6

Trong phng th nghim

Trong phn ny, cc hng dn c a ra nhm tr gip cc cn b mi vo ngh, gip h pht trin nhng k nng cn thit cho mt cn b chn on bnh cy, bao gm cc cch: dng knh hin vi phn lp cy truyn lm thun gim nh ly bnh nhn to. Vi kinh nghim ca mnh, cn b chn on bnh cy c th thay i cc quy trnh sao cho c hiu qu cao nht. Cc phng php bo qun mu nm sng, cc cng thc nu mi trng nui cy v quy tc cng nh phng php kh trng c trnh by Ph lc 3.

6.1

Kim tra mu bnh trong phng th nghim

Nhng quy trnh sau y c p dng kim tra nhng cy c triu chng ho, ci cc v bnh trn l. Bc u tin l xem xt cn thn ton b cy v so snh nhng quan st ny vi nhng quan st trn ng rung.

Phn 6. Trong phng th nghim

59

6.1.1 Ho v ci cc
Nu cy b ho v ci cc, kh nng c nguyn nhn t bnh ho do tc b mch hoc thi thn hoc r. 1. Kim tra s ha nu ca mch dn (a) Nu mch dn ha nu, c th l bnh ho do nm hoc vi khun. Kim tra dch khun. Nu c dch khun, chun b a cy khun. Nu khng c dch khun, c th l bnh ho Fusarium hoc Verticillium. Cy phn thn ha nu ra a phn lp nm. (b)

Nu mch dn khng ha nu, c th l bnh thi r hoc thn do nm thc hoc mt loi ging nm hoc tuyn trng gy ra. (Ghi ch: nn kim tra thm cc triu chng do virt bi v mt s virt gy bnh cng gy triu chng ho v ci cc.)

2. Kim tra tm hch v si nm (a) (b) Nu c hch, tm cch phn lp Sclerotinia sclerotiorum, S. rolfsii hoc Rhizoctonia spp. da vo hnh thi loi hch quan st c. Nu khng c hch, c th l bnh thi r, tuyn trng hoc sng r cy h thp t.

3. Kim tra tm bng chng bnh tuyn trng (nt sng r hoc vt lot r). (a) (b) Nu c, tch tuyn trng ra xc nh tc nhn gy bnh v gi n mt phng th nghim tuyn trng gim nh loi. Nu khng c, v c thi r (mu nu), tm cch phn lp nm hoc tc nhn ging nm gy bnh

6.1.2 Cc bnh l
Nu l c cc du hiu bnh, kim tra vt bnh di mt knh lp soi ni. 1. Kim tra tm triu chng khm, vn, l cun hoc l ci. (a) Nu c, c th l bnh do virt. Gi mu bnh n mt phng th nghim chn on virt thc vt. (Ghi ch: bin vng v m cng c th do virt gy ra, chng hn nh virt m vng u v virt m ho c chua.) Nu khng c, c th tc nhn gy bnh l nm hoc vi khun.

(b)

2. Kim tra tm m l xanh trong dng git du, chy l hoc dch khun. (a) (b) 60 Nu c, c th tc nhn gy bnh l vi khun. Phn lp bng cch cy dch khun t nha cy ln mi trng King's B. Nu khng c, c th tc nhn gy bnh l nm.

Cm nang chn on bnh cy Vit Nam

3. Kim tra tm cc cu trc nm di knh lp soi ni. Nu c thc hnh nhiu, nm sng mai, g trng (Albugo candida), phn trng v g st c th c xc nh bng cch quan st mu bnh di knh lp soi ni v knh hin vi. Do nhng nm ny u k sinh chuyn tnh, chng khng th pht trin trn mi trng nhn to. Nu c cc du hiu ca cc nm bnh khc (si nm, bo t hoc cc cu trc sinh sn), cn chun b mu lam knh quan st di knh hin vi. Cc chi gy bnh m l thng thng c th xc nh c bao gm Alternaria, Cercospora, Stemphylium, Septoria v Phomopsis. C th kch thch s hnh thnh bo t bng cch m l b bnh trong iu kin c nh sng. Kim tra l bnh hng ngy tm du hiu ca nm bnh. (Ghi ch: nm v vi khun hoi sinh mc nhanh trong mi trng m nn c th dn n vic chn on sai.) Cng nn cy m bnh mp ngoi ca vt m l ly t mu bnh mi thu thp ln mt mi trng ngho dinh dng phn lp tc nhn gy bnh (xem Phn 6.3).

6.2

Knh lp soi ni v knh hin vi

Knh lp soi ni v knh hin vi l nhng thit b khng th thiu c trong mt phng th nghim chn on v cn b chn on bnh cy cn phi lm quen vi vic iu chnh, bo dng v s dng knh.

6.2.1 S dng knh lp soi ni

Knh lp soi ni c dng kim tra mu bnh nhm tm ra cc cu trc nm nh, nh qu cnh, a cnh, khi bo t v qu th vi phng i thp (ti khong 100). Vi knh lp soi ni, c th d dng ly v chuyn nhng cu trc nm trn sang lam knh chun b cho vic quan st di knh hin vi vi phng i cao hn (ti 400). Mt knh lp soi ni cng c s dng cho cc cng vic t m nh cy n bo t ny mm hoc cy nh sinh trng ca si nm trong qu trnh lm thun mu nm, kim tra tuyn trng v chuyn chng sang mt lam knh quan st di knh hin vi. Bng cch ny c th xc nh xem tuyn trng c phi l k sinh thc vt hay khng. Knh lp soi ni cng rt hu ch trong vic kim tra cc tn nm ang pht trin (Hnh 6.1). Mi knh lp soi ni cn c mt gng gn vo c th iu chnh nghing cung cp nh sng thng v cho gc cho cc mu c tng phn nh sng thp. L tng nht l knh c gn ngun nh sng dng ri sng pha trn b mt mu, tuy nhin nu khng c cng c th khc phc bng cch dng mt ngun sng ring r bn ngoi.

Phn 6. Trong phng th nghim

61

Hnh 6.1 Kim tra cc tn nm di knh lp soi ni

6.2.2 S dng knh hin vi

Cn tun th cn thn cc ch dn i km theo knh hin vi m bo knh c dng ng cch v trnh h hi. iu quan trng l khng c vt knh b xc hoc chm vo mt thch, nm hoc cc mu nhum. Cc ming lamen thng rt mng v, nu v, c th lm t tay. iu chnh knh hin vi 1. t mt lam knh cha mu trn bn gi ca knh hin vi. 2. Bt n v iu chnh nh sng n khong 50% sng. 3. Chnh mu tht nt vi vt knh 10. 4. ng mng ngn quang trng ca knh cho nh li. 5. Chnh nt mng ngn quang trng bng cch thay i cao ca t knh. 6. Xoay hai nt chnh trung tm t sng cho n khi nh ca mng ngn quang trng trung tm - chi tit ny rt quan trng! 7. M mng ngn quang trng cho n khi va ra khi tm nhn (ngha l cho n khi va ln hn khung nhn mt cht). 8. Chnh mng chnh m nhn r mu. S chnh (s m) trn mng chnh m nn vo khong 75% ca s m vt knh ang s dng. 9. Quan st mu (Hnh 6.2 v 6.3).

62

Cm nang chn on bnh cy Vit Nam

6.2.3 Chun b mu lam knh

Cc mu bo t nm hoc cu trc to bo t nh ti qu cnh, qu th bu hoc qu th kn c th c c nh trn lam knh trong nc. C nh mu trong nc 1. Nh mt git nc ct nh ln lam knh. 2. t mu vo v tr git nc, di mt knh lp soi ni. 3. t lamen sao cho mt cnh chm lam knh gn mp git nc. 4. T t h cnh bn kia ca lamen xung sao cho lamen trm ln trn git nc - phng php ny gip y cc bt kh ra khi mu lam. 5. Dng mt ming giy thm hoc giy lc thm i phn nc tha pha ngoi lamen. Cc bo t t cy b bnh hoc mu nm c nui cy nhn to c th c ly ra dng mt que cy v t vo v tr git nc. Cc cu trc to bo t ln hn cn c kim tra di knh lp soi ni, sau c t vo mt git nc v lm dp bng cch dng mt dng c c b mt phng p nh ln lamen. Khi kim tra li bng mu lam, c th phn bit c qu cnh v qu th: qu cnh ch cha bo t phn sinh trong khi c qu th kn v qu th bu cha c ti bo t v bo t ti.

Hnh 6.2 Quan st bo t nm di knh hin vi

Phn 6. Trong phng th nghim

63

Vt knh Bn gi T knh Nt chnh m mng chnh Nt chnh trung tm t knh

Nt chnh nt

Nt chnh bn gi

Vng chnh m quang trng

Th knh

Vt knh

Bn gi Nt chnh m mng chnh

T knh Nt chnh trung tm t knh Chnh t im th v chnh xc Nt chnh bn gi

Vng chnh m quang trng

Hnh 6.3 Cc b phn ca knh hin vi

64

Cm nang chn on bnh cy Vit Nam

Cc cu trc vi m mm, nh qu th a ca Sclerotinia sclerotiorum, cn dng li dao co t hoc dao m ct thnh nhng lt mng ri t vo v tr git nc lm mu lam.

6.3

Phn lp nm gy bnh

Khng cn phi tun th mt cch chnh xc nhng k thut phn lp nm gy thi r, thn v l sau y, m c th s dng nhng k thut ny lm nh hng v thay i chng ty theo tng tc nhn gy bnh v cy trng c th sao cho c kt qu tt nht. iu quan trng l hc hi v tch ly kinh nghim bng cch th nghim cc phng php khc nhau. Cn b lm cng tc chn on bnh cy nn tham d cc kha tp hun v cc k nng trong phng th nghim nu h cha tng c tp hun v cc k nng ny trc y. iu quan trng l hc cch phn bit gia cc loi gy bnh v vi cc loi hoi sinh thng thng.

Thc hnh quy trnh phn lp v cy truyn vi mt lot cc nm bnh khc nhau.

Cc k thut phn lp c th c ci thin bng cch: thay i thi gian kh trng b mt mu bnh gt b lp ngoi mu bnh iu chnh thnh phn mi trng nui cy (chng hn nh pH, dinh dng v nng aga) thm khng sinh vo mi trng.

Cn tyl (70%) l cht kh trng b mt chun cho cc dng c phng th nghim v cc mu bnh. Dung dch Javen cng c th c dng lm cht kh trng b mt, nhng thng mt tc dng nu lu v tip xc trc tip vi nh sng. Lun lun thm kh m bnh trn giy thm c kh trng trc khi phn lp.

Phn 6. Trong phng th nghim

65

Chn m mi b bnh phn lp. Khng nn s dng cc m b bnh lu bi v trn b mt cc m bnh ny thng c rt nhiu nm v vi khun hoi sinh pht trin.

B mt ca m cy thng c nhiu nm v vi khun hoi sinh, chng cn phi c tiu dit trc khi c th phn lp tc nhn gy bnh. Nhiu loi nm v vi khun hoi sinh mc rt nhanh trn mi trng phn lp, v th rt kh phn lp tc nhn gy bnh. Khng dng mi trng thch ng khoai ty (PDA) hoc mi trng giu hydrat cacbon phn lp nm t cc m cy b bnh, nht l nu phn lp t r. Nm v vi khun hoi sinh mc rt nhanh trn cc mi trng giu hydrat cacbon v c ch s pht trin ca nm bnh mc chm hn. S thnh cng trong vic phn lp nm t m cy b bnh ph thuc vo mt s yu t: loi m b bnh (l, thn, r) phng php kh trng b mt thao tc cy mi trng phn lp cc iu kin nui cc a phn lp.

Kinh nghim qu kh l mt cng c v gi trong vic la chn quy trnh phn lp ph hp. Kinh nghim thng gip phn on loi nm gy bnh no c kh nng gy ra cc triu chng c bit no . Khi cn nghi ng, nn ly thng tin t cc c s d liu bng hnh nh, s lu thng tin mu bnh v cc ti liu pht hnh.

6.3.1 Phn lp t l v thn

Vic phn lp t thn thng c ci thin bng cch gt b phn v hoc cc m thn bn ngoi trc khi kh trng b mt. Cc bc c bn phn lp t l hoc thn 1. Lau sch bn lm vic bng cn tyl 70% 2. Nhng dng c (kp v dao hoc dao m) trong cn tyl 70% v h kh trn ngn la. (Cn mtyl c th c dng thay cho cn tyl.) 3. Ra mu l hoc thn trong nc loi b t bi v cc tp cht khc.

66

Cm nang chn on bnh cy Vit Nam

4. Kh trng b mt m l hoc thn bng cch dng giy mm (giy n) nhng cn tyl 70% lau mt l hoc bng cch nhng nhanh l dy vo cn tyl 70% trong 5 giy, ra li trong nc v trng v kh trn giy thm v trng. 5. Dng dng c kh trng ct nhng ming cy nh (khong 2 2 mm) t phn ranh gii gia m khe v m bnh, sau cy ln mi trng ngho dinh dng (nh thch nc ct [WA]) hoc mi trng chn lc, t nhng ming cy gn mp a. 6. t a cy nhit khong 25oC, l tng l trong iu kin nh sng. 7. Kim tra a cy hng ngy, khi cc tn nm pht trin t nhng ming cy, cy truyn chng (ch ct mp ngoi tn nm) sang mi trng nh PDA hoc WA c cha cc ming m cy kh trng, chng hn nh cng la cn xanh, l cm chng hoc qu u. (Cc ming m cy c kh trng kch thch s hnh thnh bo t, gip cho vic gim nh tc nhn gy bnh.) 8. Gim nh ln cui dng mu cy c lm thun t mt bo t ny mm hoc nh sinh trng ca si nm. (Cc thao tc ny c m t trong Phn 6.5.1 v 6.5.2.) Mi trng dng phn lp ty thuc vo loi nm c nghi l tc nhn gy bnh. Mi trng thch nc ct hoc PDA mt phn t mnh, cha khng sinh nu cn, l nhng mi trng phn lp ph bin nht. C th s dng mi trng phn lp chn lc nh peptone pentachloronitrobenzene agar (PPA) cho Fusarium spp. v mi trng chn lc Phytophthora (PSM) cho Phytophthora spp. Cc nm hoi sinh, nh Alternaria, Pestalotia v Cladosporium, thng pht trin trn cc m l cht. S c mt ca nhng nm ny c th gy kh khn cho vic phn lp cc loi Alternaria hoc nm l khc gy bnh, nh Stemphylium v Bipolaris. Phng php khc phn lp nm gy bnh m l 1. t c l hoc mt mnh l trn giy m trong a Petri mi trng m. 2. t mu l nhit khong 25oC di nh sng thc y vic to bo t. 3. Kim tra sau 1-2 ngy di knh lp soi ni tm bo t hoc cc cu trc to bo t nh qu cnh, a cnh hoc khi bo t. 4. Thm vo mi trng phn lp cha WA mt git axt lactic (lm gim pH v hn ch s pht trin ca vi khun) hoc khng sinh (nh trong mi trng PPA). 5. Dng mt que cy v trng cy chuyn bo t vo a.

Phn 6. Trong phng th nghim

67

6.3.2 Phn lp t r mnh, nh

Cc r con mnh, nh, v cc r ph lm nhim v hp th dinh dng cho s pht trin ca cy v quan trng cho sc khe ca cy. Cc tc nhn gy bnh nh Rhizoctonia, Pythium, Phytophthora v Phoma thng gy bnh nhng b phn ny. Nhiu nm (nh mt s Fusarium spp. v Trichoderma spp.) v vi khun hoi sinh thng xm nhp vo cc t bo ngoi ca v r. V vy, vic phn lp tc nhn gy bnh t r con c th gp kh khn. Khng kh trng b mt cc r con qu mc bi v cht kh trng c th tiu dit tt c cc nm k sinh trong r con, bao gm c nm gy bnh. Phn lp t r mnh, nh 1. Chn nhng r con c c phn khe (khng triu chng) v phn b bnh, ra chng bng nc v trng ng trong l nh, thay nc 3 ln. Thm mt git thuc ty vo l trong ln ra u tin. 2. Lau chi bn lm vic bng cn tyl 70%. 3. Nhng dng c (kp v dao hoc dao m) trong cn tyl 70% v h kh trn ngn la. (Cn mtyl c th thay th cho cn tyl) 4. Nhng qua cc r con trong cn tyl 70%, ra nhanh trong nc v trng v kh trn giy thm kh trng. Cch khc, kh trng b mt r con bng dung dch Javen 1% trong cn tyl 10% ch trong khong 10-15 giy, sau ra bng nc v trng ngay lp tc v kh trn giy thm v trng trong t cy v trng. 5. Dng dng c kh trng ct r thnh tng ming di 1-2 mm phn ranh gii gia m khe v m bnh sau cy ln WA hoc mi trng chn lc. 6. n nh cc ming cy ln mt thch sao cho chng tip xc tt vi khng sinh trong mi trng phn lp. 7. t a cy nhit khong 25oC v quan st hng ngy di knh lp soi ni kim tra nm mc t cc ming r cy. 8. Cy truyn tng tn nm ln mi trng PDA hoc WA c cha cc ming m cy kh trng, nh cc mu thn la cn xanh. 9. Lm thun nm bng cch cy nh sinh trng ca si nm (xem Phn 6.5.1) hoc cy n bo t ny mm (xem Phn 6.5.2) trc khi gim nh ln cui cng. Cn ch l vic phn lp mt s nm hoi sinh cng mt lc vi nm bnh t m r b bnh l thng xy ra. Vi kinh nghim, ch bng quan st c th nhn ra mt s nm bnh trn mi trng PDA. Phi tin hnh ly bnh nhn to trc khi khng nh nguyn nhn gy bnh.

68

Cm nang chn on bnh cy Vit Nam

6.3.3 Phn lp t r v thn g

Nm gy bnh r thng phi c phn lp t r chnh hoc gc thn ca cy thn g. Nhn chung phn lp t m thn d thnh cng hn. Thng thng c t vi sinh vt hoi sinh phn gc thn hn l phn r ha g. Vic chn la phng php kh trng ty thuc vo mc ha g ca m. Kh trng b mt cc phn thn mm hn c th ch n gin bng cch lau hoc xt cn thanol 70% trc khi cy ln mi trng nhn to. Phn lp t r v thn g 1. Ct b cc r ph (Hnh 6.4). 2. Ra mu trong nc vi mt cht nc ty ra loi b t v cc tp cht khc. 3. Gt b lp ngoi ca thn hoc r v y thng l ni cha cc vi sinh vt hoi sinh. 4. B i phn di ca thn ni tip gip vi mt t. Vic la chn m phn lp ph thuc vo mc bnh hi. Khng c phn lp t cc m bnh c. L tng nht l dng cc mu m cy ly t ranh gii gia m khe v m bnh. 5. Phun xt mu bng cn 70%. 6. H qua la t bt lng cn tha, hoc nu thn mm th cho cn t bay hi. 7. Ct nhng ming m thn mng v cy ln mi trng ngho dinh dng hoc mi trng chn lc.

6.3.4 By t
By t l mt phng php gin tip phn lp cc loi Phytophthora v Pythium t t hoc r. By nm t t bng to hoc cc qu khc 1. Lau to vi cn. 2. Dng mt ci khoan nt chai sch ct mt l c ng knh khong 10 mm t mt bn ti li. 3. B t y l v dn l bng mt ming bng dnh t khi ri ra ngoi. 4. t to nhit phng c nh sng. 5. Phn lp nm sau 1-3 ngy t phn ranh gii gia m khe v m bnh mu nu lan ra t phn t by. Phng php ny khng phi l hon ton chn lc bi v nhm nm tip hp mc nhanh cng c th gy cc vt thng tng t.

Phn 6. Trong phng th nghim

69

By t dung dch t bng l v cnh hoa Cc loi Phytophthora v Pythium c th c phn lp t t bng cch th ni l hoc cnh hoa hng sch trn dung dch t by. Nu c cc loi ny trong t, du ng bo t c hnh thnh di chuyn ln pha trn v xm nhim vo l hoc cnh hoa. y l phng php chn lc bi v n thch hp cho vic phn lp cc loi sn sinh ra du ng bo t. 1. Cho khong 100g t vo mt cc nha. 2. nc v trng hoc nc ct vo cc sao cho ngp t khong 5-10cm.

Hnh 6.4 K thut phn lp tc nhn gy bnh t cc m ha g: (a) ct ri r ph, (b) ra mu, (c) ct b phn di thn ch tip gip vi mt t, (d) phun xt mu vi cn 70%, (e) cn bay hi, (f ) ct m thn cy bnh thnh tng ming cy nh

70

Cm nang chn on bnh cy Vit Nam

3. Th vo cc nhng mu b phn ti ca cy trng mn cm vi bnh, cc vt liu by ny s ni trn mt nc. 4. t cc nguyn v tr trong vng 2-4 ngy. 5. Phn lp nm sau 2-3 ngy t mp vt bnh pht trin trn vt liu by sau khi ra trong nc v trng v kh trng b mt, dng mi trng chn lc (nh PSM). Vt liu by cng c th l cc loi cy trng k ch ca Phytophthora hoc Pythium. Vt liu c th dng by bao gm l t, cnh hoa hng, l cy c mi, cy con t, u lupin v u tng. Nu by khng t ni c, c th treo by ln np cc sao cho by l lng mt nc hoc gn vo mt ming xp hay mt vt liu ni thch hp. Phn lp t r con dng cy k ch lm by 1. Ra r con b bnh (Hnh 6.5). 2. t r con vo mt cc nha v nc v trng hoc nc ct vo cc. 3. Th ni mt l cy k ch vo cc. 4. cc nguyn v tr trong 2-4 ngy. 5. Phn lp nm sau 2-3 ngy t mp ngoi ca vt bnh pht trin trn l by sau khi ra bng nc v trng v kh trng b mt, dng mi trng chn lc (nh PSM).

6.3.5 Phng php pha long dung dch t

Phng php pha long dung dch t c s dng phn lp cc loi Fusarium t t kh bng mi trng chn lc PPA. Phng php ny c th c p dng phn lp mt s loi nm khc s dng cc mi trng chn lc thch hp.

Hnh 6.5 By Phytophthora t t bng cnh hoa v l

Phn 6. Trong phng th nghim

71

Phn lp t t kh bng phng php pha long dung dch t 1. Phi mu t cho kh, dng ci chy nghin s t (loi ci chy lm bng dng trong nu bp) v trn u. 2. Cho 10g mu t vo mt l c cha 100mL WA 0.01% hp tit trng, to dung dch 1:10. 3. 10mL dung dch sang l th hai cha 90mL WA 0.01% to dung dch 1:100 v lc u m bo t c phn tn u trong dung dch. Lp li bc ny c dung dch vi nng 1:1000, thng th long ny l ph hp cho vic phn lp Fusarium t t trng rau mu (Hnh 6.6). 4. Phn tn u 1mL dung dch t trn b mt mi trng phn lp trong mt a Petri ng knh 90mm: chun b cc a mi trng phn lp v kh trong vi ngy loi b hi nc ng trn b mt a dng pipette cn thn ht 1mL dung dch t cho vo mt pha ra ca a mi trng cm a hi nghing t mp c dung dch v lc nh vung gc vi chiu dc dung dch t u trn b mt a. 5. t cc a phn lp ny di nh sng trong 5-7 ngy cho n khi cc tn nm pht trin. 6. Cy truyn cc tn nm v lm thun chng bng phng php cy n bo t ln mi trng PDA, thch l cm chng (CLA), hoc thch nc ct c cha mt mu m cy kh trng.

10g

10mL

10mL

nghin t 100mL 1:10 90mL 1:100 90mL 1:1000

Hnh 6.6 S cc nng s dng trong phng php pha long dung dch t

72

Cm nang chn on bnh cy Vit Nam

Mc d nng pha long 1:1000 thng c s dng, long thch hp nn s dng khi cho kt qu 10-30 tn nm trn mi a (Hnh 6.7). V mt Fusarium trong t ty thuc vo lch s rung trng v loi t, c th cn phi iu chnh nng pha long c c mt kt qu mong mun.

Hnh 6.7 a phn lp t dung dch t pha long cha Fusarium spp. trn mi trng thch peptn PCNB (l tng l s tn nm khong t 10 n 30)

Nu quy trnh phn lp c thit k cung cp s liu nh lng, dng 3-5 a nhc li cho mi nng v s dng vi mu t. C th c s khc nhau ng k gia cc a nhc li v gia cc mu t. Phng php ny khng xc nh c s lng bo t trong t, m l lng 'mm bnh' ca mt loi trong t. Cc mm bnh c th gm bo t phn sinh, bo t hu v cc mnh si nm trong tn d thc vt b bnh. S lng nhng n v to tn nm (colony-forming units - CFU) ny trong mi gram t c th c tnh cho mi loi dng cng thc sau:

long s tn nm trung bnh ca loi nm trn cc a phn lp = CFU/g t

Phn 6. Trong phng th nghim

73

6.4

Cy truyn t cc a phn lp

Cy truyn l bc trung gian gia phn lp t mu bnh v lm thun vi sinh vt gy bnh. Giai on ny gip xc nh vi sinh vt no c phn lp. Quy trnh cy truyn 1. Kim tra cc a cy hng ngy di knh lp soi ni v nh gi s pht trin ca si nm t cc ming cy. 2. Xc nh xem c nhiu hn mt loi nm mc ln hay khng. 3. Cy truyn khi si nm mc c khong 5mm t ming cy. 4. Ct mt ming thch nh (2 2 mm) t ra mi tn nm v cy sang mi trng PDA hoc mt mi trng c cha gi th t nhin (nh mi trng CLA hoc mi trng thch thn la cn xanh). C mt s bnh nm m tc nhn gy bnh c phn lp mt cch d dng v cc vi sinh vt hoi sinh him khi cn tr vic phn lp. Chng hn nh Sclerotinia sclerotiorum v Sclerotium rolfsii c th c phn lp d dng t ranh gii gia m khe v m bnh thn bnh cn xanh. Vic phn lp t cc b phn khc ca cy c th kh khn hn. Khi phn lp t r b bnh, thng c khong hai loi nm hoc nhiu hn mc t r, thm ch ngay c trn mi trng chn lc (Hnh 6.8). Vn tng t c th xy ra khi tm cch phn lp nm bnh t vt m l, bi v nm hoi sinh mc nhanh trn cc m l b bnh. Cn ch cy truyn khi cc tn nm cn nh v cy truyn c thc hin d dng hn trc khi cc loi mc nhanh mc ln t cc loi mc chm. V vy, cn kim tra cc a phn lp hng ngy tm nm mc. Quan st cc ming cy xem c hin tng hnh thnh bo t khng, y c th l du hiu nhn ra tc nhn gy bnh. Tuy nhin, nn lu rng bo t hnh thnh c th t nm hoi sinh.

Thc hnh nhiu s dn n s thnh cng! Thc hnh phn lp trn nhiu loi nm bnh khc nhau ly kinh nghim v hc cch nhn bit s pht trin ca cc nm gy bnh thng thng trn cc a phn lp.

Cc nm hoi sinh thng xut hin trong m bnh v thng pht trin trn a cy phn lp (Hnh 6.9). Mt s, nh Trichoderma, cn tr qu trnh phn lp. Cc nm hoi sinh lan truyn qua khng kh, nh Penicillium v Cladosporium cng c th gy nhim cc a cy.

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Cy truyn

3mm

Hnh 6.8 S mt a phn lp t r cho thy (hnh trong) nhiu loi nm mc ra t cng mt on r

Hnh 6.9 Nhng nm tp thng thy trn cc a cy: (a) Penicillium sp. (gy tp t khng kh), (b) Cladosporium sp. (trong mu cy sch), (c) Trichoderma sp. (mc t mt mu r b bnh)

Phn 6. Trong phng th nghim

75

Mt khi cc mu cy truyn pht trin thnh cc tn nm, chng c th c nhm li theo hnh thi tn nm v cc c tnh hnh thi khc cho vic gim nh ban u. Cc nm hoi sinh ph bin c th c xc nh, loi b v cc nm c th l tc nhn gy bnh c th c lm thun bng phng php cy nh sinh trng si nm hoc cy n bo t sang mt mi trng thch hp gim nh sau ny.

6.5

Lm thun mu nm

Giai on cui cng trong vic gim nh nm gy bnh l vic lm thun mu nm. Ch mt bo t hoc mt nh sinh trng ca si nm c cy sang mi trng sch m bo nm c cy l hon ton thun.

6.5.1 Cy n bo t
Cy n bo t l qu trnh cy truyn mt bo t ny mm to mt mu nm thun (Hnh 6.10). Phng php ny thch hp cho vic lm thun nhng chi nm to bo t trn mi trng nhn to, nh Fusarium, Colletotrichum, Alternaria, Stemphylium, Bipolaris, Verticillium v Phoma. Cy n bo t 1. Kh trng que cy. 2 & 3. To dung dch bo t bng cch dng que cy ly mt lng nh si nm trn mt thch c ln bo t hoc ly mt cht bo t t khi bo t ln ca Fusarium spp. ri cho vo ng nghim cha 10mL nc v trng. 4. Lc ng nghim phn tn cc bo t v kim tra mt bo t bng cch quan st ng nghim trc nh sng hoc kim tra mt git dch bo t di knh lp soi ni. Trnh to dch bo t vi mt bo t qu cao. Bng kinh nghim, mt bo t c th c nh gi bng mt thng khi nhn ng nghim. Lm long vi nc v trng nu cn. dch bo t vo mt a Petri c cha mt lp mng mi trng thch nc ct. dch bo t tha t a Petri i. Mt s bo t s nm li trn mt thch. dng a Petri trong khong 18 gi cho n khi bo t ny mm. Kim tra a Petri di knh lp soi ni vi ngun sng pha di. (iu chnh gng trn ngun sng cn thn c s tng phn tt gia thch, bo t, v cc ng mm.) Dng mt que cy dp ct ly ra mt bo t ny mm (Hnh 6.11) v chuyn sang mt a mi trng mi (Ph lc 1 m t cch lm mt que cy dp).

5. 6. 7. 8. 9.

10.

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Kh trng que cy 1

dch bo t vo mt a mi trng thch nc ct mng 6

Co cc bo t t ra tn nm 2

b dch bo t tha 7

Cho bo t vo nc v trng 3

dng a ln trong 18 gi 8

Kim tra mt bo t 4

Dng que cy kh trng ct mt bo t ny mm 9

Lm long nu cn 5

Chuyn sang mt mi trng mi 10

Hnh 6.10 Cc bc cy n bo t

Phn 6. Trong phng th nghim

77

Mt qu cao

ng mt

Hnh 6.11 Quy trnh cy n bo t, thao tc chn la mt bo t ng cch

6.5.2 Cy nh sinh trng si nm

Cy nh sinh trng si nm l qu trnh cy truyn nh sinh trng ca mt si nm to mt mu nm thun. Phng php ny thch hp cho vic lm thun cc chi nm nh Phytophthora, Pythium, Rhizoctonia, Sclerotium v Sclerotinia. Cy nh sinh trng si nm 1. mi trng thch nc ct vo a Petri nghing sao cho phn thch mt pha ca a nng (Hnh 6.12). 2. Cy truyn mt ming nh nm t a phn lp vo mt bn a ni thch su hn. 3. t a di knh lp soi ni v iu chnh tiu im si nm ra tn nm. (Cc si nm s mc rt tha phn nng ca thch) 4. iu chnh ngun sng (gng) nhm t tng phn tt gia mi trng v si nm. 5. Dng que cy dp kh trng cy mt ming thch nh cha nh sinh trng ca mt si nm sang mt a mi trng thch hp.

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Cm nang chn on bnh cy Vit Nam

Ch cy phn nh ca mt si nm duy nht m bo mu nm c lm thun hon ton.

Hnh 6.12 Cy nh sinh trng si nm, v d u ca mt si nm Rhizoctonia sp. c ly t a mi trng thch nghing.

6.6

Nhn bit cc mu nm thun

C th c s khc nhau ng k trong hnh thi v mu sc cc tn nm ca cng mt loi. Vic chn on tr nn d dng hn khi cc cn b chn on hc cch nhn bit tn nm ca cc loi nm thng thng trn mi trng nhn to (Hnh 6.13). Cc mu nm sau c th c phn loi d dng thnh nhm cc loi nm bnh v cc loi nm hoi sinh bng mt thng. Thao tc ny c th lm gim bt s lng mu nm cn lm thun, tit kim chi ph v thi gian.

Phn 6. Trong phng th nghim

79

Pythium aphanidermatum

Pythium irregulare

Phytophthora nicotianae

Fusarium oxysporum

Fusarium solani

Aspergillus niger

Rhizoctonia sp.

Rhizoctonia sp.

Rhizoctonia sp.

Sclerotium rolfsii

S. rolfsii (tri) v S. sclerotiorum (phi) nhn gn

Sclerotinia sclerotiorum

Pestalotia sp.

Phoma terrestris

Colletotrichum sp.

Hnh 6.13 Tn nm ca mt s nm bnh thng thng trn mi trng thch ng khoai ty

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Vic gim nh cui cng cc mu sch cn da vo: cc c tnh vi hnh thi (nh qu th v tnh v hu tnh) hnh thi bo t hoc hnh thi hch nm hnh thc to bo t (nh bn cht ca t bo sinh bo t v s to thnh hay khng to thnh chui bo t). Mt s nm gy bnh quan trng khng to bo t v ch c th nhn bit qua s c mt v hnh thi ca hch nm (nh Sclerotinia spp., Sclerotium spp. v mt s loi Rhizoctonia). Nu khng c s hnh thnh cc qu th v tnh hoc hu tnh, hay cc bo t hoc hch nm th vic gim nh n loi c th gp kh khn.

6.7

Gim nh nm gy bnh

C hn 10.000 loi nm v cc loi ging nm gy bnh cy. Nhng vi sinh vt ny l tc nhn gy ra rt nhiu cc bnh, bao gm m v chy l, thi thn, lot, thi r, ho v cht mn, cc bnh cy con, qu, thi nn v ht, u sng, g st, than en v sng mai. Vic gim nh chnh xc mt tc nhn gy bnh l bc u then cht trong vic xy dng cc bin php phng tr v qun l bnh hi tng hp. Nu xc nh c tn tc nhn gy bnh, th c th tm thng tin v c tnh sinh hc, dch t hc v cc bin php phng tr qua cc ti liu xut bn hoc qua mng internet.

Vic gim nh chnh xc tc nhn gy bnh l v cng cn thit cho vic la chn ng thuc tr nm trong trng hp phng tr bng bin php ha hc. V d, mt s thuc tr nm c th phng tr bnh sng mai th khng c hiu qu i vi bnh phn trng.

Vic gim nh tc nhn nm gy bnh bt u bng vic da vo cc c tnh hnh thi, nh bo t v cc cu trc to bo t. Nhn chung, cn quan st di knh hin vi (xem Phn 6.2) c th nhn ra tc nhn gy bnh v chn on bnh. Vic gim nh hnh thi c tr gip nh cc kha phn loi, ti liu hng dn, v cng c nhiu hnh nh minh ha rt hu ch cho nhiu loi nm bnh thng thng trong Agrios (2005). Nn lu gi mt s sch v cm nang khc lm ti liu tham kho trong cc phng th nghim chn on, v may mn l nhiu tp ch khoa hc v phn loi v gim nh c th c tip cn qua mng internet.

Phn 6. Trong phng th nghim

81

Cc nm k sinh chuyn tnh ch c th mc trn m k ch cn sng (nh sng mai, phn trng v g st) v khng th phn lp c trn mi trng nhn to. V vy, vic gim nh hnh thi ty thuc vo vic kim tra cn thn cc bo t v cc cu trc to bo t trn m bnh. Nhiu nm gy bnh khc c th c phn lp v nui cy trong mi trng nhn to trong cc iu kin tiu chun. Cc cu trc to bo t v bo t ca nhng nm bnh ny c th c quan st trong mi trng nui cy cho cc mc ch gim nh. V d nh hu ht cc tc nhn nm gy bnh l u to ra cc cu trc to bo t - qu th, qu cnh, a cnh, bc bo t hoc cnh bo t phn sinh - v nhng cu trc ny u c th quan st c di knh hin vi. Kim tra s ha hp sinh dng l mt phng php khc c th c dng xc nh dng. Nghin cu kh nng kt hp trong sinh sn hu tnh cng gip tm hiu v s a dng di truyn v c th c s dng phn bit cc loi khc nhau nhng c hnh thi tng t. Tuy nhin, nhng k thut ny i hi nhiu kinh nghim v trang thit b hn l nhng g c sn trong nhng phng th nghim chn on nh.

6.8

Ti liu tham kho

Agrios G.N. 2005. Plant pathology, 5th edition. Elsevier Academic Press: San Diego, California.

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