm ln c bn m nm (vch tia). Cch ly du vt bo t nh sau: ct ly phn m nm v t ln mt tm ba mu trng trong 15 pht n mt vi gi (ch mt c bn m nm di). t mt chic hp khng (y ln trn) sao cho lung khng kh bn ngoi khng nh hng n cc du vt bo t. Nu nm c bo t mu trng th nn s dng ba mu en. Mu ca bo t cng c th c xc nh t cc du vt bo t, v ngc li, nh c mu bo t, du vt bo t cng hin ln r hn trn nn ba. 3.4 CCH GHI PHIU IU TRA MU BNH
Nhng mu bnh d tt n my nhng khng ghi phiu iu tra y th cng coi nh khng c gi tr. Cc thng s cn thit phi ghi li trn nhn i km mu bnh bao gm: Tn cy k ch v b phn cy b nhim bnh; a im chnh xc ni ly mu, x, th x/th trn, huyn, tnh, quc gia (kinh /v v cao nu bit). My nh v v tinh (GPS) l phng tin tt nht xc nh ta . GPS c th tm kim cc v tinh quay quanh qu o tri t. Vi 3 v tinh, GPS c th tnh ton chnh xc kinh v v . Vi 4 v tinh, GPS c th tnh c cao so vi mt bin. GPS xc nh c ta , v vy gip cho vic xy dng bn phn b ca vi sinh vt hi; Ngy ly mu; Tn ngi ly mu, nh s mu; Triu chng bnh v mc bnh (v d s cy b nhim bnh). Tt c cc mu bnh chuyn n phng mu u phi ghi nhn y (Hnh 7). Tn v a ch lin lc ca ngi a mu cng phi ghi li. Gii thch l do ti sao a mu n, v d vi mc ch gim nh hoc lu gi.
21
Hnh 7 V d mt nhn ly mu c s dng ti phng lu gi mu bnh BRIP
3.5
IU TRA V LY MU
Mc ch ca cc cuc iu tra l xc nh loi bnh v mc gy hi ca bnh n cy ch. iu tra bnh cn thit cho vic: Xc nh phm vi phn b v tnh trng dch hi trong mt khu vc. Trc khi tham gia cung cp hng ha cho cc th trng v xut khu, nhng khu vc ny phi c xc nh xem c bnh hi thuc i tng kim dch hay khng; Xc nh cc bnh quan trng v hng dn cch qun l dch hi gip cho nh hng cng tc bo v thc vt; Pht hin cc bnh ngoi lai v cc bnh mi xut hin; nh gi thit hi do bnh v xc nh tnh trng sc khe cy trng; Hiu bit v a dng dch hi; Xc nh k ch chnh; nh gi hiu qu ca cc bin php phng tr; Xy dng bo co v tnh trng dch hi hin ti v i chiu vi thit hi v kinh t; Pht hin thin ch t nhin. C hai dng iu tra: iu tra nh tnh v iu tra iu tra nh lng. iu tra nh tnh ch cn xc nh xem mt bnh hi no c xut hin hay khng. iu tra nh 22
lng xc nh c t l bnh v ch s bnh lin quan n mc thit hi cho cy k ch. iu tra phi chn im i din cho khu vc v ln bo m mc chnh xc. Ph thuc vo mc ch iu tra chun b thit b, dng c cng nh k nng iu tra. Lng thng tin thu thp v mc chnh xc cng ph thuc vo phm vi iu tra. Quy cch ly mu cng nn n gin, mang tnh i din v bo m tin cy. Da vo cc cuc kho st s b trc khi tin hnh iu tra c th gip cho vic c lng trc s lng mu ly v c tnh mc bnh hi vi cc s lng mu ly khc nhau. T vic so snh cc kt qu thu c trong cc ln kho st, c th la chn s lng mu ly ti thiu m vn cho kt qu tin cy v mc nhim bnh. Mt iu quan trng nn lu l d p dng phng php iu tra no cng nn tnh n s phn b ca bnh. Cy b bnh c th phn b ri rc, ng u hoc thnh tng cm. Ly mu theo phng php ngu nhin thng cho kt qu tt bi v rt nhiu bnh phn b thnh tng cm. Trong mt s trng hp, c th phi iu tra vi quy m ln hn v h thng hn. i vi cc cuc iu tra c th, cn phi thu thp thm thng tin ln k hoch iu tra t hiu qu cao nht. Cc yu t cn tnh n l thi gian tin hnh iu tra, giai on sinh trng no ca cy trng l thch hp nht cho vic th hin triu chng bnh. bit thm thng tin v cch tin hnh iu tra trong khu vc Chu Thi Bnh Dng, c th lin lc vi TS. Teresa McMaugh 7 ti Vn phng chuyn vin bo v thc vt cao cp (OCPPO), B Nng nghip, Thy sn v Lm nghip Australia (DAFF).
TS. McMaugh ang iu hnh vic xut bn mt cun cm nang v iu tra dch hi Chu Thi Bnh Dng (Guidelines for Plant Pest Surveillance in Asia and the Pacific), do Trung tm nghin cu nng nghip Quc t Australia (ACIAR) v T chc nghin cu v pht trin nng thn (RIRDC) ti tr.
23
PHNG PHP KIM TRA MU BNH
Vic gim nh mu phi bt u t cc c tnh v m m mt thng c th nhn thy c v kim tra di knh lp soi ni. Bc tip theo l dng lam knh v kim tra cu trc ca vi sinh vt hi di knh hin vi quang hc. Nu ngi gim nh cha bit vi sinh vt ny th nn o kch thc, v hnh v chp nh vi sinh vt nu c th. Nn lu gi nhng ghi chp, hnh v v nh ban u cng vi tiu bn bnh. 4.1 NHUM MU NM V VI KHUN
Hu ht nm v vi khun c th quan st c di knh hin vi bng cch t ln lam knh, nh mt git nc v dng lamen ph ln. C th s dng axit lactic lm mi trng c nh nm. Vic nhum mu thng p dng i vi cc cu trc nm khng mu sc. Hai ha cht nhum mu thng dng cho nm l thuc nhum bng xanh (cotton blue) v lacto-fuchsin.
Cotton blue (hay trypan blue) Cotton blue (trypan blue) Axit lactic Glycerol Nc ct 0,1 g 25 ml 50 ml 25 ml
Lacto-fuchsin Acid-fuchsin Axit lactic 0,1 g 100 ml
Vic quan st vi khun trong m cy bnh thng rt kh. C th dng dung dch Toluidine blue O 0,1% nhum mu vi khun. Ct mt mu nh mu bnh phn gia m khe v m bnh, t ln mt lam knh v nh mt git cht nhum mu. t lamen ln trn v quan st vi phng i 400 ln. Vi khun (nu c) thng di chuyn xung quanh phn m ct mp ngoi mu bnh. Vi khun c nhum thng c mu xanh nc bin m, m cy mu nht hn v hi pha mu xanh l cy.
Toluidine blue O Toluidine blue O Nc ct 0,05 g 50 ml
Vi khun c phn lm hai nhm chnh: Nhm c kh nng duy tr phc hp ca thuc nhum crystal violet v it m khng b ha tan trong cn ethanol (Gram dng) v nhm khng th duy tr phc hp ny (Gram dng). Nhum mu Gram gip cho vic gim nh cc c tnh ban u ca nhiu vi khun gy bnh cy. Ha cht v phng php nhum mu Gram nh sau: 24
Ha tan 2 g thuc nhum crystal violet trong 20 ml cn ethanol 95%. Ha tan 0,8 g amonium oxalat trong 80 ml nc ct. ln hai dung dch vo nhau.
Dung dch crystal violet Crystal violet Cn ethanol 95% Ammonium oxalate Nc ct 2g 20 ml 0,8 g 80 ml
Nghin tinh th it v mui kali iodide (KI) v ha tan trong nc ct, ng vo mt bnh kn, khuy u khong vi gi n khi ha tan hon ton.
Dung dch Lugol it It KI Nc ct 1g 2g 300 ml
Pha dung dch nhum mu safranin ban u bng cch ha tan 2,5 g safranin O trong 100 ml cn ethanol 95%. Pha long 1:10 dung dch ny bng nc ct trc khi s dng.
Dung dch nhum mu Safranin Safranin O 95% ethanol 2,5 g 20 g
Chun b dung dch cha cc t bo vi khun t mt khun lc ang pht trin (sau khi cy khong 24-48 gi) v nc ct. Dng que cy vi khun qut mt vt nh (khong 1 cm2) dung dch ln mt lam knh sch. kh trong khng kh sau c nh mu bng cch a i a li lam knh (mt c vi khun ln trn) vi ln qua ngn la n cn. Khng c lm cho lam knh qu nng. Bng cch c nh ny, vi khun s dnh cht vo b mt lam knh trong qu trnh nhum mu. Ngm lam knh vo crystal violet trong 1 gi sau dng vi nc ra nh nhng n khi khng thy dung dch mu b ra tri na. Ngm lam knh vo dung dch Lugol trong mt pht. Dng vi nc ra nh v thm kh. Di nh nhng ethanol 95% ln lamen trong vi giy (khng qu 39 giy) ra ht ha cht nhum ri thm kh. Kh nhum mu bng cch ngm lam knh trong safranin trong 20 giy. Ra sch bng nc sau thm kh. Quan st mu lam di knh hin vi vi phng i 1000 ln sau khi nh 1 git du dng cho vt knh du. Vi khun Gram dng c mu tm xanh trong khi vi khun Gram m c mu hng . xc nh mc tin cy ca phn ng Gram, c th dng hydroxit kali (KOH) kim tra li. Dng que cy (nn dng que tre hoc g) ly mt lng nh vi khun t 25
khun lc ang pht trin v trn u vi mt git dung dch KOH 3% trn mt lam knh. Nhc u que cy ln cch b mt ca lam knh khong vi centimet. Nu dch vi khun dng nht dnh, to thnh si khong 5 - 20 mm th vi khun thuc nhm Gram m. Nu dch vi khun ging nh nc, khng nht dnh th vi khun thuc nhm Gram dng. Cc si nht do vi khun Gram m to ra l do cc t bo b ph hy gii phng ADN c dng sn st trong nc. Ngc li, lp v t bo ca vi khun Gram dng li c kh nng chng chu vi KOH nn khng b ph hy v khng gii phng ra ADN. Phng php th ny ch nn p dng trong trng hp vn nghi ng kt qu ca phng php nhum mu. 4.2 KNH HIN VI QUANG HC
Knh lp soi ni ch c ch trong trng hp quan st mu bnh ban u. Knh hin vi c phng i ln ti 1000 ln rt cn thit cho vic quan st hnh thi ca vi khun v nm. gim nh nm, dng que cy hoc dao u nhn ly mt phn nh m c bo t v t vo mt git dung dch nhum mu, thng s dng lacto-fuchsin (0,1 g axit fuchsin v 100 ml axit lactic), hoc dung dch c nh mu nh axit lactic (100 ml axit lactic, 200 ml glycerol v 100 ml nc ct). Dng knh lp soi ni s gip cho vic ly bo t d dng hn. Nu mu cn quan st hi ln th c th dng lamen p nh cho mu phng ra. C th loi b bt kh bng cch a i a li lam knh qua la (trnh khng mui than bm vo mt di ca lam knh). Nu t lam knh qu nng s lm n hoc nt lamen. Phng php dn bng dnh rt c hiu qu i vi vic nh hng bo t kh (c chui bo t v hnh dng bo t). Ct mt mu bng dnh v dng panh gi bng dnh st vo b mt tn nm (hoc b mt l). Sau t ming bng dnh ln mt lam knh (mt dnh ln trn) c nh sn mt git dung dch nhum mu hoc dung dch c nh mu. Dng lamen ph ln trn v quan st di knh hin vi. Mt s nm c bo t dng chui rt mng manh, d dng ri ra di tc ng ca nhng lung khng kh rt nh. C th khc phc tnh trng ny bng cch dng lam knh cy nm nh sau: t mt que thy tinh gp li ln mt t giy lc thm nc di y mt a Petri. Ct mt mu agar khong 1 cm2 t ln mt lam knh tit trng ri t lam knh ny ln trn que thy tinh. Cy nm vo bn cnh ca ming agar ri t lamen ln trn. Sau vi ngy c th quan st hnh thi nm trn lam knh bng knh hin vi. p dng phng php ny c th quan st c cu trc ca nm nguyn vn khi ang pht trin. Sau , tch ming agar ra khi lamen v lam knh, t lamen ln mt lam knh mi v ngc li to thnh 2 mu quan st. i vi mt s vi sinh vt, mi quan h v khng gian gia cu trc qu th v m k ch hay v tr ca cnh bo t phn sinh trong qu th l nhng c tnh phn loi quan trng. Ct mu thnh lt mng c th quan st c cc c tnh ny di knh hin vi. t mu di knh lp soi ni, dng dao hoc my ct vi phu ct mu thnh lt mng. i vi cc mu ti hoc trong t nn dng my ct vi phu lnh. My ct vi phu lnh c gn mt tm nh t mu bnh ln, mu c th c lm lnh ngay trn mt tm bng nhit in hoc bng cht lng lm lnh nhit thp. 26
i vi vi khun, thng rt kh quan st ngay ti mu bnh. C th s dng Toluidine Blue O pht hin vi khun. Cng c th p dng phng php nhum mu Gram phn bit gia hai nhm vi khun khc nhau v mt ha hc, Gram dng v Gram m (t bo vi khun Gram dng c mu tm m trong khi t bo vi khun Gram m c mu ). C th tham kho thm v phng php ny chng 7, Gim nh vi sinh vt gy hi. duy tr tt cht lng ca knh hin vi quang hc i hi phi c bo dng. Nn bo dng knh hin vi nh k 6 n 12 thng mt ln. Trong mi trng nhit i m, nm c th mc lan vo vt knh v b mt cc b phn quang, nh hng n cht lng knh hin vi, b mt cc b phn ny c th b hng hon ton nu khng kim tra. Trong iu kin kh hu nhit i nn bo qun tt c cc knh hin vi trong phng c iu ha v my ht m. Bo qun knh hin vi trong phng c iu ha m khng c my ht m th khng gim c m cn thit ngn nga s pht trin ca nm trn mt knh. Nu khng c my ht m chuyn dng th c th bo qun knh trong t c ht m hoc ct vo hp bo qun knh nu khng s dng. Hp bo qun knh c lm bng g hoc nha, to c th cha knh hin vi v mt bng n in 25 W. Bng n c chc nng nh mt my ht m do ta nhit nng lm bc hi nc trong hp v gi hp lun kh ro. C h thng my nh cng nn bo qun trong hp nh vy. 4.3 CHP NH MU
Nu c th nn chp nh mu bnh (dng my nh thng hoc k thut s) khi ly mu trn rung iu tra. Chp nh trn rung ti thi im iu tra cung cp nhng t liu nh v triu chng bnh trong t nhin. Nn chp nhiu nh, sau chn la nhng nh tt nht v lu vo mt cun nht k c ch thch thng tin v mu bnh trnh ln ln gia nh ny vi mu bnh khc x l ti phng th nghim. Chp nh mu ti phng th nghim cho php loi b c nhng nh hng ca iu kin mi trng n hnh nh. Khi chp nh ti phng th nghim c th phi dng n nh sng n mc d nh sng n thng to thnh bng khi chp nh. Khi chp nh mu nn dng mu xm nht lm nn cho nh, mu en v mu trng c th lm cho mu bnh trong nh sng qu hoc ti qu. Hnh nh k thut s v triu chng bnh c u im l c th c gi n cc ng nghip mt cch d dng v khng b hng do nm mc hoc gim cht lng do thi gian (c bit l trong iu kin kh hu nhit i) nh phim nh. Ngoi vic s dng my nh k thut s, hnh nh k thut s cn c th c to ra bng cch dng my qut nh hoc phim dng bn, thm ch c th qut trc tip mu bnh. 4.3.1 Cch qun l v t tn file nh
My nh k thut s t ng t tn cho cc file nh, (v d IMG_001.jpg, DSCF0001.jpg). Nhng tn ny khng c ngha g khi chng ta mun tm nh trn my tnh. Dng cc phn mm qun l hnh nh (bn di) c th khc phc c 27
nhc im ny nh tnh nng i tn file nh. Nn s dng cc tn c tnh m t (v d thn th xoi.jpg). Nu s lng nh nhiu, nn nh s file nh sp xp cc nh thnh catalog. Nn bt u bng s c ch s khng trc, v d 001 thay cho 1 v phn mm my tnh khi xp th t thng 100 trc 99. Nu hnh nh minh ha triu chng bnh t mt mu tiu bn trong phng mu th nn dng tn truy cp tiu bn t tn file nh. Khng nn lu gi qu nhiu file trong mt th mc. Nu c qu nhiu file th s mt nhiu thi gian tm kim hnh nh mong mun. To cc th mc nh sp xp theo vi sinh vt, k ch, a im ly mu hoc thi gian ly mu. Mt trong nhng cch tt nht qun l cc hnh nh l s dng cc phn mm qun l nh chuyn dng. Cc chng trnh ny cho php sp xp cc hnh nh, m t chi tit, hin th ln lt cc hnh nh mt cch t ng, chn cc nh mong mun v a ln mng internet cng mt lc. C rt nhiu phn mm qun l nh c th s dng : a hnh nh t my nh vo my tnh; Hin th hnh nh; Catalog ha cc hnh nh; Chnh sa hnh nh; a cc hnh nh ln mng internet. Mt s chng trnh phn mm qun l nh ang lu hnh hin nay: Ablaze Image Manager, ThumbsPlus, Cumulus, Epsons File Factor, Piccolo, PhotoWallet, Polybytes Polyview, Thumber, PIE, ACDSee (PC v Mac), PicaView32, Image Fox, Graphic Workshop, Professional, GraphicConverter, Power Browser, IrfanView32, Photopage, Quisknailer, Compupic, Image Viewer, Photo Recall, Photo Explorer, EXIFRead, VuePrint, ImageAXS, iView Multimedia, Compupic Pro, Sony PictureGear, FlipAlbum, PictureWorks MediaCenter, JASC Media Center, Pictureshow, Qpict Media Organizer v DigiPicng s
28
PHNG PHP PHN LP NM, VI KHUN V TUYN TRNG TRONG PHNG TH NGHIM
Mu bnh sau khi em v phng th nghim c phn thnh cc nhm chnh. Nhm u tin bao gm cc mu bnh phn hy nhanh chng, v d nm ln c tht dy hoc cc mu bnh m vi sinh vt hi phi c phn lp t m cy. Nhm khc bao gm cc mu bnh c th thm mt thi gian m khng nh hng g, v d g st, than en. Cc mu thuc nhm ny c th kh bng cch p bo, sau vo t -20C trong 7 ngy, c th gim nh sau. Mu virus hi thc vt c th c bo qun tm thi trong cc l lm kh nh m t phn 3.3.1. Virus thc vt s khng c cp thm trong chng ny v chng khng c phn lp v lm sch (tch virus ra khi cc b phn cu thnh t bo) bng cc phng php thng thng trong quy trnh gim nh. Nm v vi khun thng phi c phn lp v nui cy trc khi c th gim nh. Cc vi sinh vt c kh nng pht trin hoi sinh (vi sinh vt k sinh khng bt buc hoc hoi sinh) c th c nui cy trn mi trng nhn to mc d khng phi loi no cng c nui cy d dng. Phn lp nm t m cy bnh bng cch cy nhng mu nh m bnh ln mi trng nhn to thch hp, v d mi trng agar nc ct (water agar) c trong cc a Petri kh trng. Nu vi sinh vt xut hin trn b mt mu bnh, dng que cy ly bo t trc tip t cu trc qu th v cy ln b mt mi trng. Nhiu loi nm hoi sinh v vi khun thng pht trin th sinh trn m cy b bnh. V vy, phi ht sc cn thn trnh phn lp cc vi sinh vt th sinh bng cc phng php kh trng. Tit trng b mt cc m cy bnh c th gip loi tr cc vi sinh vt hoi sinh thng pht trin trn b mt m bnh. Ly bng hoc giy n sch nhng cn ethanol 70% lau phn m bnh dng cy sau kh. Tt nht nn s dng cc mu knh hoc cc b mt cng, nhn ct mu cy bnh. Tit trng panh v dao bng cch nhng chng vo cn ethanol 95% v h qua ngn la n cn. Khng cn phi gi dng c qu lu trn ngn la v la c th lm hng chng. Cn rt d chy v vy phi ht sc cn thn khng dng c va h la qu gn l hoc cc cha cn. Tit trng que cy bng cch h cho nng trn la n cn. que cy ngui trc khi s dng. Kh trng b mt gip loi b vi sinh vt hoi sinh v to iu kin cho vi sinh vt gy bnh pht trin t do, khng b cn tr khi mu bnh c cy ln mi trng nhn to. Nhng hoc lau b mt vt bnh bng cn ethanol (70%) c th kh trng hu ht cc b mt m khng cn ra li cho ht cn v cn c th bay hi, nu cn c th h nhanh qua la n cn. Natri hypochlorite cng thng c s dng kh ph bin lm dung dch kh trng. Thuc ty trn th trng thng cha 10-14% Clo. C th pha long 10 % (dung dch pha long cha 1-1,4% Clo) v ngm mu khong 1-5 pht. Bo qun dung dch trong t lnh v qua thi gian dung dch s gim hiu lc. Thay dung dch Natri 29
hypochlorite 2-3 tun / ln. Dung dch hypochlorite c nhc im l c mi hc, nu b dy ra qun o thng li vt v lm hng qun o. La chn cch phn lp vi khun v nm nn da vo c tnh ca k ch v vi sinh vt hi. 5.1 PHN LP NM
Cc bc phn lp nm bnh t m cy bnh nh sau: 1. Ra mu bnh di nc my loi b t v bi bn; 2. Nu c nhiu vi sinh vt hoi sinh trn b mt vt bnh nn ra mu bng cn ethanol 70%. i vi mu cy thn g nn p dng cch kh trng ny; 3. Ct m vt bnh thnh mu nh ti im tip gip gia m bnh v m khe; 4. Nhng m bnh vo dung dch natri hypochlorite 1% trong cn ethanol 10% khong 1 5 pht (c th thay i nng ph thuc vo tnh cht m cy, v d mt s l cy c b mt rt xp, c th hp th dung dch kh trng lm cht vi sinh vt hi); 5. Ly mu ra khi dung dch kh trng b mt v ra li bng nc ct, sau lm kh mu bng cch t mu ln giy lc tit trng (nu c th nn trong t cy c h thng thi lc khng kh). Ct mu bnh thnh tng ming cy nh khong 2 x 2 mm sau t ln mi trng agar - nc ct hoc mi trng khoai ty - ng - agar. Vic mu kh trc khi cy c tc dng hn ch s pht trin ca vi khun ln tp. Khi t ming cy ln mi trng agar cn m v ng np hp Petri cn thn v nhanh trnh vi sinh vt ln tp t khng kh xung quanh; 6. Sau khi cy xong, t ngc a Petri trnh ng hi nc trn b mt mi trng cy. Hu ht cc nm bnh thc vt u pht trin tt 25C; 7. t c mu nm thun, khng ln tp c th dng phng php cy n bo t hoc cy nh si nm t cc tn nm mc ln t ln phn lp u tin. Phng php cy n bo t nh sau: Chun b dung dch bo t trong mt ng nghim cha nc ct tit trng ri dung dch bo t ln a Petri cha mi trng agar - nc ct (hoc mi trng khc ph hp) sao cho dung dch ph kn a. a Petri cha dung dch bo t trong bng ti nhit phng khong 24 gi. t a bo t di knh lp soi ni, dng que cy ct tng bo t ny mm v cy ln mi trng thch hp; 8. C th thay i t nhiu phng php trn da vo kinh nghim hoc tun theo ti liu tham kho. Phn lp t l Chn l c vt bnh cn tr v nm thng ang pht trin mnh cc m l ny. Cn thn dng ko hoc dao ct mu l thnh tng mu nh ti phn tip gip gia m khe v m bnh. Kh trng b mt bng cch ngm trong dung dch natri hypochlorite 10% khong 1 3 pht ri ra qua bng nc v trng. Dng panh kh trng t ming cy ln mi trng agar. 30
Phn lp t thn Trong trng hp vt bnh su hoc nm pha trong b mch ca cy bnh, c th ct ming cy cc m bn trong trnh phi kh trng b mt. Dng dao (hoc dng c c th ct thn g) tit trng qua la n cn ch dc thn bnh theo chiu t phn khe n phn bnh. Dng dao v trng cn thn ct ming cy (3-5 mm) ti im tip gip gia m bnh v m khe, chn m bnh cn mi. Dng panh v trng gp ming cy t ln a Petri cha mi trng agar - nc ct (hoc mi trng khc thch hp). i vi cc thn cy nh, vt bnh ch yu b gii hn bi phn m pha ngoi hoc trong trng hp khng th ct ming cy phn m bnh pha trong th c th dng dao v trng ct ming cy phn tip gip gia m khe v m bnh, sau kh trng b mt (1-3 pht trong natri hypochlorite), ra li bng nc v trng v cy ln b mt mi trng. Phn lp t r Ra ht phn t bm b phn r, sau t r vo mt chu thy tinh nh hoc a Petri thy tinh vi mc nc khong 2-3 cm sao cho d dng nhn thy phn r bnh. Dng panh hoc dao x r thnh tng mu nh. Dng dao hoc ko v trng ct mu r cy (di khong 5 mm) ti im tip gip gia m khe v m bnh. C th kh trng qua b mt ming cy, tuy vy i vi r v rt mng manh nn cn ra li k bng nc v trng nh sau: t cc mu r vo ry c l nh ri di vi nc sch chy lin tc trong 30 n 90 pht. Dng dao hoc panh v trng t cc ming cy ln a Petri c cha mi trng agar - nc ct (hoc mi trng khc ph hp), ch n nh cho ming cy tip xc su vo b mt mi trng. 5.1.1 Bung gi m
i khi triu chng bnh th hin rt r nhng phn lp vi sinh vt gy bnh li khng n gin cht no. Trong trng hp kh phn lp vi sinh vt hi c th mu trong mi trng m, ch s xut hin ca cc dng qu th v hnh thnh bo t. Tuy nhin, trong iu kin m, vi sinh vt hoi sinh cng rt d xut hin trn b mt m bnh. Lau nh bng cn cng nghip hoc natri hypochlorite 10% c th gip hn ch s pht trin ca vi sinh vt hoi sinh nhng li c th ph hy cu trc vi sinh vt hi trn b mt vt bnh. khc phc tnh trng ny c th ra mu bnh bng nc v trng ri lm kh trc khi m. Hp m nn gi nhit di 25C v tt nht l nn ni sng, v d trn bn th nghim, nhng khng nn trc tip di nh nng mt tri, nn gi nhit khng i trnh hi nc ngng t. Mu cn c kim tra hng ngy di knh lp soi ni vi phng i thp quan st s xut hin ca bo t. Cc cu trc bo t c th c ly ra bng que cy v trng kim tra di knh hin vi c phng i ln hn hoc cy ln mi trng agar. i vi cc mu bnh nh nh l hoc cnh nh c th dng a Petri bng thy tinh hoc nha. Cc mu bnh ln hn c th m trong cc hp nha v cc mu qu ln c th m trong cc ti nilon ln. 31
Hp hoc ti dng m phi c kh trng. Nu khng th cho vo ni hp c th phi lau sch bng cn 70% ri kh. Ti nilon phi mi cha s dng ln no. t giy lc ( c hp kh trng) vo a Petri hoc hp lm m ri dng nc ct v trng thm t giy lc. Khn giy dng trong phng th nghim c th c s dng bng cch thm t ri vo trong ti nilon hoc hp nha ln. Mu bnh phi c pha trn giy thm nc bng cch t mt li nha vo hoc bt c vt g (nhng trong cn 70% v h qua ngn la n cn) k cao mu bnh trong hp, trnh tip xc trc tip vi giy thm. 5.1.2 Phng php pha long
Nm c th c phn lp t t v b mt cc b phn cy nh l, hoa bng cch ra v s dng phng php pha long ln lt phn lp c cc tn nm trn mi trng thch hp, v d nh mi trng agar - nc my (tap water agar) (TWA). Khng nn s dng cc mi trng giu dinh dng v chng s kch thch s pht trin ca c quan sinh dng thay v vic hnh thnh bo t. C th s dng khng sinh nh streptomycin sunfat trn ln vo mi trng agar hn ch s pht trin ca vi khun. Phng php pha long ln lt c thc hin nh sau: Nghin nh hoc ry mn t hoc cc mu nh cy bnh, trn tht k ri ly mt lng nh vo l McCartney (dung tch 20 ml) cha 10 ml nc v trng. Dng pipet v trng ly 1 ml dung dch ri vo l McCartney khc cha 9 ml nc v trng. Dng pipet v trng khc ly 1 ml dung dch va c pha long ri vo l McCartney th ba cha 9 ml nc v trng. C tip tc pha long ln lt nh vy n khi dung dch t n nng mong mun. Ly mt lng nh dung dch pha long cui cng vo b mt mi trng trong a Petri v dng que thy tinh dn u sao cho dung dch bao ph ht b mt mi trng. Sau mt thi gian cc tn nm bt u xut hin, cn cy truyn ngay ln mi trng ph hp trc khi chng mc chng ln nhau. i khi ch c mt lng rt nh nm hnh thnh bo t, v d: Nm ti hoc nm dng qu cnh. Trong trng hp ny cch tt nht l p dng phng php sau: nh mt git nc v trng vo mt lam knh v trng ri gp mt qu th nm t vo git nc, i n khi bo t c gii phng khi qu th. C th iu chnh mt bo t di knh hin vi. Dng que cy a dch bo t vo thnh tng vt trn mi trng TWA. Sau 24 gi cy n bo t t TWA ln mi trng thch hp. 5.1.3 Pht trin v sn sinh bo t
i vi hu ht nm bnh, sn sinh bo t l hnh thc sinh sn ch yu, bo t sau khi sinh ra s c pht tn trong mi trng sng ca chng. S sn sinh bo t ph thuc rt nhiu vo iu kin mi trng nui cy. Hu ht loi nm c th pht trin tt nhit phng th nghim. S sn sinh bo t l yu t rt cn thit cho vic gim nh v to ngun nm ly bnh nhn to. iu kin mi trng nhn to nh: t nh n ti v cc mi trng nui cy giu dinh dng thng khng c li cho vic sn sinh bo t hu ht nm bnh. Nn s dng l k ch ( tit trng), v d: cng rm ca la m, l ng, l cm 32
chng t vo mi trng ngho dinh dng nh TWA kch thch s sn sinh bo t. C th kch thch s sn sinh bo t nhiu loi nm bng cch t di nh sng en (cc tm yu). Mc d nh sng en c th nh hng n mu sc tn nm, hnh thi i th ca tn nm, thm ch hnh thi bo t, nhng bin i ny khng nh hng n vic gim nh. Gn bng n nh sng en vo pha trn gi a cy kch thch s sn sinh bo t cc loi nm m vic hnh thnh bo t i hi mi trng nh sng ny (Hnh 8).
nh sng en Hp n hunh quang L thng hi, khong 50 mm ng knh 450 mm 350 mm
Thit b iu chnh thi gian
Mu nm phn lp
Hp n hunh quang dy 100 mm
500 mm
Hnh 8 Mt ct nghing ca bung mu nm phn lp c nh sng en
Nhng im cn ch khi s dng nh sng en kch thch s hnh thnh bo t:. Bng n hunh quang c nh sng en sn c trn th trng vi cc loi 20 W, 40 W v 80 W v chiu di khc nhau. Bng n hunh quang vi nh sng trng thng pht ra mt lng tia sng cc tm yu, v vy nu khng c n nh sng en c th dng n hunh quang vi nh sng trng thay th. C th s dng kt hp cc loi bng khc nhau (v d: mt bng nh sng en gia hai bng nh sng trng). Khng nn s dng n c cng chiu sng ln; Dng thit b iu chnh thi gian chnh nh sng lin tc sao cho c tip theo 12 gi UV li c 12 gi bng ti; Mt s loi nm i hi mt khong thi gian trong bng ti bt u s hnh thnh bo t, v vy bung mu nm phn lp nn c che sng.
33
Sau khi cy 1 n 4 ngy, bt u s dng nh sng en cho n khi nm ngng pht trin. Khng bao gi mu nm phn lp tip xc vi nh sng en ngay sau khi cy vo mi trng; i vi mt s loi nm, nh sng cc tm yu s khng c hiu lc i vi s hnh thnh bo t nu nhit cao; nh sng en c th truyn hiu qu qua a Petri nha v cc a khng mu. V vy nn dng a nha cy nm khi nh mu nm phn lp nh sng ny. 5.2 PHN LP VI KHUN
Nu bnh do vi khun gy ra, mt lng ln cc t bo vi khun thng d dng c tm thy khi vt bnh trn l hoc trong cc b mch b v. Phng php pht hin v phn lp vi khun t m cy bnh nh sau: 1. Ra phn cy b bnh di vi nc chy loi b t, bi v cc sinh vt ln tp. Nu mu bnh ban u c gi gn cn thn, khng b c xt nhiu th khng cn thit phi kh trng b mt. Ni chung, cng hn ch kh trng b mt cng tt, nht l i vi cc mu bnh kh hoc gn kh. Cc cht c tc dng kh trng thng xm nhp rt nhanh v d dng dit vi khun. Nu cn thit phi kh trng, ch nn nhng mu bnh tht nhanh vo dung dch natri hypochlorite 10%, sau ra li bng nc v trng. Nu mu bnh dy v d nh n, qu, cnh hoc nt sng th c th nhng qua cn etanol 70% v h qua la n cn trc khi phn lp. 2. Ct vt bnh thnh tng mu nh phn gia m bnh v m khe, sau t mu ln lam knh tit trng nh sn mt git nc v trng. i vi nhng m bnh dy hn nn dng que cy hoc dao cy x nh ra sau khi t vo git nc v trng. Dng lamen v trng t ln trn ming mu bnh v kim tra ngay di knh hin vi vi phng i 400 ln. C th h thp t quang hoc ng phin lc bn di mu knh hin vi nhn t bo vi khun r hn. Nu vt bnh do vi khun gy nn ta c th nhn thy mt m vi khun chuyn ng t vt ct. Cn ch khng nhm ln vi khun vi cc thnh phn khc nh: nha cy, ht tinh bt, plastid. Khi nhn thy dch vi khun chuyn ng ra ngoi t vt ct, c th b lamen ra v dng que cy vi khun vi u que cy hnh vng trn cy dch vi khun ln mi trng agar thch hp. Trong mt s trng hp c th phi ngm m bnh trong nc v trng khong vi gi m bo lng vi khun chuyn ng ra ngoi thnh dch, sau mi cy ln mi trng. 3. Cy vi khun bng cch dng que cy vi khun cha dch vi khun vch tng vch ln b mt agar. C th vch que cy theo nhiu cch: Cch 1: to thnh hnh zch zc trn mt gc phn t ca a cy, bt u t mp a. Cc gc phn t khc vch thnh cc ng thng song song, mt u trng ln vch ca gc phn t trc (Hnh 9). Que cy phi c kh trng trc mi ln vch ln mt agar. Cch 2: vch ln ton b b mt agar t trn xung di, t tri sang phi phn lp c tng khun lc ring bit. 4. Da vo d on nguyn nhn gy bnh m la chn mi trng thch hp. Mi trng phi c kh trc khi cy bng cch t ngc np a cy 34
v nghing trong 24 gi bung v trng hoc m np trong t cy v trng trong 30 pht. Cn thn trnh khng cho khng kh tp ln vo mi trng. 5. a phn lp nn t ngc trnh ng hi nc trn b mt mi trng. Hu ht cc vi khun pht trin tt nhit 25-28oC. 6. C th thay i mt s thao tc trn theo kinh nghim hoc tun theo cc bi bo.
2
4 Hnh 9 Cch vch phn lp vi khun trn mt agar: 1. Vch phn lp ban u t dch bo t. 2. Dng que cy sau khi kh trng vch thnh cc vch thng th nht ni vi vch phn lp ban u. 3. Cc vch thng th hai. 4. Cc vch thng th ba, khun lc c to thnh ring r trn cc vch ny.
Sau khi phn lp, t a cy iu kin nhit phng th nghim v kim tra hng ngy trong 4-5 ngy, hoc 10-14 ngy i vi cc vi khun pht trin chm. Da vo triu chng c th d on vi khun gy bnh v cy truyn, k c trong trng hp nhiu vi khun khc nhau cng xut hin trn mt a phn lp. Nu chn la mu bnh v mu cy tt th thng thng ch phn lp ra mt loi vi khun gy bnh. Nu t hai loi vi khun c pht hin trn mu phn lp th ch ghi chp li mc ph bin ca tng loi. Khng tnh n cc vi khun hoi sinh ph bin, nn cy truyn kim tra cc vi khun xut hin nhiu, c bit l cc a im khc nhau. Ch nn cy truyn cc khun lc ring r, tt nht ln cng loi mi trng trong ng nghim. C th p dng phng php phn lp tng t nh i vi nm. Cy trc tip m bnh ln b mt mi trng, vi khun s pht trin xung quanh ming cy. Nu vi khun hoi sinh c mt trong mu cy, chng thng ln t vi khun gy bnh c bit l i vi cc vi khun gy bnh pht trin chm. V vy, ch p dng phng php ny trong trng hp phng php cy dch vi khun khng ph hp. 5.3 PHN LP TUYN TRNG
Tuyn trng (giun trn khng phn t) l mt nhm ng vt a dng, nh, mt thng khng nhn thy c. Chng phn b hu ht mi ni c nc t do, thm ch c nhng ni rt t nc. Tuyn trng tng hp cht dinh dng t nhiu ngun khc nhau, nhiu loi c kh nng k sinh chuyn tnh cao. i a s thc vt v 35
ng vt l k ch ca t nht mt loi tuyn trng k sinh. V vy m rt nhiu tuyn trng k sinh l dch hi quan trng i vi kinh t. Ngc li, cc loi tuyn trng s dng vi khun v nm lm thc n (hay cn gi l tuyn trng sng t do) li c chc nng quan trng v mt sinh thi. V d, tuyn trng sng t do ng vai tr quan trng trong chu k dinh dng ca h sinh thi t. Tuyn trng k sinh thc vt c phn lm hai nhm chnh: (1) nhm di chuyn v ly thc n trn b mt hoc gia cc r cy (tuyn trng k sinh di chuyn) v (2) nhm khng di chuyn, kch thch cy ch sn xut ra cc cu trc bo v v d tr dinh dng nui tuyn trng (tuyn trng k sinh ti ch). Cc loi tuyn trng di chuyn thng c kh nng k sinh trn nhiu k ch khc nhau v gy thit hi v kinh t khi mt tuyn trng cao tr mt s t ging. Cc loi tuyn trng k sinh ti ch c kh nng k sinh chuyn tnh cao v tc ng n k ch mc phn t, tc ng rt ln n nng sut cy trng nhng thng gy hi trn t i tng hn l i vi cc loi tuyn trng di chuyn. gim nh tuyn trng k sinh thc vt, nn tch tuyn trng ra khi t hoc mu bnh v bo qun cn thn. 5.3.1 Mu t
i vi mc ch gim nh v lu gi mu, nn ly mu bnh vng r cy c biu hin triu chng. Nu iu tra mt khonh b nhim bnh trn mt rung th nn ly mu khu vc ranh gii gia khonh b bnh v khonh khng b bnh, v khu vc ny tuyn trng gy bnh cy c th tp trung vi mt cao nht. Ngoi ra, cng nn ly mu khu vc khng b bnh so snh. Chiu su ca im ly mu v s lng mu ly ban u cng quan trng nhng chng ti s khng cp su v vn trong phn ny. Tt nht nn ly mu t m bi v mt s tuyn trng ng ngh trong t kh thng b thng do t gy. t mu t trong ti nilon v gi ni mt m trong qu trnh chuyn ch, x l mu cng sm cng tt. Mt s tuyn trng c th c gi trong t lnh nhng khng phi l tt c cc loi u c th p dng cch ny. i vi t nh, b, c th dng ry (kch thc l ry 10mm) loi b cc mnh rc sau trn k. Phng php ny khng phi lc no cng p dng c vi t nng. Phng php tch tuyn trng ra khi mu bnh c th da vo s di chuyn ca tuyn trng (ch ng) hoc da vo kch thc v mt tuyn trng (th ng). Phng php ch ng n gin nht l dng khay Whitehead hoc phu Baermann tch tuyn trng ang hot ng. Mc d n gin, qu trnh ny c th ko di 24 ngy v mu thu c c th rt t, do tuyn trng ln di chuyn chm. Phng php th ng c tin hnh bng cch lc, gn v tch vi nhiu cch kt hp khc nhau. Trong phn ny, chng ti tp trung m t phng php ry t v cc phng php ly tm khc nhau. Phng php ry t c th dng phn lp bo nang ca cc loi Heteroderid. Tch tuyn trng bng khay B t vo mt khay c lt mt ming li th v mt lp vi hoc giy thm, dn u t ri nc vo sao cho ch va thm t t (Hnh 10). Sau 1 - 4 ngy, 36
tuyn trng ang hot ng s di chuyn xung di, xuyn qua ming vi vo trong nc di y khay. Lng t s dng ph thuc vo kch thc ca khay; khay loi ln (450 x 300 mm) c th cha mt lp 200g t mng. Lp t cng mng cng c tc dng tng hiu qu tch tuyn trng v gim thi gian tch.
t Vi Li Nc Khay
Hnh 10
Khay Whitehead dng tch tuyn trng t t v cy
Trc khi thu thp tuyn trng, cn thn nhc phn li ln hn ch t ri vo phn nc pha di. Chuyn phn nc vo mt cc ong v nh vy vi gi cho tuyn trng lng xung. Sau , tt nht nn dng pipet ly bt nc ra ch gi li khong 5-20 ml, cn thn khng lm xo trn tuyn trng lng xung di. C th thay th phng php ny bng cch trt nc c cha tuyn trng ra mt ry lc vi kch thc l ry 20 - 38 m. Tuy nhin, dng loi ry vi l ry cng nh th cng t v kh mua. Tuyn trng c th lt qua ry nhng c th khc phc bng cch bt v lc li vi ln. Phu Baermann t t vo phu c lt li di v giy thm hoc mt ming vi trn, c phu c gn ng cao su c kp. nc vo ming phu va thm t t (Hnh 11). Phng php ny ch tin hnh c vi mt lng nh t nhng c u im l tuyn trng tp trung phn ng cao su pha trn kp v c th m kp ly khong 5 n 10 ml nc cha tuyn trng vo ng nghim.
t Vi Li
Nc
ng cao su
Kp
Hnh 11
Dng phu Baermann tch tuyn trng t t v cy bnh.
37
Phng php lc t v ly tm t vo chu ri t di vi nc chy, ha khong 4 lt dung dch t - nc, trnh khng nc chy trn. Khong 10 giy sau, cc phn t t ln hn chm xung y chu. Phn nc cn li c lc qua ry (kch thc l ry 38 m) xung mt chu khc. Tuyn trng b mc li l ry s c thu hi bng cch ly tm vi mt lng nc rt nh. Tuyn trng lt qua ry c thu hi bng cch ry li (c th ry li n 3 ln). tng lng tuyn trng thu c, nn lp li ton b qu trnh t khu ha t vo nc thm 2 ln na. Tuyn trng c thu hi bng cch ly tm, cn thn rt phn nc pha trn ng nghim, ch li mt lng nc nh cha tuyn trng. Sau ha tuyn trng thu c vo nc ng c (450 g/lt) ri ly tm li. Loi b cc hp cht hu c ni ln trn ng, nc ng cng vi tuyn trng c ra qua ry thu hi tuyn trng. C th ry li 2 ln tn dng tuyn trng lt qua l ry. Khng nn tuyn trng qu lu trong dung dch ng c v ng c th kt tinh gy hi n tuyn trng. C th thay th dung dch ng bng cc dung dch c nh NaCl, MgSO4 hoc keo silica (Ludox, DuPont de Nemours). Phng php tch tuyn trng bo nang Ha tan t bng 4 lt nc x t vi, trnh chy trn. phn t lng xung sau 10 giy, phn nc c lc qua ry th (710 m) t trn mt ry 250 m. t c ra li v lc qua ry 2 ln na. Cc cht hu c ng li trn ry l nh c thu v vo phu Buchner c lt giy lc. Kim tra bo nang di knh lp soi ni. Nu dng phu tam gic v ha tan cc hp cht hu c bng cn ethanol 70% th bo nang s nm li thnh mt vng pha vnh ngoi ca giy lc. 5.3.2 Mu cy bnh
Tuyn trng c th c tm thy trong r, thn, l v ht ca cy, v vy phng php tch cng khc nhau ty tng loi mu v cc loi tuyn trng khc nhau. Cch tt nht tch tuyn trng ni k sinh di chuyn l dng thit b phun sng, ngoi ra c th dng khay Whitehead hoc phu Baermann nh m t trn. i vi tuyn trng khng di chuyn c th dng dao ct m bnh ly tuyn trng ra hoc bo qun in situ. C th tch trng ca mt s tuyn trng c nh bng cch ra trong thuc ty v lc qua ry. Tuy nhin chng ti khng m t phng php ny y v trng tuyn trng khng c ngha nhiu v mt phn loi. Tuyn trng con di chuyn v tuyn trng c hnh giun c tch bng phng php phun sng. Phng php tch tuyn trng bng phun sng t mu bnh (ct thnh tng mu di 10mm) vo phu c lt li v mt mnh vi pha trn. phu c cha mu bnh vo bung phun sng, c khong 10 pht th phun sng mt ln (Hnh 12). Nc thm qua phu c hng vo 1 ng nghim vi lng chy chm tuyn trng lng xung y ng v c ly ra sau 2- 4 ngy. Gim lng nc trong ng bng pipet (Dng pipet ht bt nc trong ng nghim). Phun sng bo m cho nc c oxy ha tt v loi b nhng c t do cc m cy bnh hoi sinh sn sinh ra, v th m tuyn trng c gi iu kin tt nht. C th p dng phng php khay Whitehead v phu Baermann nhng cc phng php ny c nhiu hn ch khi p dng vi mu cy bnh hn l vi mu t. 38
Phun sng (nh k)
R Vi Li
ng nghim
Nc
Hnh 12
Tch tuyn trng t m bnh bng phng php phun sng
Ct mu t mu vo mt a Petri. Dng dao cy nhn u hoc que cy x mu m b bnh tuyn trng thnh tng phn nh di knh lp soi ni. Tt nht nn dng kt hp nh sng truyn v nh sng ti. C th quan st tuyn trng tch ra t m bnh bng nh sng gin tip pha di knh. mu trn a Petri nh vy mt thi gian c th to iu kin cho mt s tuyn trng di chuyn ra ngoi m bnh. nh sng ti s c ch cho vic quan st giai on sng ca tuyn trng c nh nh bo nang (Heterodera spp.), nt sng (Meloidogyne spp.) u l v ht (Anguina spp.). C th quan st tuyn trng ni k sinh bng cch lm sch m bnh bng thuc ty, sau nhum mu bng nhiu cch khc nhau. Phng php ny c th c ch cho mc ch gim nh nhng mu bnh sau khi x l khng ph hp bo qun lu di. 5.3.3 Bo qun v lm tiu bn
X l nhit Trc khi c nh mu tuyn trng bng cht bo qun phi git chng gi nguyn vn cu trc. Ngm tuyn trng trong nc hoc cht c nh 60oC, sau lm lnh tht nhanh bng nc lnh hoc cht c nh. Nu khng c b cch thy duy tr nhit 60oC, c th nc si vo dung dch cha tuyn trng vi lng nc si bng lng nc cha tuyn trng to ra nhit va git tuyn 39
trng. Ngoi ra, c th t dung dch cha tuyn trng (mc nc nng) vo t sy nhit 55 60oC, kim tra mt pht/ln cho n khi tuyn trng b cht. Cn lu trong qu trnh x l nhit khng c luc hoc nhit qu cao v s ph hy cu trc bn trong ca tuyn trng. C nh mu Phng php c nh tuyn trng n gin nht l dng formaldehyde. Nh dung dch formaldehyde m c vo dung dch cha tuyn trng sao cho nng formaldehyde cui cng l 2 5%. Mu c ngm trong formaldehyde t nht 12 gi, l tng nht l 2 tun trc khi tin hnh cc bc tip theo. C th bo qun mu lu di trong dung dch formaldehyde. Cc cht c nh khc thng c s dng bao gm TAF (3% formaldehyde, 0,2 % triethanolamine trong nc ct) v FA4:1 (4% formaldehyde, 0,1% axit axetic trong nc ct). Tuy vy, cc ha cht ny khng ph hp vi bo qun lu di. Khng s dng cn ethanol c nh mu cho mc ch nghin cu v hnh thi, nhng c th s dng cn ethanol bo qun cho mc ch nghin cu v sinh hc phn t sau ny. Lm tiu bn lm tiu bn vi mc ch bo qun lu di, mu tuyn trng c ngm trong glycerol v bay hi chm. Cho mt lng nh dung dch glycerol 2 - 5 % (vi ml) vo mt a thy tinh nng, sau t tuyn trng vo. Cho a vo t sy 40oC hoc bnh ht m. lm chm li qu trnh bay hi nc khng nn ng kn np a. Quan st di knh lp soi ni gp tng mu tuyn trng vo mt lam knh hin vi. S dng que cy nhn u c dnh keo dn lng mi gp tuyn trng. Ko tuyn trng dn dn v pha b mt c th quan st c v nhc ra khi b mt bng cch di chuyn que cy. Qu trnh gp tuyn trng ra c tin hnh t a nng nhng nhiu khi phi thng xuyn thay i c ly ng knh tm tuyn trng. Mun thnh tho thao tc ny cn phi thc hnh nhiu. Gp mt vi n 10 tuyn trng cc giai on i din vo mt git glycerol khan t trn mt lam knh. Dng que cy t c nh mu chnh gia lam knh. gi cho mu tuyn trng khi b nghin nt, dng vn thy tinh, lamen v (S 0) hoc sp t xung quanh mu tuyn trng. Nh nhng t lamen (tt nht nn dng loi trn) ln trn git glycerol khan, khng glycerol trn ra mp ngoi lamen. Nu dng sp co ru hoc vng sp (nu chy ra ri ngui) th phi t lam knh ln bp in lm sp chy ra. Sau hn lamen li. Trc y glycin thng c dng hn lamen nhng hin nay glycin khng sn c trn th trng v vy c th thay bng nha epoxy hai phn (v d: 5 min. Araldite) hoc DePeX (c nhiu cng ty khc nhau). Mc d mt s ni c th s dng thuc nh mng tay chn nhng cch ny thng khng bn trong iu kin kh hu nng. 5.4 MI TRNG NUI CY
Hu ht cc mi trng nui cy thng c lm t cc ha cht c bn hoc thm ch c th mua sn t th trng. Cc mi trng bn sn trn th trng c cht lng ng u hn mi trng t chun b t cc thnh phn c bn. 40
Hn ch s pht trin ca vi khun bng cch cho thm mt lng khng sinh vo mi trng. Hu ht cc loi khng sinh u d b bin tnh iu kin nhit cao, ch b khng sinh vo mi trng sau khi hp kh trng (trc khi agar kp ng li).
Penicillin (50 n v / ml), streptomycin (50 n v / ml) v tetracycline (30 n v / ml) c th s dng ring hoc kt hp. Chloramphenicol (50 mg / l) c th hp kh trng (cho thng vo mi trng trc khi hp).
Lng agar cho vo cc mi trng (gii thiu phn di) c th khc nhau ty thuc vo nhn hiu agar v c s sn xut. Lng agar dng nu mi trng phi bo m cng c th cy vi khun bng phng php vch que cy ln mi trng. Nu pht hin trong nc my c cha cht bn hoc c t th nn s dng nc ct thay th. Mi trng khoai ty - ng - agar (PDA): y l mi trng tt cho s phn lp v pht trin ca nm. Ra sch khoai ty (khng gt v). Ct khoai ty thnh tng ming nh v luc trong khong 1 gi, sau lc khoai ty v nc luc bng ry (hoc vi mn) ri vt b phn b i. C th dng my xay sinh t xay nt hn hp. Ha tan ng dextrose (glucose) v agar vo 1 lt nc my cng vi nc lc khoai ty. Hp kh trng 121C trong 20 pht. Mi trng PDA Khoai ty Dextrose (glucose) Agar Nc my 200 g 20 g 20 g 1 lit
Cng thc ny c th c thay i mt cht bng cch dng cc loi rau v ng cc khc thay th khoai ty. C th gim hoc lng khoai ty v lng ng ty thuc yu cu s dng. Mi trng agar - nc my (TWA): y l mi trng ph bin phn lp rt nhiu loi nm. C th tit trng cng la hoc ht ng cc ri t ln mi trng kch thch nm sn sinh bo t. Trn agar trong nc v hp 121C trong 20 pht. Mi trng TWA Agar 20 g 41
Nc my
1 lit
Mi trng nc qu V-8 - agar (V-8J): y l mi trng tt cho s pht trin ca nm, c bit l Phytophthora. Lc qua nc V-8 trc khi s dng. Trn cc thnh phn vo nhau v hp kh trng 121C trong 20 pht. Nu khng c nc V-8 c th thay th bng nc sinh t c chua, c rt, cn ty ln Mi trng V-8J Nc V-8 lc CaCO3 Agar Nc ct 200 ml 3g 20 g 800 ml
Mi trng King B - agar (KBA): y l mi trng tt phn lp vi khun hi ni chung v c bit l pseudomonads hunh quang v Erwinia spp. iu chnh pH n 7,2 v hp kh trng 121C trong 25 pht. Mi trng KBA Proteose peptone Glycerol K2HPO4 (khan) MgSO4.7H2O Agar Nc ct 20 g 10 g 1,5 g 1,5 g 15 g 1 lit
Mi trng Nutrient agar (NA): L mi trng thng c s dng nui cy vi khun. iu chnh pH n 7,4 v hp kh trng 121C trong 15 pht. Mi trng NA Cht chit nm men Peptone NaCl Agar Nc ct 3g 5g 5g 15 g 1 lit
Mi trng Sucrose peptone agar (SPA): L mi trng thch hp cho s pht trin ca nhiu loi vi khun, c th s dng phn lp vi khun hi. iu chnh pH 7,2 7,4 v hp kh trng 121C trong 15 pht. 42
Mi trng SPA Sucrose Peptone K2HPO4 (khan) MgSO4.7H2O Agar Nc ct 20 g 5g 0,5 g 0,25 g 12 g 1 lit
Mi trng Malt extract agar (MEA): L mi trng tt phn lp v nui cy nhiu loi nm si v nm men. C th s dng lng mch nha khc nhau. Hp kh trng 121C trong 20 pht. Mi trng MEA Malt extract (mch nha) Agar Nc ct 2 20 g 20 g 1 lit
Mi trng Glycerol agar (GA): c s dng bo qun mu phn lp lm tiu bn kh. Hp kh trng 121C trong 20 pht. Tn nm pht trin trn mi trng agar cha kh hon ton c t ln mi trng GA ( vo mt np a Petri). a kh trong 2-3 ngy. Lp mi trng GA kh mm, do hn hu ht cc mi trng agar khc. Mi trng GA Glycerol Agar Nc my 25 ml 20 g 1 lit
43
6
6.1
CCH BO QUN V LU GI MU BNH
TIU BN MU BNH
Tiu bn mu bnh l mt mu thc vt hoc nm kh c trong mt gi c dn nhn. Tiu bn mu bnh c th bao gm cc mu nm phn lp kh, cc tiu bn lam, nh v hnh v minh ha. Gi tiu bn c th bao gm c cc bn ch thch, th t trao i v cc thng tin lin quan khc. 6.1.1 Tiu bn kh
Mu cy bnh v nm bnh c p v lm kh bng cch t chng gia cc lp giy thm hoc giy bo. Dng 2 tm ba dng ln sng p cc lp giy thm hoc giy bo cha mu li vi nhau. Dng 2 tm g (hoc 2 tm nguyn liu cng) p cht 2 mt ca khi mu ri dng dy buc cht li to ra lc p ln mu cy bnh. Mu cy bnh phi kh nhng khng c nhn hoc co li. Mu cy bnh c th t tiu chun sau vi ngy p. lm nhanh qu trnh kh (c bit l trong cc khu vc kh hu m nh cc vng nhit i) nn dng t sy hoc qut. Thay giy thm hoc bo thng xuyn. Tip tc qu trnh p n khi mu bnh kh hon ton. C th t mu p di nh nng mt tri, trong phng c my iu ha hoc gn hi nng thc y qu trnh kh. Thi gian mu kh hon ton ph thuc vo loi mu cy v m. Ly mu ra khi lp giy thm hoc bo p qu sm s lm mu b nhn hoc to iu kin cho cc loi nm pht trin trn b mt tiu bn. a s cc mu thc vt c th kh sau 5 -10 ngy. Trc khi mu vo phng tiu bn phi xng hi kh trng hoc t mu trong t (t lnh su) dit nhn v cn trng. Cch n gin nht bo qun nm Agaricales v Ascomycetes loi ln l chng kh tht nhanh. Mc d qun l tiu bn kh d dng hn tiu bn ngm, qu trnh lm kh thng nh hng n mu sc, hnh dng v kch thc mu bnh. Nm ln c th c lm kh bng t lnh su (t ) gi nguyn mu sc v hnh dng, tuy nhin phi gi mu rt cn thn v bo qun bng cch ny mu tr nn rt gin v d v. Kch thc ca nm ln thng qu to so vi cc gi tiu bn v rt d gy v, v vy bo qun mu lu di nn dng cc hp carton nh hoc cc ti nilon c cha silica gel. Nn bo qun tiu bn mu bnh kh v cc mu nm phn lp kh trong cc gi giy hoc ba lm bng cht liu khng c axt vi kch thc tiu chun (khong 1015 cm rng 10 cm di). C th t mu vo cc gi giy khng axt mng hn trc khi cho vo gi tiu bn. L tng nht l tt c cc bn ch thch, nhn, keo dn, mc, nha hoc ti nilon dng trong phng tiu bn u lm t cht liu khng c axit gi tiu bn c lu di khng b , hng. Hin nay trn th trng c bn bt th giy xem c cha axt hay khng. Mi gi tiu bn phi c dn nhn mt trc (Hnh 13) vi cc thng tin nh sau: S th t truy cp; 44
Tn khoa hc ca vi sinh vt hi; Gi th hoc tn khoa hc ca k ch; a im ly mu, bao gm: nc, tnh, kinh v v ; Tn ngi ly mu v s hiu mu; Ngy ly mu; Tn ngi gim nh mu; Ti liu tham kho trong c cc bi bo cp n tiu bn.
Hnh 13
V d mt nhn dn trn gi tiu bn s dng ti phng tiu bn BRIP
Mi tiu bn trong phng mu u c nh s truy cp phn bit vi cc mu khc. S truy cp tiu bn c in trn nhn ca gi tiu bn (Hnh 13). S kt hp ca s truy cp v tn vit tt ca c quan lu gi mu trong cun Index Herbariorum bo m rng bt k mu tiu bn no cng c mt tn duy nht, phn bit vi tt c cc tiu bn khc trn th gii. Nu mt tiu bn hm cha hn mt n v phn loi th c th thm cc k hiu (a, b, c..v.v..) i din cho mi n v phn loi vo sau s truy cp. Tiu bn mu bnh kh v mu vi sinh vt hi c phn lp t mu tiu bn phi dng chung mt s truy cp. Chun b sn cc gi tiu bn ging ht nhau trong trng hp cn thit phi chia tiu bn thnh nhiu gi nh. Cc gi ny cha cng mt lng thng tin ging ht nhau v c chung mt s truy cp, c th c s dng gi hoc trao i tiu bn vi cc phng mu bnh khc. Tiu bn nm chim s lng ln trong s cc mu tiu bn ca mt b su tp bnh cy. C th sp xp tiu bn nm thnh nhm theo th t tin ha. Hu ht tiu bn nm thuc lp nm ti Ascomycotina hoc nm m Basidiomycotina, cc tiu bn trong mi nhm ny li c sp xp theo vn abc ca tn chi. Mt s nhm vi sinh vt hi ph bin c th c xp ring, v d nh g st (Uredinales), than en (Ustilaginomycetes) v phn trng (Erysiphales), cc mu tiu bn trong cc nhm vi 45
sinh vt ny li c sp xp theo vn abc tn k ch (v nhiu loi g st, than en v phn trng u k sinh chuyn tnh trn cc k ch khc nhau). Nm giai on v tnh (Deuteromycetes, Fungi Imperfecti, asexual fungi, conidial fungi) nn s dng tn hu tnh nu c. Hn na, mt s nm c th c t nht 2 tn v tnh (synanamorphs) vi cng mt tn hu tnh. Nn dng tn hu tnh t cho tiu bn thm ch giai on hu tnh ca nm khng c mt trong tiu bn. Trong trng hp ny cn ch thch r v s vng mt ca giai on hu tnh trn nhn tiu bn. i vi nhng tiu bn qu ln khng ch xp ng v tr theo th t tin ha c th thay vo v tr bng mt ming ba hoc mt gi tiu bn rng trn ghi r v tr t tiu bn c th tm c d dng. Tng t c th dng mt ming ba t vo v tr ca cc tiu bn ang cho mn v c ch thch r rng. 6.1.2 Tiu bn chun
Tiu bn chun (mu gc) l mt tiu bn mu hoc hnh minh ha duy nht ban u c s dng xc nh tn khoa hc cho mt loi mi. Khi mt loi nm c m t v nh tn, tiu bn mu ca loi mi phi c chuyn n mt phng mu bnh v c cng nhn l mu chun cho tn loi nm . Tiu bn chun ca nm phi c bo qun v lm kh vnh vin. Tuy nhin, cc mu nm chun cng c th c bo qun trng thi ng ngh bng phng php lnh kh hoc lnh su. Cc mu chun (mu ban u) thng c gi trong cc gi mu mu hoc nh du c th nhn ra chng d dng trong phng tiu bn. C cc loi mu chun sau: Holotype - Mt tiu bn hoc hnh minh ha c tc gi dng nh tn mi; Isotype - Bt c tiu bn no c nhn ln t holotype; Lectotype - Mt tiu bn hoc hnh minh ha c ch nh nh mu ban u (mu chun) nu ti thi im cng b khng c mu holotype hoc mu holotype b mt hay c pht hin l thuc hn mt n v phn loi; Isolectotype - Bt c tiu bn no c nhn ln t lectotype; Syntype - Bt c tiu bn no c cp trong ln xut bn (cng b) u tin m trong khng c holotype no c xc nh hoc c th l t 2 tiu bn tr ln cng ng thi c xc nh nh mu chun; Paratype - Bt c tiu bn no c cp n trong ln xut bn (cng b) u tin m khng phi l holotype, isotype hay mt trong cc syntypes; Neotype - Mt tiu bn hoc hnh minh ha c coi nh mu chun theo danh php nu tt c mu ban u dng nh tn b mt hoc b hng; Epitype - Mt tiu bn hoc hnh minh ha c la chn nh mt mu chun khi m holotype, lectotype hay neotype c quy nh trc , 46
hay tt c cc mu ban u c cp n trong ln xut bn (cng b) u tin khng r rng c th gii thch v khng c xc nh r rng theo n v phn loi; Topotype - Bt c tiu bn mu no c thu thp ti cng a im vi holotype. 6.1.3 Mu vi sinh vt kh
Cc c tnh hnh thi ca tn nm c ngha quan trng trong nghin cu v nm hc. Mu tn nm kh l cch duy nht bo qun lu di. Cc mu tiu bn c phn lp t t, nc hoc k ch ng vt. Cc mu nm kh cn c tc dng h tr b sung thng tin cho cc tiu bn cy bnh vi t bo t. Mu nm phn lp c lm kh v bo qun cng vi tiu bn bnh trong gi tiu bn. Mu nm phn lp kh c chun b bng cch nui cy nm trn mi trng thch hp trong mt a Petri n khi nm pht trin ti giai on mong mun. Mu nm c lm kh trong a Petri cho n khi tn nm khng dnh cht vo a Petri na. C th lm kh tn nm bng cch a Petri trong bung cy 2 3 ngy. Sau khi tn nm hon ton kh c th ly ra khi a Petri v t vo gi tiu bn. Cc mu nm phn lp vi cu trc nm mng manh c th c bo qun trong cc a Petri mng. Thng thng, mu nm phn lp c lm kh nh trn c th b cong, nt v v rt kh c th ly ra khi a Petri m khng b v. C th khc phc nhc im ny bng cch ly mu nm ra khi a Petri trc khi kh hon ton v t ni trn mi trng glycerol agar (GA) nng (xem phn mi trng nui cy, chng 5) c vo np a Petri trc . Khng y np Petri cho n khi mu kh hon ton. Bng cch ny mu nm phn lp c lm kh m vn mm do, c th bc d dng ra khi a Petri v t vo gi tiu bn. Phng php lm kh nh trn khng phi lc no cng git cht c nm. Tt nht, nn git cht mu nm phn lp v mt s nm sn sinh ra rt nhiu bo t c th pht tn ra trong khng kh lm ln tp cc mu nm khc trong phng th nghim. Git nm bng cch t cc mu kh trong bnh lm kh cha formalin hoc bng cch t ngc a Petri cha mu nm phn lp, nh khong 1,5 ml formalin vo np a ri trong bung thot kh 1 3 ngy. 6.1.4 Tiu bn lam
Tiu bn c nh bng axit lactic c th c hn bng cch dng thuc nh mng tay qut 2 3 lp ln mp lamen. Cch ny c th gi cho tiu bn lam khng b kh trong vi nm. Dng polyvinyl alcohol (dng bt) ha tan trong axit lactic to ra cht keo gn, cch ny c th gi tiu bn c lu di. C th p dng qu trnh loi nc trong trng hp mun gi tiu bn lam lu di. Qu trnh loi nc i hi phi ngm mu cy hoc nm lin tc trong chui cc dung dch cn vi nng tng dn (v d 40%, 50%, 60%, 70%, 80%, 90% v cn nguyn cht 100%), mi nng ngm vi pht. Bng cch ny c th loi b ton 47
b hm lng nc trong mu. Lm sch mu bng xylene C6H4(CH3)2 v dn vo lam bng cc mi trng nh bm Canada. Tt nht khng nn gi tiu bn lam trong cc gi tiu bn m dng cc hp ng tiu bn lam chuyn dng. Ch nn cc tm ba nh ghi r s truy cp ng v tr v ch thch ni lam tiu bn. 6.1.5 ng gi v vn chuyn tiu bn
Tt c cc tiu bn bnh cy lu gi trong phng tiu bn phi c gi trong cc gi giy hoc ba lm bng cht liu khng c axit (Hnh 14). Nhng mu c kch thc ln c gi trong cc hp carton trnh va p. Mi mt vt trong gi tiu bn, v d nh bn ly mu, bn ch thch, hnh v, tiu bn lam u phi c in s truy cp tiu bn r rng. Tiu bn mu vi sinh vt phn lp c t ln giy lc v gi trong cc gi giy trong sut, c gi cng vi tiu bn cy bnh, tt nht nn gi tiu bn lam trong hp ng tiu bn lam bng ba cng hoc bng nha trnh v, gy.
Hnh 14
Cc gi tiu bn Bo tng mu bnh Ustilaginales Vnky (HUV) v Bo tng bnh cy ca QDPI&F (BRIP)
Khi vn chuyn, tiu bn phi c gi bn trong cc gi giy hoc ba. Cc gi tiu bn c b li thnh tng b ri dng giy bo hoc ti nilon xp bc li trc khi t vo hp carton ln hn vn chuyn. Nn dng bng gi mu vi sinh vt sng c bo qun lnh kh (trong cc ng ht chn khng) v t vo trong cc hp giy hnh tr. 6.2 MU VI SINH VT PHN LP
Mu nm hoc vi khun c lu gi vi mc ch duy tr trng thi sng ca chng m khng lm thay i c im hnh thi, sinh l cng nh di truyn trong mt giai on nht nh. Mu vi sinh vt hi c gi trng thi sng t nht l trong qu trnh nghin cu hoc c th c gi v thi hn, nht l i vi cc mu vi sinh vt chun (ban u) hoc cc mu c cp n trong cc bi xut bn. Nu khng c cc mu vi sinh vt c gim nh v lu gi, c bit l vi khun, th khng th tin hnh so snh, phn loi nh danh n v phn loi mi. K t nm 1995, Cng c v a dng sinh hc c 180 nc trn Th gii k kt c hiu lc i vi tt c cc mu vi sinh vt lu gi. Cng c ny xc nh ch 48
quyn ca cc nc c xut x vi sinh vt, vi mc ch bo m s cng bng v phn chia li nhun bnh ng i vi nhng nc mun khai thc li ch kinh t t mu vi sinh vt . Vic trao i mu vi sinh vt gia cc trung tm mu bnh vi nhau thng i hi phi c vn bn tha thun chuyn giao mu - Material Transfer Agreement (MTA). Tuy nhin, vn bn tha thun chuyn giao mu gia cc trung tm mu bnh l khng r rng.Trn thc t nhiu trung tm lu gi mu, vic thc thi cng tc ny vn cn cha tin trin. Khng phi tt c cc phng php bo qun m t sau y u ph hp vi cc loi nm v vi khun pht trin trn mi trng nhn to. i khi phi th nghim v chp nhn ri ro tm ra phng php tt nht cho vic bo qun mu v thi hn. 6.2.1 Nui cy trn mi trng agar
Mu vi sinh vt thng c gi trong l McCartney 20 ml c c rng c th d dng cy truyn khi cn. Nn s dng cc mi trng nui cy thch hp, v d nh PDA i vi nm hoc mi trng NA i vi vi khun. Mt s nm v vi khun i hi phi s dng cc mi trng c bit. Mi trng thng c n xp x l trc khi tit trng. Sau l c t nghing khi mi trng cn nng khi ng li mi trng to thnh mt phng nghing. Cy nm hoc vi khun vo mt nghing ca mi trng, sau khi vi sinh vt pht trin nh mong mun, bo qun mu trong mi trng sch, khng bi bm 5 8oC. i vi mt s nm v vi khun nhy cm vi mi trng lnh, nn bo qun 15C. Mu vi sinh vt phi c theo di thng xuyn v mi trng agar c th b kh rt nhanh trong iu kin m thp. Khi bo qun nm phi rt cn thn trnh nhn xm nhp. Tt c cc l gi mu phi c hn kn ming bng mng nha hoc nilon siu mng trong sut qu trnh bo qun mu. Mu vi sinh vt phi c cy truyn sang mi trng mi 6 thng 1 ln. Phi tht cn thn khng lm mt cc mu vi sinh vt c gi tr mc d cy truyn nhiu ln c th dn n nhng thay i v hnh thi, mt kh nng k sinh v gim kh nng hnh thnh bo t. 6.2.2 Bo qun bng du khong
Phng php ny c bit c ch cc vng nhit i v n c th gi cho mu vi sinh vt khi b kh v trnh c s xm nhp ca nhn. Cy mu vi sinh vt nh trn nhng dng lng agar t hn. Mun s dng phng php ny, tiu chun quan trng i vi mu vi sinh vt l phi pht trin khe mnh, i vi nm phi sn sinh rt nhiu bo t. du khong ln trn mu vi sinh vt, ngp trn nh mt agar khong 1 cm. Tit trng du khong trc khi s dng bng cch hp 2 ln 121C trong 15 pht. Chia du khong thnh nhiu lng nh trnh ln tp. C th dng l McCartney c np nha, tuy nhin ch khng lm l v du c th chy ra ngoi. K thut ny n gin, khng i hi dng c v ha cht t tin. Phng php ny c th bo qun mu t 2 - 40 nm, tuy nhin l tng nht l nn cy truyn sang l mi 5 nm 1 ln.
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6.2.3
Bo qun trong nc v trng
Ct cc ming agar ti nh ca cc tn nm pht trin sau 1 tun nui cy ri t vo nc ct v trng trong l Wheaton 5 ml hoc l McCartney 20 ml. Np c vn cht v hn bng nha hoc bc nilon siu mng. Bo qun l mu nhit phng. Phng php ny c th bo qun mu trong vi nm, c bit ph hp vi bo qun Pythium spp. v Phytophthora spp. Phng php ny cng c th p dng i vi vi khun. Dng que cy vi khun v trng ly cc khun lc mc sau 24-48 gi t vo nc ct v trng. 6.2.4 Bo qun ng kh
C ch ca phng php ny l loi b hm lng nc trong mu vi sinh vt bng cch gim p sut trng thi ng bng, qu trnh ny c gi l thng hoa. Thng hoa xy ra khi mt dung dch ng bng chuyn ha trc tip sang trng thi kh, b qua trng thi lng. Mu vi sinh vt sau khi ng kh c hn li v bo qun trong cc ng hoc l thy tinh nh. Cc nm sn sinh nhiu bo t l cc nhm ph hp nht vi phng php bo qun ny. Phng php ny cng rt thch hp vi vi khun. Mu vi sinh vt vn c th sng st sau 10 nm hoc hn. Tuy nhin thit b lm ng kh kh t v vic duy tr bo qun bng phng php ny cng kh tn km. i ng cn b cng yu cu phi c kinh nghim chun b mu v vn hnh h thng lm ng kh. Mt trong nhng u im ca phng php ny l mu nm c ng kh khng cn bo qun lnh m c th bo qun nhit phng. Sau khi lm ng kh, ly mt ng thy tinh t mi mu vi sinh vt m ra v cy truyn kim tra kh nng sng st v thun v khng phi tt c cc loi u sng st qua qu trnh ny. Nhiu nm bo qun ng kh c th c phc hi bng cch cy mt mu mu nm kh ln mt a mi trng agar mi. Vi khun v nm men bo qun ng kh cn phi c thi gian lm m li trong nc ct v trng (khong 30 pht) trc khi cy ln mi trng agar. 6.2.5 Bo qun trong t
t c ry mn, vo cc l McCartney 20 ml v sy tit trng bng nhit (hoc hp 2 ln 121C trong 15 pht). Dung dch bo t c vo t v trng v gi 20-25C trong khong 5-10 ngy. Sau gi mu vi sinh vt trong t lnh. Mu vi sinh vt c th sng st trong t trong khong thi gian kh di. Cy li vi sinh vt bng cch dng dao cy v trng ly mt t t cy ln mi trng thch hp. Phng php ny c th s dng bo qun Fusarium spp., v sau mt thi gian trong mi trng agar Fusarium thng b bin i. 6.2.6 Bo qun bng silica gel
To dung dch bo t nm v t bo vi khun t sa khng bo 5% (w/v) sau dung dch nm hoc vi khun ln silica gel v trng ngui. Dng 6-22 li silica gel tinh lc khng ch th vo l thy tinh c np vn v tit trng trong t sy. Lm kh silica gel nhit phng cho n khi cc ht tinh th tch ri nhau sau 14 ngy. Vn cht np v gi l trong t lnh pha trn silica gel ch nh vi 50
nhit khong 4- 6oC. Khi cn cy li, t mt vi tinh th silica ln mi trng ph hp. Phng php ny c th duy tr s sng ca vi sinh vt ti 11 nm ty thuc tng loi. Phng php ny ph hp cho cc vi sinh vt sng st trong mi trng bo qun ng kh, bao gm c cc dng si nm sn sinh ra th hch hoc cc hu bo t. 6.2.7 Bo qun trn giy thm
t mt ming giy thm ln 1 a Petri v thm t ming giy bng vi git nc ct tit trng. Ct cc mu agar t mi trng nm thun t ln trn ming giy, sau t a Petri vo t nh n 25C cho n khi nm pht trin bao ph ton b ming giy thm. Khi ming giy thm kh hon ton (khong 2-4 tun trong t nh n), dng ko v trng ct ming giy thnh tng mu ri vo cc l ng mu nh, c np. Bo qun mu trong t lnh 4oC. Khi mun cy li mu, gp 1-2 mu giy lc cy ln mi trng PDA. Sau 2 4 ngy c th nhn thy nm pht trin. Phng php ny thng p dng bo qun cc loi Fusarium. 6.2.8 Bo qun lnh su
Bo qun vi sinh vt trong t lnh su nhit thp t -20C ti 85C (bo qun lnh su) l phng php bo qun u vit i vi hu ht loi nm, vi khun v virus. Mt trong cc h thng bo qun lnh ph bin nht v n gin nht i vi nm v vi khun l vic s dng cc ht gm r (cryobeads) t vo dung dch cht lng bo qun lnh, nh glycerol, trong cc l nha nh. Sau khi cy li, dng pipet v trng ht bt cc dung dch tha ri t l dng mu vo t lnh su. Khi mun cy li vi sinh vt, ch cn ly mt trong cc ht gm v cy ln mi trng lng hoc qut ln b mt ca mi trng rn thch hp. 6.2.9 Bo qun bng nit lng
Bo qun vi sinh vt nhit cc thp t 190C n -196C trong hoc trn nit lng (cng l mt dng ca bo qun lnh su) l phng php bo qun tt nht i vi hu ht cc loi nm v vi khun. X l mu vi sinh vt phn lp, dch m hoc dch bo t vi sinh vt bng cht bo qun lnh, v d nh glycerol 10% (trn ln vi dung dch cht giu dinh dng nu x l vi khun), trc khi chuyn sang ng v trng v lm lnh ti nhit cc thp trong giai on bay hi nit lng. Tc lm lnh c ngha rt quan trng trong ton b qu trnh, vi sinh vt t t l sng st cao nht khi qu trnh lm lnh din ra chm. nhit cc thp, qu trnh trao i cht b c ch, nu vi sinh vt sng st sau bc lm lnh u tin th mu c th c lu gi v thi hn bng cch ny. Phng php ny i hi phi c trang thit b t tin v ngun nit lng sn c. i ng cn b lm vic ny cng cn phi c kinh nghim duy tr cc iu kin ti u cho cng tc bo qun mu vi sinh vt.
51
GIM NH VI SINH VT GY HI
Nhiu loi vi sinh vt gy hi khng c kha phn loi sn c. Hn na, cc ti liu tham kho v vi sinh vt hi li rt nhiu v phc tp n ni ch c cc chuyn gia v bnh cy mi c th tip cn thng xuyn c vi cc thng tin v phn loi hc ca cc nhm vi sinh vt hi nht nh. Tuy nhin, cng tc gim nh cng bt kh khn hn nu chng ta lm quen vi cc kha phn loi, ti liu tham kho v phng php kim tra mu bnh. 7.1 NM
Nm l cc vi sinh vt nh a bo, thng c dng si v mang bo t. Nm khng c dip lc, thnh t bo cha kitin hoc cellulo hoc c kitin v cellulo. Tn nm l th sinh dng ca nm bao gm mt tp hp nhiu si nm sinh trng to thnh. S pht trin ca ca h si nm xy ra cc nh ca cc si nm. Nm sinh sn bng bo t. Bo t l cc th sinh sn c bit bao gm mt hoc vi t bo. Bo t c th c sn sinh v tnh hoc hu tnh. i vi cc nhm nm nguyn thy, bo t v tnh c sinh ra trong cc ti gi l bc bo t. Mt s bo t trong nhm ny c th chuyn ng v c gi l du ng bo t. Cc nhm nm khc sn sinh ra cc bo t v tnh gi l bo t phn sinh (conidia) t cc si nm c bit gi l cnh bo t phn sinh. mt s nhm nm, bo t v tnh (conidia) c sn sinh bn trong cc cu trc c vch dy gi l qu cnh. Hu ht cc nhm nm u c giai on sinh sn hu tnh. Trong mt s trng hp, hai t bo (giao t) ha nhp vo nhau sn sinh ra hp t gi l bo t tip hp (zygospore). Trong mt s trng hp khc, hp t c gi l non bo t (oospore). i vi lp nm ti, bo t hu tnh c sn sinh ra trong t bo hp t gi l ti bo t (ascus). Bo t bn trong ti bo t gi l bo t ti (ascospore). i vi lp nm m, t bo hp t c gi l m (basidium) v bo t trong m c gi l bo t m (basidiospore). Khong hn 250.000 loi nm c kh nng gy bnh cho cy. Hu ht tt c nm gy bnh cy u sng mt phn vng i trn cy k ch v mt phn vng i trong t hoc tn d thc vt. Cc vi sinh vt truyn thng c cc nh nm hc nghin cu u thuc nhm Nm (Fungi) nhng mt s thuc nhm ng vt nguyn sinh (Protozoa) v Chromista. Protozoa bao gm c cc loi gy mc, nht. Chromista bao gm lp nm trng Oomycetes trong c c nm sng mai, Pythium v Phytophthora. C 4 lp nm chnh: Zygomycota, Chytridiomycota, Ascomycota and Basidiomycota. Bnh do nm gy ra thng c th nhn dng t b phn b bnh v dng triu chng. Cc bnh ph bin thng gp l cht cy con, thi r, ho, sng mai, phn trng, m l, chy l, g st, than en, thn th, u sng, cht cnh v cc bnh sau thu hoch (xem Bng 1).
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7.1.1
Nm gy bnh trn r
Nm xm nhim b r thc vt lm cn tr qu trnh ht nc v trao i dinh dng, kt qu l lm cho cy b ci cc, ho v l b vng. Cc r cn non rt d b nhim nm bnh v cc vt thng c gii phn r trong qu trnh trng, canh tc thng gp phn lm bnh trm trng thm. Tng t, nu t ngho dinh dng (P hoc K), mui v pH khng cn bng cng c th lm gim tnh khng ca cy i vi cc bnh v r. Cc bnh thi r thng rt kh chn on v nguyn nhn c th do c mt phc hp cc loi nm, v d nh Fusarium, Pythium, Macrophomina cng vi tuyn trng hoc mt chui nm cng gy hi. Phytophthora v Pythium thng ph bin nht trong cc khu vc t t. Rhizoctonia v Fusarium th li c mt ch yu trong iu kin m hn v trong t vi hm lng cht hu c cao. Mc d Fusarium v Phytophthora c th gy bnh r mt s cy thn g, a s cc bnh thi r ca cc cy thn g li c nguyn nhn t vic ph hy h thng cellulo ca lp nm Basidiomycetes nh Armillaria, Ganoderma, Rigidoporus v Phellinus. Mun tm hiu thm thng tin v cc bnh do Phytophthora gy ra cn tham kho thm ti liu ca Erwin and Ribeiro (1996). 7.1.2 Nm gy bnh trn thn
Ho b mch Cc vi sinh vt hi gy ho thng xm nhim hn ch trong h thng mch dn (mch g). Triu chng thng gp l cy bnh mt sc cng, l ho, bin mu (vng l), trong trng hp bnh nng th cy v cht. Ch sau khi cy bnh cht th nm mi di chuyn sang cc m khc ca cy v sn sinh bo t. Bn chi nm thng gy bnh ho l Fusarium, Verticillium, Ceratocystis and Ophiostoma. Tham kho thm thng tin v Ceratocystis v Ophiostoma trong ti liu ca Wingfield v cng s (1999). Fusarium xm nhim h mch dn v gy ho nhiu loi rau, hoa, qu, cc cy trng ly si. Hu ht cc loi gy ho nm trong nhm Fusarium oxysporum complex. Nhm Fusarium oxysporum complex c rt nhiu dng chuyn ha (forma specialis, f. sp.) khc nhau, mi dng gy hi trn mt nhm k ch nht nh v thng bao gm rt nhiu chng c kh nng gy bnh. tham kho thm thng tin v cc k thut trong nghin cu Fusarium xem Burgess v cng s (1994). Lot Cc vt lot trn nhng cy thn tho thng do nm gy ra nh Colletotrichum v Phomopsis, cc chi nm ny cng gy hi trn c l v qu. Rhizoctonia solani v Corticium rolfsii l cc tc nhn quan trng gy cc vt lot phn gc cy thn tho, c bit trn u . Thng thng c th nhn thy cc si nm trn b mt cy bnh. Phytophthora v Fusarium thng gy hi trn cy thn g mc d triu chng bn ngoi rt kh pht hin. U sng
53
U sng l triu chng pht trin khng bnh thng v kch thc hoc sng ln ca cc m thc vt do tc ng kch thch ca cn trng, vi khun, virus v nm gy bnh v d nh Exobasidium v Synchytrium. Chi rng Triu chng to thnh do s pht trin khng bnh thng ca rt nhiu ngn v r cy to thnh cc bi nh chi rng. V d, nm Crinipellis pernicosa gy bnh chi rng trn ca cao. Bnh nm hng Nm m Corticium salmonicolor to ra cc lp vy mu hng trn cnh ngn v cnh nhnh ca rt nhiu thc vt thn g nhit i v bn nhit i, gy ra bnh mc hng. Nm xm nhp vo v cy v phn g lm cht cnh ngn, cnh nhnh v lm l cy co li. 7.1.3 Nm gy bnh trn l
Triu chng trn l c tm quan trng c bit vi vic chn on bnh cy. Mt s triu chng trn l do vi sinh vt hoi sinh gy ra, mt s khc do nm bnh k sinh chuyn tnh gy ra. Triu chng ph bin trn l thng do nhiu nm bnh gy ra nhng cc triu chng in hnh thng do mt nhm vi sinh vt chuyn ha gy ra. Trn l, rt nhiu vi sinh vt c th gy cc vt m v cc mn hoi t vi cc hnh dng khc nhau. Cc c tnh thun li cho vic chn on l s c mt ca cu trc qu th nm, tui l v kch c ca vt bnh. m l thng c gii hn trong mt phm vi nh ca cc m b hoi t. Trong mt s trng hp, m bnh khng cht m ch bin mu do s c mt ca tc nhn gy bnh. Rt nhiu nm bnh (v cn trng hi) c th gy triu chng m l. Xung quanh vt m l c th c cc qung mu vng. m vng l triu chng kt hp ca rt nhiu vng trn ng tm biu hin bng cc vin trn mu m hoc nht; m gc cnh do b gii hn bi gn l v m mt cua thng c hnh gn ging ht u vi mt chm m gia. m thng l t dng m t bnh m l trong trng hp phn hoi t gia b rng ra khi l. Hin tng ny l nguyn nhn ca phn ng bo v t cy ch nhm hn ch s ly lan ca bnh. Thn th Triu chng ca bnh thn th l vic hnh thnh cc vt m hoi t mu m, lm xung, ng vin vt bnh c th ni g ln. Trong nhiu trng hp, c th nhn thy a cnh bo t nm (acervular conidiomata) tp trung thnh tng vng trn trn vt bnh. Triu chng thng tp trung trn l, thn v qu. Khi bnh nng c th gy ra triu chng cht cnh ngn v cnh nhnh. Nhiu nh bnh cy thng s dng t thn th ch cc bnh (khng phi tt c) do Colletotrichum gy ra. G trng G trng thuc b Peronosporales. Triu chng th hin in hnh bi s xut hin cc chui bc bo t sn sinh ra t cc ti bo t di biu b l. Triu chng ban 54
u l cc m nh phng rp ln di biu b. C th pht hin bo t trng trong m l. V d: g trng trn ci bp do Albugo candida gy ra. Tn li Thut ng tn li dng m t hin tng l, hoa, ngn, qu, thm ch ton b cy bnh co li rt nhanh v cht. Cc m cy cn non thng b ph hy trc. Hin tng tn li do nhiu nm bnh gy ra bao gm Colletotrichum gloeosporioides (tn li hoa xoi) v Phytophthora colocasiae (tn li l khoai s). Bng Triu chng bng l l hin tng xut hin cc vt bnh trn l ging nh b di nc nng. Vt bnh thng b mt mu, mt phn vt bnh c mu sng c. Thng triu chng ny khng i km vi hin tng a vng. V d: bnh bng l la (Gerlachia oryzae) v bng la mch (Rhynchosporium secalis). Chy l Chy l l hin tng to thnh cc mng hoi sinh mu nht trn l do nm v vi khun gy ra. Ngoi nguyn nhn do vi sinh vt hi, triu chng tng t c th do iu kin thi tit khng thun li, cy b c ch hoc do cn trng gy ra. V d: bnh o n trn la v mt s loi ha tho do Pyricularia oryzae gy ra. So So l triu chng cc vt bnh ri rc, g gh, li lm trn b mt l bnh. Triu chng c to thnh do hin tng dy ln mt cch khng bnh thng ca tng m ngoi, c th xut hin vt bnh trn b mt lp bn (lie). Nguyn nhn gy bnh so thng l cc chi nm Elsino, Fusicladium, Sphaceloma, Venturia v Cladosporium. So cng c th xut hin trn qu v thn. Sng mai Do nm thuc b Sclerosporales (cc loi gy bnh trn c) v Peronosporales (cc loi gy bnh trn cy hai l mm). Hu ht cc loi nm di chuyn xm nhim cy k ch thng qua nc b mt. Bnh sng mai c th ph hy hon ton cy trng trong iu kin m cao. V d: bnh sng mai k do Sclerospora graminicola gy ra. Triu chng xut hin nh hoa trng mt di l bnh. C th phn bit cc chi nm gy bnh khc nhau thng qua hnh dng cnh bo t phn sinh. Tham kho thm v bnh sng mai trong ti liu ca Spencer (1981). Phn trng Nm gy bnh phn trng l nm k sinh bt buc thuc h Erysiphaceae, v d Blumeria graminis gy bnh phn trng trn ng cc v c. Triu chng bnh phn trng c th hin bi s xut hin ca h si nm mu trng v cc bo t trng ging bt trng trn b mt l. C th xut hin nhiu cc qu th ti nh hnh cu pht trin trn b mt l. Vi ht k sinh c hnh thnh trong t bo biu b; cnh bo t phn sinh c hnh thnh t nhnh si nm v bo t khng vch ngn c sn sinh ra dng chui pht trin t ngn n gc. Nhiu loi nm phn trng bao gm cc dng chuyn ha (f. sp.) khc nhau k sinh chuyn tnh trn cc loi k ch khc nhau. Nm phn trng thch nghi vi iu kin kh hu kh, thc t y l nhm nm k sinh thc vt duy nht m bo t c kh nng ny mm c trong iu kin khng c nc t do. C th c thm v bnh phn trng trong ti liu ca Braun (1987). 55
Mui en Mui en do cc loi trong b Capnodiales v Chaetothyriales gy ra. Chng to thnh cc lp mui en trn b mt l v thn ti do pht trin trn cc cht tit ra t cn trng. Mui en thng c mt cng vi s xut hin ca cn trng ht nha cy nh rp. Mui en lm gim ng k kh nng quang hp, gim kh nng pht trin v gim nng sut ca cy. C th tham kho thm thng tin v mui en trong Hughes (1976). Mui en Meliolales Nm mui en thuc b Meliolales thng d nhm ln vi mui en trong 2 b Capnodiales v Chaetothyriales. y l bnh trn l ph bin cc rng ma nhit i, ch yu lm mt tnh thm m ca cy. c im ca triu chng l cc si nm vi cc ti bo t v bo t ti mu ti ni ln trn b mt biu b. Tham kho thm thng tin v b Meliolales trong Hansford (1961) v Hansford (1963). 7.1.4 Nm gy bnh trn qu v ht
Mt s vi sinh vt hi ph bin thng l nguyn nhn ca bnh thi qu. Mt trong s nhng loi ph bin nht l Colletotrichum gloeosporioides (giai on hu tnh Glomerella cingulata) gy thn th trn qu. Vt bnh thng b nt sau mt thi gian. Cc loi Phytophthora cng gy bnh thi trn nhiu loi qu, ca cao v da l nhng v d in hnh. Phomopsis v Fusicoccum cng gy ra triu chng thi cung nhiu loi qu ca cy nhit i. Qu c bit rt d b nhim cc bnh sau thu hoch. Cc bnh ny pht trin trong qu trnh ng gi, bo qun v vn chuyn. Vi sinh vt gy hi trn qu sau thu hoch thng c mt trn ng rung t trc nhng khng biu hin triu chng. Bnh c th tip tc pht trin ngay c trong iu kin bo qun lnh v triu chng c th xut hin bt c lc no trong qu trnh bo qun v vn chuyn sau thu hoch, c bit khi gp iu kin mi trng thch hp. V d mt s bnh sau thu hoch: Thi (Rhizopus stolonifer), mc xanh (Penicillium expansum), mc xm (Botrytis cinerea), thi en qu (Aspergillus spp.) thi cung (Phomopsis spp. v Fusicoccum spp.). Tham kho thm thng tin v bnh sau thu hoch do nm gy ra trong ti liu ca Pitt v Hocking (1999). Nm ca (Ergots) Nguyn nhn ca bnh nm ca l do nm thuc b Clavicipitales, bnh thng xy ra trn c v hu ht cc cy ng cc. Bo t phn sinh giai on Sphacelia ca nm ca xm nhim vo nhy hoa. Bo t c trong mt hoa cy k ch (to thnh t phn ng ca k ch trc s xm nhim ca nm) c pht tn i khp ni nh cn trng. Hch nm cha alkaloids c ln tp trong ht ng cc c hi cho sc khe ngi v ng vt nu n phi. bit thm thng tin v nm Clavicipitalean c ti liu ca White v cng s (2003). 7.1.5 Nm g st
Nm gy bnh g st thuc b Uredinales. Triu chng c th hin bng s xut hin cc mn bt trn l v thn. Bo t c th mu vng, da cam hoc nu trng ging nh g st. V d: g st ng Puccinia polysora, g st la min Puccinia 56
purpurea v g st u tng Phakopsora pachyrhizi. c thm thng tin v nm g st trong ti liu ca Cummins v Hiratsuka (2003). Nm g st l nhm vi sinh vt gy bnh cy c tm quan trng c bit v kinh t. Hn 7.000 loi thuc 160 chi c m t, trong chim s lng nhiu nht l chi Puccinia. t nht 30 chi ca nm g st ch c mt loi i din (n chi n loi). Nm g st c th c ti 5 giai on bo t trong mt vng i. Cc giai on bo t c k hiu bng cc ch s La m: O I II III IV n bo t (Spermatia) - sn sinh ra t ti n bo (spermogonia). Bo t xun (Aecidiospores) - sn sinh ra t ti bo t xun (aecidia). Bo t h (Urediniospores) - sn sinh ra t cm bo t h (uredinia). Bo t ng (Teliospores) - sn sinh ra t cm bo t ng (telia). Bo t m (Basidiospores) - sn sinh ra t m (basidium) do bo t ng ny mm to thnh. Nm g st sn sinh 5 giai on bo t gi l nm g st c chu trnh ln. Nhiu nm g st khng c kh nng sn sinh ra mt hoc vi giai on bo t, v d nm g st c chu trnh nh ch sn sinh ra giai on bo t III v IV. Nhiu nm g st c 2 k ch khng lin quan trong vng i, sn sinh ra giai on bo t II, III v IV trn k ch chnh v sn sinh ra giai on bo t O v I trn k ch trung gian. Nm g st c 2 k ch khng lin quan trong vng i gi l d ch. Nm g st c duy nht mt k ch trong vng i gi l n ch. Sau y l vng i ca mt nm g st d ch c chu trnh ln: 1. 2. Bo t m ny mm xm nhim trn l cy k ch. Ti n bo t (Spermogonia) c hnh thnh t l, gii phng ra cc n bo t nh (spermatia) di dng git nh v c pht tn bi cn trng. T mt ti n bo t s sinh ra si nm th sinh c kh nng tip nhn (th tinh) mt n bo t khc gii. Ti bo t xun v bo t xun c sn sinh ra v pht tn nh gi hoc cn trng. Bo t xun ch c th xm nhim k ch trung gian. Sau khi xm nhim vo k ch, si nm hnh thnh cm bo t h v bo t h di dng tng m bi nh c mu da cam hoc nu. Bo t h pht tn nh gi. Trong mt ma v cy trng, nhiu th h bo t h c th tip tc xm nhim trn cng mt k ch. Cm bo t ng cng vi bo t ng c hnh vo cui v sinh trng ca cy k ch. Trong mt s trng hp, bo t ng v bo t h c th sinh ra t cng mt bo t. Bo t ng c chc nng ng ngh. Mi t bo ca bo t ng c kh nng hnh thnh 1 m vi 3 vch ngn, t bo t m c sn sinh ra trn cc cung bo t. Bo t m c pht tn nh gi. 57
3. 4.
5.
Khi kim tra mu bnh g st, cn ch cc chi tit sau: Cm bo t h (Uredinia) v cm bo t ng (telia) - xut hin mt no ca l (hoc b phn khc ca cy); Bo t h (Urediniospores) hnh dng v cch biu hin triu chng trn b mt cy bnh; Si nm v tnh (Paraphyses) nu xut hin c hnh dng v kch c nh th no; L mm (Germ pores) - s lng v v tr trn bo t h; Bo t ng hnh dng, s lng vch ngn, c cung hay khng. 7.1.6 Nm than en
Bnh than en do nm than en thuc lp Ustilaginomycetes gy ra. Triu chng in hnh ca bnh l s xut hin ca cc khi bo t nh bt en. Cc bo t xut hin trong hoc trn r, thn, l, hoa, bao phn v nhy. Nm than en l vi sinh vt hi quan trng trn cy trng v cy cnh. C khong 90 chi nm than en bao gm 2000 loi khc nhau. S phn loi ca nm than en cng tri qua nhiu thay i trong nhng nm gn y da trn nhng nghin cu mc phn t v siu cu trc (s phn loi trc y thng ph thuc ch yu vo phng thc ny mm ca bo t). Tham kho thm Vnky (2002). c im d nhn bit nht ca nm than en l s hnh thnh cc m bo t t cc bo t cc b phn nht nh ca cy. Khc vi bo t ng ca nm g st, bo t teliospore ca nm than en khng c cung (pedicellate). Bo t nm g st thng c pht tn nh gi. Bo t teliospores ny mm v sn sinh ra bo t m basidiospores (sporidia) pht trin ging nh cc tn nm men trn mi trng nui cy. Trong t nhin, hai bo t m ny mm tip hp vi nhau hnh thnh si nm xm nhim. Thng thng nm ch c th xm nhim vo nhng b phn nht nh ca cy k ch, v d: nhy hoc hoa. Nhng thay i gn y trong h thng phn loi nm than en dn n s thay i tn ca nhiu loi nm than en. Sau y l mt s v d: Sporisorium c dng gii hn cho nhm Ha tho, bao gm nhiu loi nm g st trc y thuc v cc chi khc, v d: Sphacelotheca v Sorosporium; Sphacelotheca c dng gii hn cho k ch trong h Polygonaceae, nay c phn vo nhm Urediniomycetes (nhm cha nm g st); Sorosporium c dng gii hn cho hoa ca k ch thuc h Caryophyllaceae;
58
Ustilago c dng gii hn cho c trong khi phn ln cc loi nm than en gy hi trn cc cy k ch khc (ch yu cy 2 l mm) thuc chi Microbotryum trong nhm Urediniomycetes. Khi kim tra mu bnh than en, cn ch cc chi tit sau: B phn cy k ch b nhim bnh; M cy b phnh to; S c mt ca cc bc bo t (spore balls) v cc bo t ri (loose spores); Hnh dng, kch thc bo t v cch biu hin triu chng trn b mt; S c mt hoc vng mt ca cc t bo v tnh trong m bo t; S c mt hoc vng mt ca cc cung v v bc bo t trong cc bo t. 7.2 VI KHUN
Vi khun l vi sinh vt nguyn sinh n bo, khng c dip lc v c kh nng sinh sn rt nhanh. Vi khun c mt mi ni v rt a dng v sinh l, chng c th sinh sng rt nhiu h sinh thi rng ln. Bnh cy do vi khun gy ra cng xut hin mi ni trn th gii. Vi khun thch hp vi iu kin sng m v m nn chng c bit gy hi nhiu cc khu vc nhit i, bn nhit i v n i m. Hu ht cc vi khun c th sng st trong tn d cy trng, trong t hoc trn ht ca cy k ch. Vi khun xm nhim thng qua cc vt thng c gii hoc cc l h t nhin v d nh kh khng v b khng. Cc ht ging v cy con b nhim vi khun, nc, cn trng v my mc trn ng rung l cc tc nhn cho s lan truyn ca vi khun. Khng c mt h thng phn loi chnh thc no dnh cho vi khun nhng tt c cc tn ca vi khun u c quy nh. Kha nh danh vi khun Quc t International Code of Nomenclature of Bacteria (Bacteriological Code) c cc quy tc ch r cch s dng tn vi khun. Nm 1975, Bacteriological Code (bn sa i nm 1975) a ra cc khi nim v vic dng tn vi khun trong xut bn. S ra i ca bi bo v danh mc tn vi khun c cng nhn - Approved Lists of Bacterial Names (International Journal of Systematic Bacteriology, 1980, 30, 225-420) t nn mng ban u cho vic nh danh vi khun. Kha nh danh vi khun Quc t International Code of Nomenclature of Bacteria (bn sa i nm 1990) l nn tng c s cho h thng nh danh vi khun. Tn ca mt n v phn loi c hiu lc khi c xut bn (cng b), v vy n cng c ch ng trong h thng nh danh nu p ng mt trong cc tiu ch sau: Tn vi khun c trch dn trong Danh mc tn vi khun c cng nhn (Approved Lists of Bacterial Names); Tn vi khun c xut bn trong cc bi bo ca Tp ch Quc t v phn loi hc vi khun International Journal of Systematic Bacteriology 59
(IJSB) hoc Tp ch Quc t v tin ha v phn loi vi sinh vt hc International Journal of Systematic and Evolutionary Microbiology (IJSEM) v p ng cc yu cu do kha nh danh vi khun ra. K t thng 8 nm 2002, IJSEM quy nh rng tt c cc tc gi ca loi mi, chng mi u phi chng minh rng mu vi khun chun (ban u) c gi n t nht hai phng mu vi sinh vt hi c tn tui hai nc khc nhau; Tn vi khun ch c hiu lc xut bn khi c cng b trong danh mc vi khun c cng nhn. Danh mc vi khun c cng nhn l danh mc cc tn vi khun c xut bn trong tp ch IJSB hoc IJSEM hoc c xut bn ni khc nhng c cc tp ch ny cng nhn. Danh mc tn vi khun c xc nhn (Approved Lists of Bacterial Names) bao gm 2.212 tn chi, loi hoc phn loi v 124 tn ca cc n v phn loi cao hn. Cng vi vic p dng cc phng php hin i trong phn loi vi khun, n nm 2002 trong danh mc c 5.806 loi vi khun trong 1094 chi c xc nh, trong c 132 loi vi khun thuc 29 chi l c kh gy bnh cy. Cc chi vi khun hi cy ch yu l Agrobacterium, Clavibacter, Erwinia, Pseudomonas, Streptomyces, Xanthomonas v Xylella. C ti vi trm vi khun hi nm trong cc chi ny, trong c nhiu bin th gy hi chuyn tnh trn cc loi v chi k ch nht nh. V d: Pseudomonas syringae c hn 40 bin th khc nhau, Xanthomonas campestris c ti hn 123 bin th. Nhiu vi khun hi thc vt c vng i bao gm 2 giai on: giai on k sinh ngoi cy ch v giai on k sinh gy bnh. Nhiu vi khun hi khng trc tip k sinh bn trong t bo m c nhn ln khong khng gia cc t bo k ch. Vi khun xm nhim qua cc l h t nhin nh kh khng, thy khng v cc vt thng. Vi khun qua qu trnh tin ha c kh nng sn sinh ra rt nhiu c t khc nhau k c cc enzym ngoi bo, hormone thc vt v polysaccarit ngoi bo. chn on c vi khun gy hi thc vt phi kim tra cc triu chng tht cn thn v xem xt tt c cc yu t quan trng. Khi khng c du hiu ca cn trng v nm, nu thy dch chy ra t vt ct trn mu bnh th c th kt lun nguyn nhn gy bnh l do vi khun. Phng php pha long thng c s dng phn lp vi khun. Mc ch ca phng php ny l thu c cc khun lc n, v vy m c th bo m mu vi khun thu c hon ton thun khit, ch c mt loi duy nht. S dng mi trng nui cy thch hp v b mt mi trng phi kh ro trc khi cy. Phi thu c mu vi khun thun khit trc khi bt u gim nh v nu vi khun khng thun, ln ln cc loi vi nhau th mi c gng gim nh ch l v ngha. Nhiu vi khun gy bnh c th c gim nh ngay bng mt s phng php th n gin. Nn kim tra thun khit ca vi khun phn lp trc khi gim nh. Th thun ca vi khun bng cch pha long dch bo t trong nc v trng ri dng que cy vch dch vi khun ln mi trng agar kh b mt. Kim tra thng xuyn trong vi ngy xem cc khun lc c hon ton ging nhau hay khng. Cc khun lc khi khng pht trin cng nhau trng c th ging nhau nhng khi pht trin gn nhau th li c th nhn r s khc nhau. Nu nghi ng v thun khit 60
ca vi khun sau khi kim tra th nn cy truyn li n khi phn lp c vi khun thun hon ton. Khng bao gi gim nh trn mu vi khun khng thun khit. C th dng a vi khun kim tra thun quan st hnh thi khun lc. Ghi chp li cn thn cc c tnh hnh thi khun lc nh: hnh dng, kch thc, kt cu, du vt trn b mt khun lc, hnh thi dim khun lc, ng nht, mu sc, trong, c, t l mc Nn ch c cc du hiu v s thay i mu sc, to thnh kt ta hoc kt tinh trong mi trng. Kha phn loi lng phn (Dichotomous keys) l mt trong nhng kha phn loi ra i sm nht p dng cho phn loi vi khun. C th v vic gim nh vi khun nh l qu trnh tin trin tng bc mt dc theo mt con ng phn nhnh ngon ngoo. Mc d kha phn loi khng c tin cy trong lnh vc phn loi vi khun ni chung, chng vn c cc nh gim nh bnh cy ni ring s dng thnh cng cho vic gim nh vi khun gy bnh. Cng cn phi nhn mnh l khi s dng kha phn loi phi ht sc cn thn. C qu trnh phn loi c bit l nhng bc th ban u u phi c tin hnh cn thn. Ngoi vic s dng kha phn loi, c th s dng cc phng php khc nh phn tch axid bo, phn loi bng th thc khun, khng th n clone, gen th (probes) axit nucleic. Cc c tnh hnh thi c ngha khng nhiu i vi vic phn loi vi khun. Kch thc khun lc, t l mc, mu, kt cu v tnh phn quang khng cung cp cc thng tin cn thit gim nh mt vi khun. V vy, vic gim nh vi khun ph thuc rt nhiu vo chui cc phng php th khc nhau, thng lin quan n s c mt hoc vng mt ca mt s enzym nht nh. 7.3 PHYTOPLASMA
Phytoplasma, hay trc y gi l vi sinh vt th Mycoplasma, l cc vi sinh vt tin nhn thuc lp Mollicutes. Chng tng t nh vi khun nhng khng c mng t bo cng v khng th sng t do trong mi trng, cho n nay phytoplasma cha tng c nui cy trn mi trng nhn to. Phytoplasmas xm nhp vo b mch libe ca cy, hu ht c a vo thng qua mi gii l cn trng chch ht. Phytoplasmas k sinh bt buc v ton b vng i ca chng l k sinh trn m k ch. Chng gy bnh trn rt nhiu cy k ch. Triu chng thng gp l l bin vng, cy mc cn ci, thm ch cht kh, l nh, c th xut hin triu chng ging chi rng, ha l, ha xanh v hoa khng l. Cho ti gn y, phng php chnh phn bit cc loi bnh phytoplasma l triu chng hc, phm vi k ch, vector chuyn tnh v quan st lp ct m bnh di knh hin vi in t. S pht trin ca phng php sinh hc phn t vi cc on DNA mi sn c ca on gene 16S ribosomal RNA (rRNA) tng cng kh nng pht hin v gim nh phytoplasmas. 7.4 VIRUS V VIROID
Virus l vi sinh vt cc k nh b, k sinh bt buc, ch c th quan st c di knh hin vi in t. Khng ging nh vi khun v nm, virus khng c cu to t bo m ch bao gm cc v protein hay cn gi l capsid bao quanh axit nucleic (RNA hoc DNA). Virus ch c th nhn ln trong t bo sng ca k ch da vo 61
tn hiu ca qu trnh di truyn t cy k ch. Vt cht ca t bo c hon ton chuyn hng s dng cho vic sinh sn virus. S xm nhim ca virus lm suy yu cc chc nng bnh thng ca cy k ch v d nh chc nng quang hp v sinh trng. Cn trng ht nha cy nh rp v ve su thng l mi gii truyn bnh virus. Cy trng c nhn ging v tnh cng l ngun ly lan virus. Virus c th sng st trong k ch trung gian.
Viroid l cc phn t RNA c trng lng rt nh b, dng vng, khng c v protein bao bc. Chng xm nhim t bo thc vt, sao chp trc tip v gy bnh. Viroid c ly lan trc tip qua cc bin php c gii nh bm ngn, ta cnh, qua ht ging v cc bin php nhn ging v tnh nh ghp cnh.
7.4.1
Cc loi virus v thng tin lu gi
Cc loi virus khng bt buc phi c tiu bn i km v chng l cc vi sinh vt gy hi mc t bo. Cc loi virus hi thc vt, theo quy nh ca y ban v phn loi virus Quc t International Committee on Taxonomy of Viruses (ICTV) (http://www.ncbi.nlm.nih.gov/ICTV/), phi c m t v xc nh da trn s kt hp duy nht ca mt vi c tnh nh sau: Loi thc vt nhim virus ngoi t nhin; Triu chng cy bnh ngoi t nhin cc giai on xm nhim khc nhau; Dng truyn bnh, v d: tip xc hay c xt c gii, qua ht, phn hoa, mi gii; Cc loi d nhim bnh trong iu kin th nghim; Hnh dng ca virus; c tnh sinh ha ca protein v axit nucleic virus; So snh trt t nucleotide ca gene vi cc virus khc; Huyt thanh. Cc h s v virus gy bnh cy thng khng c tiu bn chun nh km l do hin nay vn cha c phng php no c th bo qun mu virus sng. Hu ht virus u khng n nh khi bo qun k c bng phng php ng kh. Nhng tin b v khoa hc hin nay cho php bo qun virus lu di mc d kh phc tp, v d nh phng php cy b gen virus vo t bo vi khun. V nhng l do trn, hu ht trong cc h s lu tr virus u khng lu gi tiu bn mu. Thay vo l lu gi cc c tnh hnh thi, v d: cc on ghi chp triu chng, nh, s liu th nghim, trt t nucleotide ca on gen v phn ng huyt thanh. Thng tin v hu ht cc loi virus gy bnh cy c th c tm thy trn h thng c s d liu trao i thng tin gim nh virus VIDE (Virus Identification Data Exchange) (http://image.fs.uidaho.edu/vide/refs.htm#descriptions). 62
Virus hnh gy Cc virus hnh gy, trong c virus khm l thuc l Tobacco mosaic virus (TMV) thng c kch thc ng knh 3 - 25 nm v chiu di 150 - 2000 nm ph thuc vo chiu di ca phn t RNA. Virus loi ny c th thng, cong hoc gp li. Cu trc bao gm RNA v protein xp theo ng xon c. Virus i xng Virus i xng xut hin n l hoc thnh tng cp c ng knh 20 - 70 nm. Khi quan st di knh hin vi in t, nhng vi rt ny c hnh dng ging nh mt cu trc hnh hc vi 20 mt i xng (12 nh v 20 mt hnh tam gic). V d: virus khm l xp l Cauliflower mosaic virus (CaMV). Virus hnh que Virus hnh que c hnh dng tng t vi khun thuc chi Bacillus. Chng khng hoc c th c mng bao bc. V d: virus khm l c linh lng Alfalfa mosaic virus (AMV) v virus hnh que hi ma Sugarcane bacilliform virus (SCBV). C th p dng mt s phng php gim nh virus gy bnh. Cc nh virus hc thng trng cc cy ch th trong nh knh chng cn trng nghin cu s xm nhim ca virus v tm ra cc cy c kh nng l k ch. Cc cy ch th thng biu hin triu chng khc nhau khi c ly nhim bng cc loi virus khc nhau. Tuy nhin, ch quan st triu chng khng thi th khng y m phi kt hp s dng cc phng php trong phng th nghim nh: huyt thanh, dng knh hin vi in t, phn tch axit nucleic. 7.4.2 Triu chng bnh virus thc vt
i vi nhng ngi lm cng tc gim nh c kinh nghim, cc triu chng bnh virus thng c th c xc nh kh d dng. C 2 loi triu chng virus ph bin: loi triu chng xut hin do kt qu ca ln xm nhim u tin vo t bo k ch, v d: vt m v loi triu chng xut hin do kt qu ca ln xm nhim th sinh hoc xm nhim c h thng, v d: khm l. Khng ging nh cc nm bnh, virus ch c th xm nhp vo t bo thc vt thng qua cc vt thng nh vt gy ca lng biu v, l thng hoc cc vt try xc nh trn tng biu b do cn trng chch ht gy ra. Triu chng ban u pht trin t im xm nhim ca virus vo t bo thc vt c gi l triu chng cc b v thng to thnh tng vt bnh c gii hn gi l vt m. Cc vt m do virus gy ra thng c kch thc to nh khc nhau, c th mt mu (do mt dip lc) hoc hoi t (nu t bo cht). Vt m thng biu hin r sau khi virus theo dng nha xm nhp vo b mt l cy hoc dng t ph bin hn l sau khi cn trng chch ht n nha l. Trong mt s trng hp, do mi tng quan gia k ch v k sinh m virus khng th lan rng ra ngoi im xm nhim. Lc vt m ti im xm nhim l triu chng duy nht quan st c. Loi phn ng ny gi l phn ng mn cm. Nu virus khng b hn ch bi phn ng ca cy k ch, chng s lan khp phn tht l. Khi nhim n h thng mch dn, virus s ly lan rt nhanh ti ton b cc b phn 63
ca cy k ch, gy hin tng xm nhim th sinh hay xm nhim h thng. Hu ht cc virus c kh nng di chuyn trong libe. Triu chng xm nhim th sinh hoc xm nhim h thng dn n nhng thay i bn ngoi m mt thng c th nhn thy c (v d nh hin tng bin mu v ho) v nhng thay i bn trong ch c th quan st c di knh hin vi in t (nh s hnh thnh cc t bo c cu trc d thng). Triu chng khm l l hin tng nhng t bo nht nh trong b phn cy b bnh b nhim virus v bin mu trong khi cc t bo khc vn pht trin bnh thng. Cc t bo b nhim virus thng c mu xanh l cy nht do lng dip lc b gim. Dng triu chng khm khc nhau ty thuc vo cc loi cy k ch khc nhau. i vi cc cy mt l mm, triu chng thng xut hin di dng khm sc hoc vt. i vi cc cy hai l mm, cc phn bin mu thng c hnh trn v xut hin triu chng nh cc vt vn, lm m, loang l, thm ch g gh. Trong mt s trng hp tng tc gia virus v cy k ch, ton b l bin vng do suy gim qu trnh sn xut dip lc v lc lp b ph hy. y l triu chng in hnh ca bnh vng l virus, vng l c ci ng v vng ln la mch. Hin tng vng l bt u t s bin vng ca phn m xung quanh gn l, phn m gn h thng mch dn c th vn xanh, tng phn vi cc phn m khc trn l. Mt s virus c th gy triu chng vng gn l v trong gn l, nh trng hp bnh gn ln rau dip v bnh khm virus turnip (Turnip mosaic virus). m vng l triu chng cc m b bnh b gii hn thnh tng vng cc t bo nhim virus. Cc t bo nhim virus c th bin mu hoc cht hoi. Cc vng c th tp trung li di dng vng trn ng tm (Hnh 15). m vng c th xut hin trn thn v qu mc d ph bin nht l trn l. V d mt s virus gy m vng: Virus m ho c chua Tomato spotted wilt virus (TSWV) v virus m vng u Papaya ringspot virus (PRSV). T bo hoi t c th tp trung thnh cc m cc b xung quanh im xm nhim hoc tp trung mt cch h thng trong cc phn c bo v khc ca cy bnh nh qu, ht hoc l. V d: virus khm l turnip gy ra cc m hoi t cc lp l ci bp pha trong. Hin tng gim kch thc cy bnh (ln, ci cc) l triu chng kh ph bin do virus gy ra, thng xut hin cng vi cc triu chng khc. Hin tng ci cc c th xy ra trn ton b cy bnh hoc ch gii hn nhng b phn nht nh ca cy nh nh sinh trng. Triu chng ny thng kh nhn thy, tr khi cy b nhim virus pht trin ngay bn cnh cy khe. Virus khm l u (Bean common mosaic virus) v virus m vng du ty (Strawberry latent ringspot virus) l nhng v d v virus gy hin tng cy bnh pht trin khng bnh thng (l v thn bin dng). S pht trin khng bnh thng xy ra do s mt cn bng hormon trong l. Ngoi ra s bin dng v pht trin khng bnh thng cn biu hin bng s tng qu nhanh s lng t bo, v d virus sng chi cacao Cacao swollen shoot virus. S tng nhanh v s lng t bo c gi l hin tng hyperplasia; s tng nhanh v kch thc t bo gi l hin tng 64
hypoplasia. V d hin tng lm trn thn cnh cam qut do virus tristeza hi cy c mi gy ra.
Hnh 15 Triu chng bnh virus (t tri qua phi): vng bin mu, m vng v khm l
Mt s virus gy hin tng tng trng qu nhanh to thnh u bu trn l v r; S pht trin qu nhanh trn l to cc mu li nh mn cm c mt trn v mt di ca l cy bnh. Virus khm bin dng u Pea enation mosaic virus (PEMV) gy hin tng u li trn l. Ging nh hin tng bin dng thn v l, u bu l kt qu ca s mt cn bng v hormon, gy hin tng tng nhanh t bin ca t bo. Hin tng bin mu cnh hoa tulip l mt trong nhng bnh virus u tin c m t vo th k 17. Bnh do virus khm l tulip gy ra, hoa tulip xut hin cc m mu sc khc nhau. C cy hoa tulip nhim virus c ngi trng hoa H Lan rt qu, thm ch bnh vn c khai thc cho n ngy nay. Virus khm turnip v virus khm vng u c th gy hin tng t bin mu trn hoa lay n. Virus c th gy triu chng mo m qu, qu t v nh. V d virus khm l da chut (Cucumber mosaic virus) gy triu chng qu nh v bin dng. Tng t, virus khm l rau dip lm gim qu trnh to ht, phn hoa bt th v suy yu. S ph hy lc lp v tng s lng t bo mt cch khng bnh thng c cp phn trn. Ngoi ra virus cn gy ra nhng bin i v m v t bo, v d th vi c hnh thnh do tc ng ca virus. Mt s virus k sinh trong nhn t bo thc vt. Nhiu virus lm bin i lc lp, phn ln trong s lm suy gim h thng sinh ha v cu trc t bo dn n hin tng mt mu sc v bin i hnh dng. Cc thay i khc v m bao gm s tng hoc gim s lng t bo, cht hoi t bo bn trong, s ha g, thoi ha v cht ca t bo libe. Virus c th tch ly vi s lng ln trong t bo to thnh cc th vi. Th vi c th c hnh thnh trong nhn nhng ph bin nht l trong t bo cht. Virus trong th vi c sp xp cnh nhau, ni ui nhau hoc chng ln nhau theo khng gian ba chiu. Nu cy khng xut hin triu chng khng c ngha l khng nhim virus. Virus c kh nng xm nhim nhng k ch nht nh v nhn ln trong t bo k ch m khng biu hin triu chng. Xm nhim tim tng kh ph bin i vi cc thc vt 65
hoang di v c di. Virus c th tn ti trn k ch trung gian v ti xm nhim cy trng sau mt thi gian vi s gip ca cn trng chch ht mi gii. S pht trin ca triu chng ph thuc vo virus v mc c ca gen. Bn thn cy k ch c th khng bnh, d nhim bnh hoc dung np bnh. Tng t, tui cy v thi gian xm nhim ng vai tr quan trng trong vic biu hin triu chng. Nhn chung, cy cng non th cng d nhim bnh v cy cng gi cng d dung np bnh. Thi im xm nhim cng sm cng thit hi v nng sut nhiu hn so vi thi im xm nhim mun. Triu chng bnh virus thng pht trin chm trong iu kin nhit cao bi v iu kin ny virus thng sinh sn chm. Tuy nhin, nhit cao cng c th lm gim kh nng khng bnh ca k ch v v th, khi nhit h xung, qu trnh xm nhim thng din ra rt nhanh. Cy pht trin trong iu kin cng nh sng ln thng t mn cm vi bnh hn l trong iu kin nh sng yu. Cy trng trn t giu dinh dng thng mn cm vi bnh hn l trong mi trng ngho dinh dng. Hm lng nit cao c th lm tng kh nng nhim bnh ca cy. Triu chng bnh do virus gy ra thng ging vi triu chng thiu dinh dng hoc hu qu ca cht c ha hc v d nh tc ng ca thuc tr c. C hai cch loi tr s ri lon dinh dng v mt cn bng ha hc: Quan st s phn b ca cy bnh. Thng thng trong trng hp ri lon dinh dng, s phn b ca cy bnh s c lin quan vi loi t hoc v tr p dng thuc ha hc. Virus thng lan truyn nh mi gii nn s phn b thnh tng m cc b hoc c mi lin quan vi ngun xm nhim, v d nh c di; M t li qu trnh lan truyn ca virus v pht trin triu chng bng cch ghp hoc chuyn nha c gii t cy bnh sang cy khe di iu kin th nghim. y l bc u tin trong vic p dng quy tc Koch xc nh nguyn nhn gy bnh. 7.5 TUYN TRNG
Phng php gim nh tuyn trng vn c dng ph bin t trc n gi l phng php so snh c tnh hnh thi ca mu tuyn trng vi cc ti liu m t xut bn, cng vi s tr gip ca kha phn loi. Mu tuyn trng c bo qun v c nh quan st v o kch thc di knh hin vi c cng nh sng cao. i vi nhiu loi tuyn trng, s dng khong 5 10 tuyn trng ci v c gim nh v mt s c tnh c trng mang tnh nh lng v s sai khc trong phm vi loi l kh ph bin. Trong nhiu trng hp, phn loi tuyn trng thc vt mc chi c th thc hin vi mt s c tnh hnh thi chung, kt hp vi nhng hiu bit v cy k ch v qun th tuyn trng ti im ly mu. Trong mt s trng hp, c th da vo cc thng tin ny phn loi tuyn trng mc loi. Tuy nhin, mt s loi tuyn trng rt kh xc nh k c khi c cc thng s v hnh thi v s o hnh th chnh xc. V vy m xu hng gim nh tuyn trng hin nay l p dng cc phng php ha sinh v sinh hc phn t. i vi mt s 66
chi tuyn trng, vic m t loi mi i hi phi c s liu v trt t nucleotide ca mt on DNA nht nh. Nhng nghin cu v phn loi da trn c tnh ha hc gip cho vic tm ra s c mt ca cc loi n m vic s dng cc c im hnh thi khng th phn bit c. Tuy vy, vic tm ra nhng sai khc v c im hnh thi cng b sung cho cc c tnh v ha hc. 7.5.1 Nhng yu cu v gim nh
S tip cn cng nh cc k nng chuyn mn cn thit trong phn loi tuyn trng ph thuc vo mc ch gim nh. Cc mu tuyn trng lu gi trong cc b su tp mu Quc gia v Quc t phi c xc nh cn thn hoc t nht l phi c mt chuyn gia v phn loi tuyn trng khng nh li. Tuy nhin, i vi nhng n v phn loi kh, cc chuyn gia v phn loi tuyn trng c th a ra cc kt lun khc nhau v trong mt s trng hp ch da vo c im hnh thi cng khng th gip g c. i vi mc ch iu tra, nhng ngi lm cng tc gim nh tuyn trng c th t kt lun, ch cn tham kho kin chuyn gia v phn loi i vi nhng loi mi cha c m t trc . Nu xut hin loi mi mu tuyn trng phi c chuyn n cc phng mu bnh Quc gia c kim tra li trong trng hp cn thit. Nu khng th vn chuyn mu v l do v kim dch hoc v thng mi, cng nn c chuyn gia kim tra li trc khi xut bn (cng b). i vi cng tc qun l hoc nghin cu v sinh thi, vic chn on cng ch cn dng li phm vi gim nh trn mu tuyn trng sng kt hp vi cc thng tin lin quan h ng vt v k ch ti im nghin cu. Tuy nhin, kt qu c th tin cy hn, nn chn mt s mu i din cc chuyn gia khng nh li. Thng i vi mc ch ny ch cn gim nh n chi l . 7.5.2 Nhn dng tuyn trng k sinh thc vt
Trong thc t sn xut, iu quan trng cn thit u tin l phi phn bit c tuyn trng k sinh thc vt vi tuyn trng k sinh trn cc gi th khc. Tuyn trng s dng thc n thc vt c cc kim ht (b phn ht thc n rng, c th th ra tht vo) trong khoang ming. Nu kim tra di knh lp soi ni cht lng tt c th xp tuyn trng ang sng thnh cc nhm dinh dng theo phng on. Hnh 16 minh ha nhng bin i v hnh thi pha trc ca tuyn trng lin quan n thi quen n ung.
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Hnh 16
Hnh thi mt trc ca mt s nhm tuyn trng. A, B, C tylenchid, aphelenchid, dorylaimid tuyn trng c kim ht, ht dinh dng t thc vt, nm v to, mt s c kh nng ht dinh dng t ng vt; D, E - rhabditid, cephalobid dngthc n vi khun; v F mononchid loi n tht.
Ngoi cc loi tuyn trng c kh nng ht dinh dng thc vt, mt s loi c cu to kim ht c th ht dinh dng t nm, to, a y, mt s khc c kh nng k sinh cc ng vt nh trong t. V vy, cn ch n hnh thi hc v s tng h vi k ch c th nhn dng nhm tuyn trng k sinh cy trng mt cch t tin. 7.5.3 Gim nh n loi
Gim nh hnh thi Tuyn trng c gim nh da vo hnh thi v cu trc gii phu bao gm c h sinh sn v s o kch thc, t l s o kch thc. Kha phn loi v cc ti liu v m t hnh thi u ch r cc c tnh gip cho vic gim nh n chi v loi. Hin nay c cc kha phn loi in t (dng phn mm my tnh). C th tham kho hai kha phn loi dng hai chng trnh khc nhau sau: Cc chi tuyn trng hi thc vt (xem www.lucidcentral.org) v Tuyn trng Australia (xem www.ento.csiro.au). Gim nh bng sinh hc phn t v ha sinh Hin nay, cc phng php DNA da trn phn tch trnh t chui nucleotide, u d, on ct DNA pht trin v t c nhiu tin b gii quyt nhng kh khn trong cng tc gim nh v phn loi. Mt s phng php da trn vic tch v nhn on DNA t cc c th tuyn trng. Mt s phng php khc c th nhn bit nh lng mt s loi tuyn trng c bit trong cc mu t. Mt hn ch ng lu l vic phn loi c th hn ch trong phm vi mt loi hoc mt nhm loi nh m s lng mu ly c th khng i din cho cc bin i trong phm vi loi. Tuy nhin, vic p dng cc cng ngh tin b ny chc chn s pht trin nhanh chng. Cc phng php phn loi da trn c tnh ha hc cng kh pht trin, bao gm phn tch isozyme, protein, huyt thanh nhng khng c p dng ph bin. Nhn dng cc loi tuyn trng nt sng bng phng php phn tch isozyme kh thc t bi v vic gim nh da trn hnh thi nhiu khi khng chnh xc. Vic p dng cc 68
phng php DNA v ha hc trong gim nh tuyn trng i hi phi c trang thit b hin i nhng mt khc li gim s cn thit phi c cc nh phn loi c k nng trong gim nh tuyn trng hng ngy. 7.6 7.6.1 CC K THUT PHN LOI Knh hin vi in t qut (Scanning electron microscopy - SEM)
Khng ging nh knh hin vi quang hc s dng nh sng quan st mu, knh hin vi in t qut (SEM) dng cc in t to hnh nh ca mu vt. SEM c phn gii ln hn rt nhiu ln so vi knh hin vi quang hc do cc in t c bc sng nh hn 100.000 ln so vi bc sng nh sng. SEM cng rt c ch trong vic nghin cu cu trc bn ngoi ca cc bo t nm. phn gii tt nht ca mt knh hin vi quang hc l 0,2 m hay 200 nm. phn gii ca SEM c th t ti 3 - 6 nm, tt hn 100 ln so vi knh hin vi quang hc. Knh hin vi in t qut cung cp cc hnh nh b mt chi tit, r nt v c chiu su hn knh hin vi quang hc, iu cng c ngha l cng mt lc c th quan st c nhiu mu hn. SEM dng mt chm in t qut b mt mu vt, to ra cc hnh nh khng gian ba chiu ca mu vt. Cc in t c kch thc rt nh v d dng i lch hng do s tc ng ca cc phn t kh trong khng kh. V vy, cc in t c th tip cn mu vt, h thng ng bn in t v bung mu vt u c bo qun trong mi trng chn khng. gi nguyn vn cu trc ca mu sinh hc trong iu kin chn khng, phi lm kh mu cn thn bng carbon dioxide (CO2) lng trong h thng my gi l my lm kh ti hn (critical point dryer). Thng thng, mu c c nh trn mt mu kim loi (thanh g mu) dng bng dnh 2 mt c bit, sau tiu bn c m mt lp kim loi mng, v d nh vng kch thch tnh dn in. Bo t nm g st v than en c thnh dy khng cn phi lm kh ti hn v c th c m kim loi ngay sau khi mu c c nh trn thanh g mu. 7.6.2 K thut ha sinh v phn t
Nu khng thy triu chng bnh xut hin trn cy khng c ngha l cy hon ton sch bnh. Trong trng hp , cc nh bnh cy nn s dng cc bin php k thut phn t v ha sinh pht hin vi sinh vt. Indexing l khi nim dng ch bt c quy trnh no c s dng kim tra s c mt ca vi sinh vt c bit n, c bit l virus k sinh trong thc vt. Indexing gip thc hin nhanh cc bin php phng tr v hn ch kh nng pht dch. Indexing cng rt quan trng trong vic thc hin kim dch hn ch s xm nhp ca bnh ngoi lai v h thng chng ch trong sn xut cy ging v nhn ging cy sch bnh. 7.6.3 Huyt thanh (min dch)
Trong k thut huyt thanh, cc khng th c hiu c to ra (nh s c mt ca khng nguyn sn sinh t vi sinh vt hi) c khai thc cho mc ch chn on. 69
Cc khng th bao gm khng th a dng polyclonal (hn hp cc khng th c sn xut ra t h min dch ca ng vt sau khi tim cht tit vi sinh vt vo mu ng vt) hoc khng th n dng (t bo ty spleen sn sinh ra mt loi khng th duy nht t h min dch ng vt c nhn v tnh bng nui cy m t bo). Mt trong nhng phng php chn on bng huyt thanh ph bin nht l k thut min dch lin kt men (enzyme linked immunosorbent assay - ELISA). Khng th c t vo cc ging trn a microtitre. Dung dch kim tra c nh vo cc ging, nu khng nguyn c mt th chng s tng tc vi khng th. Ra sch ging, t enzyme tip hp khng th vo ging v ra li ln na, sau cho gi th enzyme vo. Nu khng nguyn c trong mu, s t hp khng th - enzyme s lm bin i mu gi th. Chn on bnh bng phng php huyt thanh c rt nhiu u im. Mc d sn xut ra khng th i hi thi gian vi tun nhng nu bo qun ng phng php khng th c th duy tr c trng thi n nh trong mt thi gian di v cho kt qu nhanh chng. Phng php huyt thanh c th c p dng c trong phng th nghim v ngoi ng rung. 7.6.4 K thut nucleic axit
Cc vi sinh vt chng c cng nhiu kiu gen nhng nhn chung, cc gen c cng chc nng cc n v phn loi (taxon) khc nhau s c trnh t chui sp xp chui DNA khc nhau. S sai khc ny c th c khai thc trong gim nh bng cc k thut phn t nh: lai nucleic axit v phn ng trng hp chui (PCR). Cc k thut phn t hin i phn tch nucleic axit nhy cm n mc m trong nhng iu kin l tng, lng DNA c th c pht hin n picogram. Mt chui phn ng PCR in hnh lin quan n qu trnh lm nng DNA, enzyme DNA polymerase, on mi (DNA primers) v dNTP trong mt dung dch m ti nhit 90C lm bin tnh DNA, sau lm lnh ti khong 50-60C gn cc on mi (primers) vo cc chui DNA n sau tng nhit ln 72C, nhit ti thch cho phn ng trng hp gn cc chui DNA b sung vo nhau, bt u t v tr on mi. C sau mi vng bin tnh, gn mi v trng hp, lng DNA li c nhn i. Sau 30 vng c nhn ln theo cp s m, lng DNA tng nhiu n mc c th nhn thy c trong agarose sau khi nhum bng ethidium bromide. K thut nucleic acid p dng cho gim nh c li th l nhanh v nhy vt xa phng php huyt thanh. Nhc im ca phng php ny l phi c trang thit b, ha cht t tin, khng tin hnh c vi nhiu mu mt lc nh phng php huyt thanh v s qu nhy cm cng ng ngha vi vic l nu mu b tp th kt qu s b sai nghim trng.
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8
8.1
QUN L H S MU BNH
H THNG C S D LIU (DATABASE)
Vic nhp thng tin v h s bnh cy vo h thng c s d liu (database) in t l rt quan trng bi v nh c h thng ny, cc cn b ca phng mu c th tip cn thng tin mt cch nhanh nht m khng cn phi lc tm trong c ng ti liu. S liu c sp xp c h thng nn c th d dng tm kim, khi phc, x l v cp nht khi cn thit. Thng tin lu gi trong database c th c s dng to bn phn b vi sinh vt hi, v th rt quan trng trong cng tc kim dch v nh gi nguy c dch hi. Mt database c th ch n gin nh mt bng biu xy dng t phn mm Microsoft Excel, hoc c th l cc chng trnh c s d liu phc tp hn nh Microsoft Access, Oracle, BioLink hoc KE EMu. Nhng chng trnh ny cho php qun l cc hnh nh k thut s v ng thi c cc cng c cho php vic trao i thng tin mt cch nhanh chng. H thng Database nh KE EMu (Hnh 17) c th ni l v gi v n cho php ngi s dng tm kim cc thay i. iu ny c bit c ch khi ngi s dng mun thay i ngay lp tc cc thng s tm kim nh tn ca vi sinh vt hi v tn k ch.
Hnh 17
Mun Catalogue ca KE EMu database
Database khng ch lu gi cc hnh nh k thut s m c cc phng tin khc nh: file vn bn, file PDF, html v phim video. Ngoi ra, database cn cho php lu gi cc thng tin chi tit v ngi ly mu, ngi gim nh, ngi trng cy, v d 71
nh a ch, s in thoi, s fax, email, tiu s, ti liu tham kho. Database cn c th gip cc nhn vin phng mu ln lch, t gi cho mt s cng vic nht nh. Ngi nhp thng tin vo database phi bo m rng cc thng tin lu gi trong database l ht sc chnh xc. Hin nay c kh nhiu database v phn loi sn c trn mng Internet (Hnh 18).
www.indexfungorum.org - CABI Bioscience Database of Fungal Names
www.ipni.org - The International Plant Names Index
Hnh 18
a ch Internet v trang ch ca hai database phn loi tin cy
Trong phm vi mt Quc gia, cc database ca cc phng mu bnh phn b ri rc cc a im khc nhau c th c kt ni vi nhau qua mt h thng mng to thnh mt bo tng mu o. V d: Database v dch hi thc vt Australia Australian Plant Pest Database (APPD, http://appd.cmis.csiro.au/) l mt h thng c s d liu dch hi cy trng o, mang tnh Quc gia. APPD hp nht h s mu dch hi t hn 9 im nt khc nhau phn b trn ton lnh th Australia, to iu kin cho vic tm v tr mu dch hi mt cch nhanh chng v tm kim thng tin mt cch hiu qu. Trn phm vi ton cu, Ngun thng tin a dng sinh hc ton cu - Global Biodiversity Information Facility (GBIF, http://www.gbif.org/) c mt h thng database c xy dng vi mc ch a cc thng tin ch yu v a dng sinh hc trn th gii ln mng internet cho tt c mi ngi. GBIF s dng cng chnh ca n truy cp vo mt s databases v vi sinh vt hi v cc vn c lin quan n a dng sinh hc trn ton cu.
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9
9.1
QUN L B SU TP MU
IU KIN PHNG MU
Mt phng mu bnh phi c mt v tr an ton v vnh cu lu gi mu. Phng mu phi c kh nng chng cn trng, chng chy v chng ma dt. Mt phng mu bnh thc vt ln va phi (lu gi khong 50.000 mu) cn s dng mt din tch ti thiu 9 m2, c bit nu s dng t nhiu ngn sp xp mu tiu bn. Gi v t tiu bn bng kim loi c kh nng chng cn trng hn l lm bng g. Phng gi mu nn c h thng iu khin nhit (20-23C) v m (40-60%) khng kh. Trong iu kin nhit v m nh vy c th hn ch c s ph hy ca cn trng hi, c bit khi kt hp vi bin php t mu mi vo t trc khi lu gi trong phng mu. Nn s dng my iu ha khng kh iu khin nhit v dng my ht m gim m. Lun lun ng ca ra vo v ca s ngn nga cn trng xm nhp. Dng rm che ca s trnh nh nng mt tri. 9.2 PHNG TR B CNH CNG HI TIU BN MU
cc khu vc nhit i, nhit v m cao rt ph hp vi s pht trin ca cn trng ph hi. Mt s cn trng, c bit l b cnh cng, thng n mu tiu bn kh v nhanh chng ph hy b su tp mu. khc phc tnh trng ny nn t mu bnh vo t lnh su -20C hoc thp hn trong 7 ngy dit ht ngun su hi. Cc mu bo qun trong phng cng nn lun phin t vo t lnh su trong mt khong thi gian nht nh. Trc khi mu c a vo t lnh su, nn gi mu trong ti nilon hoc hp xp y np cht trnh ng hi nc trn mu. Sau khi ly ra t t lnh su, mu trong phng c iu ha mt thi gian cho n khi mu t n nhit phng bo qun mu. Mu bnh cn ti thng c chuyn n phng mu gim nh. Khng nn gi cc mu ti ny gn phng bo qun mu m nn kim tra mu khu vc xa phng bo qun mu. Tt c cc mu bnh khi ly ra cho mn hoc kim tra u phi vo t lnh su trong 7 ngy trc khi t tr li vo v tr c trong phng mu. Nhiu phng mu bnh c ngi chuyn trch vic xng hi phng mu mt nm mt ln bng cc ha cht kh trng c php lu hnh, v d nh methyl bromide, carbon bisulphide, carbon tetrachloride, ethylene dichloride, hydrocyanic gas, lindane, dichlorvos strips hoc paradichlorobenzene. Phi ch khi lm vic vi cc cht kh trng ny v chng c hi cho sc khe con ngi v thng d chy. Tuy nhin, nu ch kh trng xng hi khng thi th cha phng tr cn trng v bin php ny thng khng hiu qu i vi trng cn trng v nhn.
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9.3
PHNG TR NHN HI MU VI SINH VT
Vn nhn n nm phn lp trn mi trng nhn to l vn kh ph bin v nguy him cc phng mu vi sinh vt. Nhn xm nhp vo phng th nghim thng qua mu bnh ti, giy dp, qun o, cn trng v cc mu vi sinh vt phn lp t cc phng th nghim khc. Chng pht trin mnh trong iu kin nhit i. Khng ch n mu vi sinh vt phn lp, nhn cn mang bo t nm v vi khun trong rut,trn c th ri ly nhim sang cc mu khc. Nu khng c pht hin sm, nhn c th gy ha ln trong phng th nghim do chng c kh nng pht trin rt nhanh, di chuyn t a cy ny sang a cy khc trong t hoc trn bn mu cy. Nu nhn k a cy bng mt thng, c th pht hin nhn di dng cc chm trng nh trn b mt mi trng. Thng thng, nhn c pht hin qua cc vt b chng li trn mi trng nm v vi khun hoc qua cc vt b chng li trn cc git nc ng np a Petri (Hnh 19). Khi c th pht hin nhn bng mt thng cng l khi nhn bng pht. Ch ngn nga s xut hin ca nhn, khng nn n khi nhn bng pht mi tm cch dit tr. C th phng nhn nh sau: Kim tra tt c cc mu vi sinh vt chuyn n phng th nghim xem c nhn hay khng. Nu mun gi mu vi sinh vt, nn cy truyn v b mu ban u vo ni hp dit trng dit ht ngun nhn tim tng; t a cy thun v a cy phn lp ban u vo hai t hoc gi khc nhau; Hp tit trng cc mu phn lp c v cc mu cy bnh sau khi s dng cng sm cng tt; Thng xuyn lau mt bn phng mu bng cn 70% v lau bn th nghim cng nh mt trong ca t mu cy bng thuc dit cn trng khng gy hi cho nm; Bc a cy bng parafin hoc nilon siu mng, tuy nhin cch ny ch hn ch phn no v cui cng nhn vn c th xm nhp vo a cy bc; Loi b tt c cc mu vi sinh vt b nhim nhn bng cch hp tit trng. Nu mu vi sinh vt rt c gi tr v khng c mu thay th th c th mu vo t lnh su trong 24 gi dit ht nhn trng thnh v trng. Sau cy truyn vi sinh vt ln mi trng mi v hy b mu c. Mt s loi nm khng th sng st sau khi lnh.
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Hnh 19
Vt b ca nhn trong cc git nc ng trn np a Petri
9.4
CHO MN MU
Cc mu bnh lu gi c th c cho mn trong thi gian ngn cho mc ch nghin cu khoa hc. Tuy nhin, cn phi ra cc quy nh i vi ngi mn bo m s an ton ca mu bnh. Tt nht ch cho cc phng mu bnh khc mn mu vi iu kin h phi bo m an ton cho mu khi vn chuyn cng nh khi lm vic vi mu. Ngi lu gi mu c quyn quyt nh khng cho mn mu nu xem xt thy vic s dng mu l khng cn thit, khng ph hp vi mc ch nghin cu hoc cm thy mu c th b s dng khng ng ch hay b hng. Mu chuyn ra nc ngoi trong mt s trng hp phi qua x l kim dch khi n ni. Phng php x l c th l bng nhit, xng hi hoc x l bng tia phng x gamma. Cc phng php x l ny c th lm hng mu hoc tc ng n cu trc DNA ca vi sinh vt. V vy, ngi lu gi b mu phi c trch nhim p dng mi bin php bo m rng mu cho mn lun c gi trong iu kin tt. Nu nghi ng v cch x l mu hay hu qu ca vic x l mu th khng nn cho mn mu. Thi gian cho mn mu thng l 6 - 12 thng, c th ko di thi gian cho mn ty thuc vo yu cu ca ngi mn. Ngi cho mn nn yu cu ngi mn tr mu li ngay sau khi hon thnh cng vic nghin cu. Ngi mn c th tr li mt phn mu (khng phi ton b mu) nhng phi tha thun trc vi ngi chu trch nhim trng gi b mu. Mt trong nhng iu kin khi cho mn mu l mu phi c bo qun trong iu kin an ton. Khng nn gp, b hoc bp gi mu v s lm hng mu. Lun lun gi mu trong gi mu nhng lc khng dng n. Tt c cc mu u phi c gi cn thn trong qu trnh bo qun v phi gi nguyn trong gi mu ban u khi tr v phng mu c. Cc mu cho mn ch c s dng cho mc ch nghin cu nh kim tra mu tiu bn v quan st hnh thi vi sinh vt hi di knh hin vi. Ngi mn mu khng c php ly mu lu gi ring di bt k hnh thc no hay chuyn mu cho mt t chc th ba m khng c vn bn cho php ca ngi ph trch 75
phng mu cho mn. C th tch mt phn tiu bn bnh hoc tch DNA cho mt s mc ch nghin cu c th. Tuy vy, phi ht sc cn thn khi tch mu tiu bn v ch tch b phn tiu bn cn thit trong iu kin vn cn tiu bn gi li trong phng mu ban u. c bit, phi ht sc cn thn khi ct tiu bn gc (tiu bn chun). Cc bn ch thch phi c s dng ghi chp nhng thng tin lin quan n mu bnh, bao gm c nhng thng tin v danh php v phn loi. Bn ch thch phi c lm t giy lu tr cht lng cao. Di y l tt c cc thng tin cn thit khi ghi giy cho mn. Nn chun b 2 bn giy cho mn, mt bn gi cho ngi mn cng vi mu cn ngi ph trch phng mu gi bn sao: 1. 2. 3. 4. 5. S cho mn (1 s duy nht cho mi mu cho mn khng b ln). Tn v thng tin lin lc ca ngi mn mu. Mc ch mn mu (phm vi nghin cu v phn loi v danh php). Ngy mn v ngy tr mu. Mu vt cho mn (mt danh mc tt c cc tiu bn v mu cho mn).
Tt c cc mu cho mn phi c ngi qun l phng mu chng nhn, k vo v ghi ngy c th trc khi chuyn n cho ngi mn mu. 9.5 BO M AN TON CHO MU BNH
Gi an ton cho b mu di hai dng. Th nht l m bo an ton c hc cho mu v th hai l trch nhim hp php v o c ngh nghip ca ngi qun l b mu. An ton c hc bo m an ton c hc cho b mu bnh, khng nn mu ni m ai cng c th vo c. B mu nn c bo qun mt ni an ton, khng chu nh hng ca thi tit, tt nht nn khu vc c kha an ton v c bo v c ngy ln m. Nn bo qun tiu bn mu bnh trn gi hoc trong t lm bng kim loi. Nn c h thng cha chy t ng tt nht l dng kh (v nc c th lm hng mu tiu bn kh). Nn t bnh cu ha c nhn ri rc trong khu nh v i ng cn b phi c hun luyn s dng h thng cha chy ny. m bo an ton cho thng tin lu gi cng ht sc quan trng. Nn thng xuyn copy cc file my tnh cp nht vo a (c th mt ngy mt ln). Nhng cn b s dng cc chng trnh qun l d liu phc tp nh EMu ( c tho lun chi tit chng 8) cn phi c k nng c bn v my tnh c th xem thng tin v hiu nh h s mu. Trch nhim hp php v o c ngh nghip Bt c c quan no lu gi mu bnh c gi tr i vi khoa hc u phi c trch nhim hp php v o c ngh nghip bo m rng b mu lun c quan 76
tm bo v v bo qun trong iu kin tt nht. V vy, c quan lu gi mu phi hn ch ti a vic s dng cc bin php k thut khng c c s khoa hc, cc iu kin mi trng khng thch hp bo v tiu bn cho mc ch s dng hin ti v tng lai. C quan lu gi nn ra cc chnh sch v quy nh bng vn bn v vic qun l, chm sc v s dng mu bnh. C quan lu gi mu phi bo m mi iu kin tt nht v c s vt cht, cn b c chuyn mn cho vic lu gi lu di mu bnh v h s c lin quan. Chnh ph mt s ni nhn ra gi tr ca b su tp mu bnh v thng qua mt s lut v bo v mu su tp. V d, phng mu DAR c bo v theo lut v mu khoa hc nng nghip Agricultural Scientific Collections Trust Act 1983 (NSW).
77
10 TI LIU THAM KHO CHN LC

Ti liu chung Crop Protection Compendium. 2004. CAB International, Wallingford, UK. Disease Compendium Series. Minnesota, USA. American Phytopathological Society, St. Paul,
Greuter, W., McNeill, J., Barrie, F.R., Burdet, H.M., Demoulin, V., Filgueiras, T.S., Nicolson, D.H., Silva, P.C., Skog, J.E., Trehane, P., Turland, N.J. and Hawksworth, D.L. 2000. International Code of Botanical Nomenclature (Saint Louis Code). Regnum Vegetabile, 138. Koletz Scientific Books, Germany. Holiday, P. 2001. A Dictionary of Plant Pathology. 2nd Edition. University Press, UK. Cambridge
Holmgren, P.K., Holmgren, N.H. and Barnett, L.C. (eds) 1990. Index Herbariorum. Part 1: The Herbaria of the World. 8th Edition. Regnum Vegetabile, 120. New York Botanical Garden. Ploetz, R.C. 2003. Diseases of Tropical Fruit Crops. CABI Publishing, Wallingford, UK. Walker, J. 1975. Mutual responsibilities of taxonomic mycology and plant pathology. Annual Review of Phytopathology, 13: 335 355. Waller, J.M., Lenn, J.M. and Waller, S.J. (eds) 2002. Plant Pathologists Pocketbook. 3rd Edition. CABI Publishing, Wallingford, UK. Waller, J.M., Ritchie, B.J. and Holderness, M. 1998. Plant Clinic Handbook. IMI Technical Handbook No. 3. CAB International, Wallingford, UK.
Vi khun Bradbury, J.F. and Sadler, G.S. 1997. Guide to Plant Pathogenic Bacteria. 2nd Edition. CAB International Mycological Institute, Surrey, UK. Fahy, P.C. and Persley, G.J. 1983. Plant Bacterial Diseases. A Diagnostic Guide. Academic Press, Sydney. Goto, M. 1992. Fundamentals of Bacterial Plant Pathology. Academic Press, San Diego, USA. Schaad, N.W., Jones, J.B. and Chun, W. 2001. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd Edition. APS Press, St Paul, Minnesota, USA. Swings, J.G. and Civerolo, E.L. 1993. Xanthomonas. Chapman & Hall, London, UK.
78
Nm Agrios, G.N. 2005. Plant Pathology. 5th Edition. Elsevier Academic Press, USA. Ainsworth, G.C., Sparrow, F.K. and Sussman, A.S. 1973. The Fungi. An Advanced Treatise. Vols. IVA, IVB. Academic Press, New York. Arx, J.A. von. 1981. The Genera of Fungi Sporulating in Pure Culture. 3rd Edition. J. Cramer, Lehre. Barnett, H.L. and Hunter, B.B. 1998. Illustrated Genera of Imperfect Fungi. 4th Edition. APS Press, St Paul, Minnesota, USA. Barron, G.L. 1968. The Genera of Hyphomycetes from Soil. Wilkins Company, Baltimore, USA. The Williams &
Boerema,G.H., de Gruyter, J., Noordeloos, M.E. and Hamers, M.E.C. 2004. Phoma Identification Manual. CABI Publishing, Wallingford, UK. Braun, U. 1987. A monograph of the Erysiphales (powdery mildews). Hedwigia 89. Nova
Braun, U. 1995. A Monograph of Cercosporella, Ramularia and Allied Genera (Phytopathogenic Hyphomycetes). Vol. 1. IHW-Verlag, Mnchen, Germany. Braun, U. 1998. A Monograph of Cercosporella, Ramularia and Allied Genera (Phytopathogenic Hyphomycetes). Vol. 2. IHW-Verlag, Mnchen, Germany. Burgess, L.W., Summerell, B.A., Bullock, S., Gott, K.P. and Backhouse, D. 1994. Laboratory Manual for Fusarium Research. 3rd Edition. Fusarium Research Laboratory, Department of Crop Sciences, University of Sydney. Carmichael, J.W., Kendrick, W.B., Connors, I.L. and Sigler, L. 1980. Genera of Hyphomycetes. University of Alberta Press, Edmonton, Canada. Crous, P.W. and Braun, U. 2003. Mycosphaerella and its anamorphs: 1. Names published in Cercospora and Passalora. CBS Biodiversity Series 1. Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands. Cummins, G.B. and Hiratsuka, Y. 2003. Illustrated Genera of Rust Fungi. 3rd Edition. APS Press, St. Paul, Minnesota, USA. Domsch, K.H., Gams, W. and Anderson, T.-H. 1993. Compendium of Soil Fungi. Vols. I, II. Academic Press, New York. Ellis, M.B. 1971. Dematiaceous Hyphomycetes. CMI, Kew, UK. Ellis, M.B. 1976. More Dematiaceous Hyphomycetes. CMI, Kew, UK. Ellis, M.B. and Ellis, J.P. 1997. Microfungi on Land Plants. Richmond, London, UK. Erwin, D.C. and Ribeiro, O.K. 1996. Phytophthora Diseases Worldwide. APS Press, St. Paul, Minnesota. Hansford, C.G. 1961. The Meliolineae. Sydowia, Beih. 2:1-806. 79
Hansford, C.G. 1963. CCLXXXV.
Iconographia meliolinearum.
Sydowia, Beih. 5: pls. I-
Hawksworth, D.L. 1974. Mycologists Handbook. Commonwealth Agricultural Bureaux, UK. Hughes, S.J. 1976. Sooty moulds. Mycologia 68:693-820. Index of Fungi. CAB International Mycological Institute, Surrey, UK. Kirk, P.M., Cannon, P.F., David, J.C. and Stalpers, J.A. (eds) 2001. Dictionary of the Fungi. 9 th Edition. CABI Publishing, Wallingford, UK. McLaughlin, D.J., McLaughlin, E.G. and Lemke, P.A. 2001. The Mycota. Vol. VII. Systematics and Evolution. Springer-Verlag, Berlin. Mueller, G.M., Bills, G.F. and Foster, M.S. (eds) 2004. Biodiversity of Fungi. Inventory and Monitoring Methods. Elsevier Academic Press, USA. Nag Raj, T.R. 1993. Coelomycetous Anamorphs with Appendage-Bearing Conidia. Mycologue Publications, Waterloo, Canada. Pitt, J.I. and Hocking, A.D. 1999. Fungi and Food Spoilage. Aspen Publishers, Gaithersburg, Maryland, USA. Rossman, A.Y., Palm, M.E. and Spielman, L.J. 1987. A Literature Guide for the Identification of Plant Pathogenic Fungi. The American Phytopathological Society, St Paul, Minnersota, USA. Sivanesan, A. 1984. The Bitunicate Ascomycetes and their Anamorphs. J. Cramer, Vaduz, Liechtenstein. Spencer, D.M. 1981. The Downy Mildews. Academic Press, London. Sutton, B.C. 1980. The Coelomycetes. CMI, Kew, UK. Smith, D. and Onions, A.H.S. 1994. The Preservation and Maintenance of Living Fungi. 2nd Edition. IMI Technical Handbook No. 1. CAB International, Wallingford, UK. Vnky, K. 2002. Illustrated Genera of Smut Fungi. 2nd Edition. APS Press, St. Paul, Minnesota, USA. White, J.F., Bacon, C.W., Hywel-Jones, N.L. and Spatafora, J.W. 2003. Clavicipitalean Fungi: Evolutionary Biology, Chemistry, Biocontrol, and Cultural Impacts. Mycology Series 19. Marcel Dekker, New York. Wingfield, M.J., Seifert, K.A. and Webber, J.F. 1999. Ceratocystis and Ophiostoma: Taxonomy, Ecology and Pathogenicity. APS Press, St. Paul, Minnesota.
80
Tuyn trng Anon. 2005. Interactive diagnostic key to plant parasitic, freeliving and predaceous nematodes. University of Nebraska - Lincoln Nematology Laboratory, http://nematode.unl.edu/key/nemakey.htm Bell, M. 2004. Plant Parasitic Nematodes: Lucid key to 30 genera of plant parasitic nematodes. http://www.lucidcentral.com/keys/nematodes/ Eisenback, J.D. 2002. Identification guides for the most common genera of plantparasitic nematodes. Mactode Publications, Blacksburg USA. Hodda, M. 2005. Key to the nematodes of Australia. http://www.ento.csiro.au/science/nematode.html Hunt, D.J. 1993. Aphelenchida, Longidoridae and Trichodoridae : their systematics and bionomicng s CAB International, Wallingford, UK. Nickle, W.R. 1991. Manual of agricultural nematology. M. Dekker, New York. Nobbs, J.M. 2004. Plant parasitic nematodes of Australia [CD]. South Australian Research and Development Institute, Adelaide. (available from nobbs.jackie@saugov.sa.gov.au) Shurtleff, M.C and Averre, C.W. 2000. Diagnosing plant diseases caused by nematodes. APS Press, St. Paul, Minnesota, USA. Siddiqi, M.R. 2000. Tylenchida: parasites of plants and insects. CABI Publications, Wallingford, UK. Stirling, G.R., Nicol. J. and Reay, F. 1999. Advisory services for nematode pests: operational guidelines. Rural Industries Research and Development Corporation, Barton, A.C.T. viii, 111 p. (http://www.rirdc.gov.au/reports/Ras/99-41.pdf)
Phytoplasma Whitcomb, R.F. and Tully, J.G. (Eds). 1989. The Mycoplasmas. Vol. 5. Academic Press, New York.
Virus Brunt, A., Crabtree, K., Dallwitz, M., Gibbs, A. and Watson, L. 1996. Viruses of Plants: Descriptions and Lists from the VIDE Database. CABI Publishing, Wallingford, UK. Hull, R. 2002. Matthews Plant Virology. Academic Press, London, UK.
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Lacto-fuchsin Acid-fuchsin Axit lactic 0,1 g 100 ml

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