TRNG I HC LM NGHIP KHOA LM HC ----------------***------------------

BO CO KHA LUN TT NGHIP

Tn kha lun : PHN LP V TUYN CHN MT S CHNG BACILLUS THURINGIENSIS C C TNH CAO I VI CN TRNG HI CY TRNG

NGNH : CNG NGH SINH HC M S : 307

Gio vin hng dn : Ths. Nguyn Th Thu Hng : Ths. H Vn Ging Sinh vin thc hin : Trn Vn Tin Kha hc : 2006 - 2010

H Ni, nm 2010

LI CM N Ti xin by t lng bit n su sc n Ths. Nguyn Th Thu Hng B mn Ging v Cng ngh Sinh hc Khoa Lm hc Trng i hc Lm nghip tn tnh trc tip hng dn v ch bo ti trong qu trnh thc hin kha lun ny. T i xin gi li cm n n thy c gio trng i hc Lm nghip ging dy cho ti nhng nn tng kin thc vng chc c th tip thu tt hn nhng kin thc khoa hc mi v to cho ti c hi hc hi qu gi ny. Ti xin chn thnh cm n Ths. H Vn Ging ng gp kin qu gi cho ti c th hon thnh bn bo co Kha lun mt cch tt nht( CH NY C CHO VO KHNG C). Cng vi lng bit n su sc gi ti ton th cc thy, c v cc anh, ch trong B mn Ging v Cng ngh Sinh hc, gia nh, bn b, nhng ngi gip , dy v ng vin ti trong sut qu trnh hc tp. H Ni, thng 5 nm 2010 Sinh vin

Trn Vn Tin

MC LC

DANH MC CC BNG TRONG KHA LUN STT 1 2 3 4 5 6 7 8 K hiu bng Bng 1.1 Bng 1.2 Bng 1.3 Bng 2.1 Bng 2.2 Bng 3.1 Bng 3.2 Bng 3.3 Bng 3.4 Bng 3.5 Bng 3.6 Bng 3.7 Bng 3.8 Tn bng Mt s type huyt thanh v cc chng i din tng ng Mt s chng Bt thng c ng dng trong sn xut ch phm Mt s nghin cu v vi khun Bt Ngun thu thp vi khun Bt t cn trng Ngun thu thp Bt t cc mu t khu vc Trng HLN Bt phn lp c t cc mu su Bt phn lp c t cc mu t Hnh dng, mu sc v ng knh khun lc ca cc chng Bt Hnh dng t bo, bo t, tinh th c ca cc chng 9 Bt S lng bo t/ml dch ln men (thu hi sau 60h ln 10 men) T l phn trm su n l da chut v da hu cht 11 bi ch phm Bt T l phn trm su n l ci bp v su ho cht bi 12 13 ch phm Bt Kt qu bo qun cc chng vi khun Bt phn lp Tran g

DANH SCH CC T VIT TT STT 1 2 3 4 5 6 7 8 K hiu vit tt Bt G6 T5 KTX KHV k C h Gii thch Bacillus thuringiensis Hi trng G6 Nh thc hnh T5 K tc x Knh hin vi ng knh i chng Gi

DANH MC CC HNH NH TRONG KHA LUN K hiu Tn hnh hnh Hnh 1.1 Hnh nh v vi khun Bacillus thuringiensis Hnh 1.2 Qu trnh xm nhp v gy c ca Bt Hnh 2.1 Hnh nh cc ngun mu thu thp vi khun Bt Hnh 2.2 Hnh nh v su th hot tnh Hnh 3.1 Mt s hnh nh trong qu trnh phn lp Bt Hnh 3.2 Hnh 3.3 Hnh dng t bo, bo t, tinh th c cc chng Bt Hnh nh v hiu qu dit su n l da chut v da
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STT
1 2 3 4 5 6

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hu

Hnh 3.4 Hnh nh v hiu qu dit su n l ci bp v su ho

T VN Vit Nam c kh hu nhit i gi ma, khng kh vi m cao l iu kin thun li cho cc loi su hi cy nng - lm nghip pht trin. Khi mun bo v nng sut cy trng, theo thi quen, ngi nng dn thng s dng thuc ho hc vi nng cao phun ngay sau khi dch su hi bng pht. i vi cy lm nghip, mi khi dch xut hin vn m, cc cnh rng trng th s lng ho cht phi dng l rt ln. Trung bnh mi ha cy trng phi phun t 5 7 kg thuc. iu qu thc l mt vn nghim trng i hi cc nh khoa hc ni chung v cc nh bo v thc vt ni ring cn nghin cu v xem xt mt cch y , bi thuc ho hc tuy dp tt c nn dch nhanh nhng cng l con dao hai li, s trc tip ph hu mi trng sng khu sn xut , nh hng nghim trng n sc kho ca con ngi, lm gim s lng sinh vt c li cho con ngi nh chim chc, tm, c, nhng k sinh thin ch nh b ra, ong k sinh Tc hi ca vic s dng thuc tr su ho hc rt r rng. Trong khi vic s dng thuc tr su sinh hc c ngun gc t vi khun, vi nm, virus, cn trng, tho mc c chng minh rt an ton i vi ngi v gia sc, khng gy nhim mi trng, khng git cht thin ch su hi v sinh vt c ch, c th duy tr cn bng sinh thi, khng nh hng n cht lng nng, lm sn... Trong khi , thuc tr su sinh hc, k c thuc tr su c ngun gc t Bacillus thuringiensis (c tiu th nhiu nht trong s cc loi thuc tr su sinh hc) vn cha ph bin. Nguyn nhn l do thi quen s dng thuc tr su ho hc n su vo tim thc ngi nng dn nc ta; thuc tr su sinh hc sn sut trong nc c hiu lc cha cao, tnh n nh cn thp; thuc tr su sinh hc nhp ngoi th c gi thnh cao; vic s dng thuc tr su ho hc va tit kim hn v chi ph, va t c hiu qu tiu dit su nhanh hn

Bi nhng l do nn thuc tr su sinh hc vn khng phi l la chn hng u ca ngi nng dn khi mun dp tt nn dch su hi. t c li ch kinh t trc mt, ngi nng dn thng s dng thuc tr su ho hc. Do vy, gp phn a thuc tr su sinh hc ni chung v thuc tr su c ngun gc t vi khun Bt ni ring n vi ngi nng dn, th vic ph bin nng cao tm nhn thc ca ngi dn v thuc tr su sinh hc kt hp vi vic cc nh khoa hc nghin cu sn xut c nhiu hn cc loi thuc tr su sinh hc hot lc cao, gi thnh h l rt quan trng. Trong cng nghip sn xut thuc tr su vi sinh, yu t ging vi sinh vt gi vai tr quyt nh nng sut, cht lng, sn lng v gi thnh sn phm nn cng tc phn lp, tuyn chn v bo qun chng ging c ngha rt quan trng. V vy, cn tip tc pht trin hng nghin cu phn lp, tuyn chn, ng dng, phn loi, nng cao c tnh dit su cng nh thc y s dng thuc tr su Bt trong nng lm nghip. tip tc hng nghin cu phn lp v la chn cc chng Bacillus thuringiensis c c lc cao, ng dng c hiu qu vo thc tin, ti tin hnh kho lun Phn lp v tuyn chn mt s chng Bacillus thuringiensis c hot lc cao vi cn trng hi cy trng

CHNG 1 TNG QUAN VN NGHIN CU 1.1. Lch s nghin cu v ng dng Bacillus thuringiensis Lch s nghin cu v ng dng Bacillus thuringiensis (Bt) ra i v pht trin cng vi s pht trin khoa hc k thut ca nhn loi, Bt c nghin cu v ng dng trn nhiu lnh vc khc nhau [21],[2]. Nm 1901, Ishiwatari Shigetane - nh sinh vt hc ngi Nht - khi ang thc hin mt cuc iu tra nghin cu nguyn nhn gy bnh Sotto gy cht t ngt, git cht nhiu qun th tm, phn lp c mt loi vi khun (chnh l Bacillus thuringiensis) l nguyn nhn gy bnh, v ng t tn loi vi khun l Bacillus sotto. y chnh l ln u tin con ngi pht hin ra vi khun Bt. n nm 1911, Ernst Berliner - nh sinh vt hc ngi c - phn lp c vi khun git hi mi Mediterranean flour. ng pht hin ra y chnh l loi vi khun m Ishiwatari Shigetane cng b, v t tn li cho loi vi khun ny l Bacillus thurigiensis (hay Bt), xut pht t a danh Thurigia l mt th trn nh c, ni pht hin ra con mi . Nm 1915, Ernst Berliner tip tc a ra bo co v mt loi c t c bn cht l protein c Bt sn sinh ra trong c th, v theo ng chnh l nguyn nhn khin cc con mi b git hi. Tuy nhin, tc dng v c ch hot ng ca loi protein ny vn cha c khm ph. Nm 1920, nhng ngi nng dn cc trang tri ln ti cc nc pht trin bt u s dng sinh khi vi khun Bt phun cho cy trng nh mt loi thuc phng tr su bnh. c bit l nc Php, sm bt u ch to cc loi thuc c ngun gc t bo t v xc ca Bt, gi l Sporine. Nm 1956, Hannay, Fitz-James v Angus nghin cu v pht hin ra tc nhn chnh quyt nh kh nng tiu dit mi v su b ca Bt l cc phn t protein c sn sinh trong c th vi khun Bt, t m ra hng nghin cu mi ca cc nh khoa hc v tc nhn, c ch dit su v di truyn ca Bt.
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T nm 1958, cc ch phm thuc tr su c ngun gc t Bt bt u c s dng rng ri M, Anh, c Ti nm 1961, Bt c nh gi l mt loi thuc tr su thn thin vi con ngi v mi trng trong chin lc pht trin nng nghip ca t chc bo v mi trng EPA (Environmental Protection Agency) ca M. Nm 1977, c 13 loi vi khun Bt c pht hin v cng b. Cc nghin cu v khm ph v ph khng ca Bt cho thy Bt khng ch gy c duy nht vi mt giai on nht nh ca cc con u trng ca b cnh vy, m cn gy c c vi u trng ca b cnh cng. T nm 1980 tr i, thc ca con ngi vi vn mi trng sng tng cao, kh nng khng c ca su bnh vi cc loi thuc ho hc k c cc loi cc c nh DDT v 666 ngy cng gia tng. ng thi, con ngi cng pht hin c lng tn d cc ho cht phng tr su hi c tch lu v gia tng dn trong mi trng sng gy nh hng nghim trng ti h sinh thi v sc kho con ngi. gii quyt nhng vn , cc ch phm thuc tr su c ngun gc t Bt ngy cng c s dng rng ri bi tinh th c do Bt tit ra c bn cht l mt loi protein, d dng b phn hu nhanh chng trong mi trng, khng nh hng n sc kho con ngi, vt nui ... Ngy nay, cc nh khoa hc pht hin ra hn 1000 s bin dng ca c t trong c th Bt. Bn cnh , s pht trin nhanh chng ca sinh hc phn t gip cc nh khoa hc ng dng cng ngh GMO (Genetically Modifie Organisms) trong chuyn gen iu khin s sn sinh c t ca Bt vo c th ca cy trng, gip cy trng chuyn gen c th tit c t dit su n l hoc c thn. Ng v la l hai ging cy trng u tin c chuyn gen Bt do t chc EPA ca M thc hin nm 1995. Cho ti hin nay, cng ngh GMO chuyn gen khng su ng dng thnh cng cho nhiu loi cy trng khc nh: Khoai ty, bng, khoai lang, u... [8],[2]. 1.2. i cng v vi khun Bacillus thurigiensis 1.2.1. c im hnh thi ca Bacillus thurigiensis Bacillus thurigiensis l vi khun t, gram (+), h hp hiu kh hoc hiu kh khng bt buc. T bo hnh que, kch thc khong 3 - 6m, c tim
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mao ph mng, c kh nng di ng, t bo ng n c hoc xp thnh chui. Bacillus thurigiensis c kh nng sinh ni bo t v tinh th c. Bo t hnh trng vi kch thc khong 1,5 2 m. Tinh th c c kch thc khong 0,6 x 0,02 m v c nhiu hnh dng nh hnh van, hnh lp phng, hnh sao, hnh trng, hnh kim ... Khi t bo sinh dng b ph v, ni bo t v th vi c gii phng (xem Hnh 1.1) . Ging nh bo t ca cc loi vi khun khc thuc chi Bacillus, bo t ca Bt rt bn, c kh nng khng li nhit, bc x, ho cht cao. C th quan st thy bo t, tinh th c, t bo sinh dng ca Bt di knh hin vi in t, knh hin vi quang hc [1], [7]. 1.2.2. c tnh sinh ha ca Bacillus thurigiensis Bacillus thurigiensiss sinh trng ti u nhit 28 30oC, pH 6,8 7,2. Trong qu trnh sinh trng, vi khun chuyn ho thnh phn ng ca mi trng thnh acid acetic, lactic, pyruvic Sau nhng cht ny li c c th s dng tip. Do pH ca mi trng lc u th tng (pha logarit), v sau th gim. Kh nng sinh bo t ca Bacillus thurigiensis ph thuc vo nhiu yu t m c bit l cc yu t dinh dng. Vi khun c kh nng nitrit ho, khng c kh nng ln men ng arabinoza, xiloza, manitol v pht trin yu trong mi trng k kh [7],[3].

Hnh 1.1: Hnh nh v vi khun Bacillus thuringiensis

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1.2.3. Cc c t ca Bacillus thurigiensis Bacillus thurigiensiss c kh nng to 4 loi c t: + Ngoi c t ( - exotoxin) + Ngoi c t ( - exotoxin) + Ngoi c t ( - exotoxin) + Ni c t ( - exotoxin) Trong 4 loi c t, ni c t c tc dng mnh nht n nhiu loi cn trng v c ng dng rng ri trong bo v thc vt nh mt loi thuc su khng c hi vi mi trng, con ngi v ng vt [6],[2]. 1.2.3.1. Ngoi c t Ngoi c t c pht hin gia nhng nm 1950 t vi khun Bacillus thurigiensis vanenlesti bi C. Toumaroff. Ngoi c t cn gi l enzyme Leucintinase, c hot tnh phospholipase - tc ng ch yu ln gc phospholipit ca mng t bo su, gip cho vi khun gy bnh d xm nhp vo cc xoang trong c th cn trng. Do vy, enzyme Leucintinase trc tip tham gia vo vic tn cng, gy tn thng t bo thnh rut. Ngoi c t c trng lng phn t thp, tan tt trong nc, hot ng pH 6,6 7,2 v khng bn nhit nn cn c gi l ngoi c t khng bn nhit, tc ng c hiu n cc loi nh: ong cn l, su thuc b cnh cng, cnh vy ...[5], [9]. 1.2.3.2. Ngoi c t Do Hall v Arkavc pht hin nm 1959 khi nui u trng mui bng thc n cha Bt. y l mt c t bn nhit c bn cht l nucleotide. Khi x l 120oC trong 15 pht vn gi c hot tnh. C ch tc ng ca ngoi c t l cnh tranh ATP vi cc enzyme ARN polymerase, dn n km hm hot ng ca enzyme, ngng tng hp ARN, gy ri lon sinh tng hp protein. Mt khc, khi ngoi c t cng hng tc ng vi ni c t c th khin cn trng cht nhanh chng. Ngoi c t gy c cho nhiu loi cn trng thuc b cnh cng, hai cnh, c bit khi cn trng giai on u trng, su non. Ngoi c t gy
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cn tr s lt xc ca cn trng. Nu c s dng nng cao, c t cn tiu dit c trng cn trng [7]. 1.2.3.3. Ngoi c t Ngoi c t l mt phospholipase, c bn cht l mch peptide ngn v mt s axit amin t do. c t ny tan tt trong nc, mn cm vi khng kh, nh sng, nhit v xi nn t hu dng trong thc t ng rung. C ch tc dng ca ngoi c t cng tng t nh ngoi c t [10]. 1.2.3.4. Ni c t Ni c t c hnh thnh trong khong 3h ca pha cn bng. Tinh th ny bn nhit (ti 80oC), khng tan trong nc, dung mi hu c nhng tan tt trong mi trng c pH kim. Mi t bo Bt c th sinh ra mt hoc hai tinh th c. Khi lng tinh th c chim ti 30% khi lng t bo mang n. exotoxin cn c gi l protein tinh th c do n tn ti dng tinh th cng vi nhiu protein tinh th khc nh cu ni disufua v lin kt k nc. V bn cht c t ny l mt protein vi 1180 gc axit amin trong ch yu gm cc loi nh glutmic, asparaginic nhng axit amin ny chu trch nhim cho c tnh im ng in thp ca tinh th. Cystein tuy chim t l thp hn nhng n gp phn quan trng trong vic gi vng cu trc tinh th, lm tinh th khng c kh nng ho tan. Ngoi ra, trong tinh th cn tp ln mt s hydrocacbon vi t l khong 5,6%. V thnh phn nguyn t, toxin cha ch yu cc nguyn t C, H, O, N, S. Cc nguyn t Ca, Mg, Si, Fe chim t l nh hn. Ni, Ti, Zn, Mn, Cu chim t l rt nh, cn P hu nh khng c [11],[13],[2]. 1.2.4. Tnh a dng ca Bt. Cho n nay cc nh khoa hc phn lp, phn loi c mt s lng ln cc chng Bt da theo cc c im sau: - Kh nng hnh thnh enzyme leucitinase. - Cu trc tinh th, kh nng gy c. - c tnh huyt thanh hc (khng huyt thanh tim mao H).

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- Phn ng ngng kt ca cc t bo sinh dng vi cc khng huyt thanh. Phng php phn loi theo c tnh huyt thanh l ph bin. Theo phng php ny, da vo 50 type huyt thanh chun ngi ta chia cc chng Bt phn lp c ra lm 63 loi ph (subspecies) [14],[3],[2] . Mt s type huyt thanh v cc chng i din tng ng (xem Bng 1.1). Bng 1.1: Mt s type huyt thanh v cc chng i din tng ng Type huyt thanh 1 Chng i din B. thuringiensis THU Bliner (1915), Heimpel& Vit tt Ngi nghin cu

Angus (1958) 3a, 3b 4a, 4b B. kurstaki B. sotto KUR SOT De Barjac & Lemill (1970) Ishiwata (1905); Heimpel & Angus (1958) 5a, 5b 8b, 8d B. candensis B. nigeriensis CAN NIG De Barjac & Bonnefoi (1972) Weiser (1984) 14 30 33 46 48 49 50 B. israelensis B. medellin B. leesis B. chanpaisis B. balearica B. muju B. navarrersis ISR MED LEE CHA BAL MUJ NAV De Barjac & Cs (1992) Orduz & Cs (1992) Lee & Cs (1944) Chainpaisang (1994) Iriate Garcia (1995) Park (1995) Iriate Garcia and prasertphon

Gn y, nh s pht trin mnh ca cng ngh gen, cc nh khoa hc gii m c hu ht cc gen m ha cho tinh th c. T c s , mt phng php phn loi khc c a ra: phng php phn loi da vo
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lp gen Cry. Vi phng php ny, cc nh khoa hc a ra 20 lp gen vi cc c im protein tinh th c, type huyt thanh, cn trng ch khc nhau. 1.3. Cc yu t hnh thnh tinh th c Bao gm tt c cc yu t nh hng n sinh trng, pht trin ca vi khun Bacillus thurigiensis nh: nhit , ngun C, N, P ... iu kin ti u cho Bt sinh trng l 28 30oC. Nu nhit l 15oC th bo t khng hnh thnh v s lng tinh th c cng gim i ng k. Ngun dinh dng C, N, P nh hng mnh n s hnh thnh tinh th c. Mt s acid amin nh leucin, isoleucin gy c ch qu trnh sinh trng ca vi khun nn gim lng tinh th c. Cc yu t cn tr s trao i acid acetic cng lm c ch s hnh thnh bo t, tinh th c. Cc chng t bin mt kh nng sinh protease ngoi bo th khng c kh nng sinh tinh th c v bo t [15], [16]. 1.4. C ch tc ng ca tinh th c ln cn trng 1.4.1. Qu trnh hot ho tinh th c Theo Angus (1907), protein tinh th c ca Bt hot ng trn t bo biu m rut gia ca cn trng pH kim. Tinh th tan vo mi trng kim ca rut gii phng ra protein tinh th, cht ny sau b phn ct bi protease. Kt qu l t mt protein 135 kDa b mt i mt na cn li phn li xp x 60 kDa bn vi protease. Nhng th nghim ch ra rng protein 135 kDa cha b ct bi protease khng c kh nng gy c vi cn trng gi l protoxin" . cn li 60 kDa gy c vi cn trng gi l toxin. Qu trnh bin i t tinh th c protoxin thnh toxin c gi l qu trnh hot ho tinh th c. Qu trnh hot ho tinh th c gm hai giai on: Giai on mt lin quan ti s gii phng protoxin t tinh th c. Giai on ny din ra do s ct t cc lin kt disulfua (ni gia cc phn t protein) trong mi trng bi cc nhn t kh disulfua. Giai on hai l s tiu ho protoxin thnh toxin. Nm 1990 Choma v cs bo co rng bin phn t protoxin CryIAc 135 kDa thnh li toxin 60 kDa phi tri qua by bc m mi bc phn t protoxin s b mt i mt mnh khong 10 kDa, trong qu trnh ny s ct b din ra khng theo mt trt t no. Tuy nhin, n nay c ch ca s ct b bin
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protoxin 135 kDa thnh toxin 60 kDa vn cha c lm sng t, v cc nh khoa hc vn cha xc nh c protoxin b ct t c hai u C v N hay b ct tun t t u C [17],[3]. 1.4.2. C ch hot ng ca toxin trn t bo biu m rut gia cn trng Nhng nghin cu v hnh thi sau khi b cht bi Bt ca cn trng cho thy rut ca cn trng b tn thng. C th l do hnh thnh cc l hng trn cc t bo biu m, dn n gy ri lon hot ng hp th dinh dng ca rut. C ba m hnh gii thch c ch hot ng ca toxin nh sau: - M hnh 1, thng c gi l m hnh dung gii ho tan thm thu keo bao gm s tng hp cc l khng c trng bi phn t c t. Khi c t lin kt vi cc receptor trn mng s hnh thnh mt phc h. Phc h ny to nn mt l xuyn qua mng t bo biu m cho php s qua li t do ca nhiu phn t vi kch thc khc nhau dn n gy ri lon chc nng ca rut [10], [14]. - M hnh 2, cho rng l hng c hnh thnh bi c t Bt l c trng rt cao i vi ion K+. Thuyt ny c a ra t bng chng l s vn chuyn axit amin ph thuc K+ b c ch bi c t tinh th. C th, nhng l ny d cho K+ i vo, sau lm gim gradient in th mng lm ri lon s vn chuyn amino axit trong c th su [14], [17]. - M hnh 3, cho rng c t Bt hot ng trn nhiu knh hin c ca t bo biu m rut. S c mt ca cc c t dn n cc knh cho php K + trn vo khng gii hn t lm c ch s hp th axit amin [19]. 1.4.3. Tc ng chn lc ca c t Bt ln cn trng S d c t do Bt tit ra c tc ng chn lc ln cn trng bi tinh th c sau khi xm nhp vo rut cn trng, c th gy cht cho vt ch th phi c hot ho thnh toxin v toxin phi gn c ln t bo biu m rut. Tuy nhin, qu trnh hot ho tinh th c ph thuc rt nhiu vo pH rut v h enzyme. Nu pH < 8 th hu ht cc tinh th c khng gii phng ra protoxin. Mt khc khi pH ph hp (pH > 8) nhng h enzyme khng ph hp th toxin cng khng c to ra. Mt iu na, khi tinh th c c hot
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ho thnh toxin nhng nhng toxin ny khng bm c ln mng t bo biu m rut, c t v tc dng. V d: su xm hi rau c pH rut l 9,5 v c kh nng hot ho tinh th c nhng khng b tiu dit, chng hon ton kho mnh khi c nui vi thc n cha ch phm Bt v trn mng rut ca chng khng cha cc th th toxin ca Bt bm vo [12]. Hnh nh v qu trnh xm nhp v gy c ca Bt( xem Hnh 1.2).

Hnh 1.2: Qu trnh xm nhp v gy c ca Bt 1.5. u nhc im ca thuc tr su Bt * u im - Khng gy c hi cho ngi v cc ng vt mu nng v pH ng rut ca cc i tng ny thp khng th hot ho c tinh th c. - Tc ng ln cn trng mt cch chn lc nn trong nhiu trng hp ngi ta c th s dng nhng chng Bt xc nh tiu dit cn trng ch m khng nh hng n cn trng v hi khc. - Ch phm Bt khng nhim mi trng nh cc loi thuc tr su ho hc. ng thi n khng tc ng xu ln cht lng nng sn [21], [4]. * Nhc im - Ch phm Bt khng c kh nng dit cn trng bn trong thc vt (thn, r...)
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- c t Bt lng ng nhanh trong nc v d dng b hp th bi cc hot cht ho hc lm cho lng c t n c cn trng gim i. - Khi c t ri xung t d b vi sinh vt phn gii; trong iu kin b chiu sng v di tc ng ca mt s nhn t mi trng khc c t Bt rt d b mt hot tnh. - Hiu qu cha tht cao do din bin chm khi gp iu kin thi tit bt li th kh t kt qu tt. - Kh cn ong ngoi ng rung, thi gian bo quan ngn thng t 1 2 nm iu kin lnh kh. - Gi thnh cao [1], [2],[3]. 1.6. nh hng ca thuc tr su Bt n con ngi, ng vt v thc vt 1.6.1. nh hng ca Bt n sc kho con ngi Khi cc ch phm Bt c ra i v ng dng rng ri, ny sinh cho con ngi mt mi lo ngi do khi s dng Bt phn ln s dng bnh xt thuc hoc vi, v th m Bt d dng tip xc vi da ca ngi nng dn, gi thit t ra l ngi ngi phun mt lng thuc ng k c th gy ng c. Ngoi ra cn c th gy pht tn bo t. Tuy vy, nhiu bo co nghin cu cho thy khng h c s kch ng i vi da ca con ngi cng nh khng xut hin nhng nh hng khng tt i vi sc kho ca con ngi [20],[18]. Trc khi c a ra th trng, cc ch phm v cy trng mang gen Bt phi tri qua rt nhiu th nghim qun l nghim ngt trong bao gm cc nghin cu v c tnh v kh nng gy d ng. Cc Bo v Mi trng Hoa k (US Environmental Protectin Agency US-EPA) trin khai nhng nh gi c t v thm ch cc protein Bt c th liu lng cao hn. Theo Extension Toxicology Network (Extoxnet), cc d n v thng tin thuc tr su mt s trng i hc ca Hoa k cho thy Kt qu cuc th nghim trn 18 ngi mi ngy n 1 gram Bt thng mi trong vng 5 ngy, v trong cc ngy khc nhau khng gy ra chng bnh g. Nhng ngi n 1 gram Bt/ngy trong 3 ngy lin tc hon ton khng b ng c hay nhim bnh.

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Hn na, mc phn t protein nhanh chng b phn hy bi dch v d dy (trong iu kin phng th nghim) [Extoxnet, 1996]. Nhiu th nghim trn con ngi vi nhng ngi tnh nguyn c thc hin bi t chc y t th gii (WHO) v t chc mi trng th gii (UNEP), kt qu thu c cho thy: mc d nhng ngi tnh nguyn thng xuyn n v ht vo mt lng ln ch phm Bt v Btk, nhng khng h thy bt k mt biu hin bt thng no xy ra trn c th con ngi. T nhng nghin cu c th ca cc t chc uy tn trn th gii, cc nh khoa hc cng nhn Bt v cng ngh Bt hon ton v hi vi con ngi v sc kho cng ng. Nm 1995, chng trnh Cc vn hp tc quc t cho s qun l cht ho hc (IOMC) hp tc gia cc t chc UNEP, ILO, The Foof and Agriculture Organization of the United Nations, WHO nhm thc y hp tc pht trin kinh t, chia s v phi hp trong cc chnh sch chung nhm qun l cht thi c nh hng nguy hi ti i sng ca con ngi. Chng trnh khuyn khch vic pht trin s dng Bt thay cho cc loi thuc tr su hi ho hc kinh in khc. 1.6.2. nh hng ca Bt ti cc loi ng vt Tinh th c do Bt sn sinh ch yu tiu dit cc loi u trng gy hi i vi cn trng nh cc loi cn trng thuc b cnh cng, cnh vy, cc loi mi mt, mui gy bnh Cc loi tinh th c ca Bt c tnh c hiu nht nh i vi cc loi nht nh v ngay c mt loi th cng ch gy c mt giai on sinh trng nht nh l giai on su non hay u trng, theo nguyn tc cha kho v kho. Cc tc nhn gy c ca Bt ch c th gy hi khi tc ng vi th th ph hp trn c th u trng, i vi cc loi protein khng ph hp khc th s khng khp c vi loi protein ca vt ch lm cho c t khng pht huy c tc dng. Thm vo , mi trng khc nhau trong thnh rut cc loi sinh vt khc nhau v trong giai on sinh trng khc nhau cng l yu t to ln quyt nh tnh chuyn bit ca protein c t. Trong lch s nghin cu v Bt trn th gii, nhiu nghin cu trn cc loi ng vt hoang d c thc hin: Nm 1989, Bendell tin hnh cc
19

nghin cu theo di tc ng ca Bt ti qun th cc loi ng vt c v nh mt s khu rng gn trang tri sn xut nng nghip Canada. Kt qu thu c khng h c s bin i s lng qun th ny k c cc loi thuc b gm nhm, h chut ng l nhng sinh vt thng xuyn n c ven rng v trong cnh ng Beavers v cng s, 1989; Lattin v cng s, 1990; Beavers, 1991 tin hnh nhng nghin cu trn cc loi chim trn th gii: Cc loi chim c chn nghin cu c x l bng ch phm Bt v theo di, tnh ton s bin ng trong qun th. Kt qu nghin cu cho thy rng khng h c s biu hin ca bnh trn chim, v khng c s suy gim s lng c th chim trong qun th. Nhiu nghin cu khc ca cc nh khoa hc thuc cc quc gia tin tin trn th gii cng tin hnh nhm iu tra nh hng ca ch phm Bt v cy trng mang gen Bt i vi cc loi sinh vt t v cc loi cn trng khc c xem l c ch trong nng nghip. Kt qu phn tch trong mt thi gian di cho thy, Bt v cng ngh s dng cy trng chuyn gen Bt khng gy ra nh hng bt li khng ch i vi cc sinh vt t c ch m cn vi cc sinh vt t khng phi l ch tn cng ca chng, thm ch ngay c khi cc sinh vt ny c x l Bt vi liu lng cao hn nhiu so vi thc t c th xy ra trong iu kin trng trt m con ngi c th a vo. T cho thy khng c s thay i no trong qun th vi sinh vt t gia cc cnh ng s dng thuc tr su Bt v cnh ng khng s dng [Donegan v cng s, 1995]. Cc cuc th nghim ca Extoxnet nm 1996 c tin hnh trn ch, chut, chut lang, th, c, ch, k ging v chim cho thy: protein Bt khng gy ra nh hng xu hay bin i s lng qun th. Cng cn nhn mnh rng, c t cng hon ton khng gy nh hng n cc loi cn trng c ch hoc ng vt n tht nh ong mt v b cnh cng. Hu ht cc ch phm ca Bt nh Bt, Btg, Btt, Btte qua nghin cu u cho kt qu m tnh vi nhng tc ng bt li n th gii cc loi ng vt. Ch nhng loi ng vt gy hi

20

chuyn bit mi b tc ng ca Bt, cc loi ng vt khng phi l ch ca Bt th khng h chu bt k mt tc ng bt li no t Bt [22]. 1.6.3. nh hng ca Bt n cc loi thc vt Khi cng ngh Bt hin i pht trin, cng vi s pht trin ca sinh hc phn t v khoa hc k thut cho php con ngi chuyn gen sn sinh protein c hiu ca Bt vo trong c th ca cc loi cy trng. K thut ny c p dng trong cc loi cy trng nh Ng, La, Bng Tuy nhin vn t ra gy lo ngi cho con ngi y chnh l cy trng mang gen Bt cho sn phm cht lng nh th no, c nh hng ti h sinh thi v qun th cy trng trn cc cnh ng khng? Vn ny tr thnh mt ch nng bng khi vo nm 1999, mt bo co khoa hc cng b v nh hng c hi ca ht phn t cy ng mang gen Bt n u trng ca loi bm c li Monarch. Bo co ny a ra gy mt mi quan tm c bit v lo ngi v nhng ri ro m thc vt chuyn gen Bt c th gy ra i vi sinh vt c ch v sinh vt trung lp trong t nhin. Tuy nhin, nhng nghin cu trong thi gian gn y ca cc nh khoa hc cho thy loi ng chuyn gen sn sinh c t ca Bt gy nh hng khng ng k i vi qun th bm Monarch trn cnh ng. Mt chng trnh hp tc nghin cu gia cc nh khoa hc Hoa K v Canada cung cp nhng thng tin nhm xy dng qu trnh nh gi ri ro tiu chun v nh hng ca ng Bt i vi qun th bm Monarch. Cc nh khoa hc i n kt lun rng, hu ht cc ging lai thng mi th protein c t Bt xut hin vi nng thp hn nhiu trong ht phn v khng h gy nh hng c hi n cc loi bm trn cnh ng. Qun th cc loi sinh vt c ch v trung lp khc khng h b suy gim trn cc cnh ng ny [22]. Nh vy, cy trng s dng cng ngh cy gen Bt (cng ngh GMO) khng h tc ng n a dng sinh hc trn cc cnh ng, cng nh sn phm ca cy trng Bt khng gy tc ng no i vi cc sinh vt s dng chng cng nh i vi con ngi.

21

1.7. Tnh hnh nghin cu, sn xut v s dng thuc tr su Bt 1.7.1. Tnh hnh nghin cu, sn xut v s dng thuc tr su Bt trn th gii T khi pht hin ra vi khun Bt, con ngi ngy cng nhn thc c r hn v tc dng v hiu qu ca loi vi khun ny. Mt lot cc nghin cu cp quc t v cc nghin cp quc gia v ang c ra nhm pht trin cng ngh s dng Bt mt cch rng ri trong cng ng vi mc ch bo v mi trng sng ca con ngi v mi trng t nhin trong sch [21], [3]. Nm 1938, ch phm Bt c sn xut v bn ra u tin t phng th nghim ca Php. Tip sau , mt lot cc nh sn sut ca cc nc khc trn th gii sn xut thuc tr su c ngun gc t Bt vi cc tn thng phm nh: VMP, Pipel, Biobit.. Trung Quc, sn lng thuc tr su Bt tng ln rt nhanh t 26 tn nm 1983 ln 260 tn 1986. n nm 1990 sn lng t 900 tn. Nga t nm 1987 t sn lng 6100 tn/nm, phc v cho 110 ha rung. Ti M, lng ch phm Bt c s dng ln ti hn 1000 tn/nm. Ngoi vic ng dng bo v cy trng, ch phm Bt cn c nhiu nc s dng trong cng tc v sinh dch t nh dit tr cc loi mui, rui.. bo v sc kho con ngi. 1.7.2. Tnh hnh nghin cu, sn xut v s dng thuc tr su Bt Vit Nam Bt c du nhp vo nc ta t nhng thp nin 1970 nhng do iu kin khch quan v kinh t m nc ta cha c iu kin p dng cng nh quan tm n vn ny. Trong vi nm tr li y, khi mt lot cc vn mi trng trong nng nghip c khm ph gy nhiu d lun bc xc, lo ngi cho sc kho con ngi. Vic nghin cu v ng dng Bt c xem nh l mt cu cnh trong lnh vc phng tr su hi. Tuy vy, vic s dng Bt nc ta hin nay cn kh nhiu hn ch, c th l trong lnh vc trin khai, nc ta cn rt thiu cc iu kin, phng tin nghin cu, c rt t cc nh khoa hc i chuyn su trong lnh vc ny. Bn cnh cc c s thc nghim v cng ngh sinh hc ca nc ta cn nh hp, s lng t i, l mt trong nguyn nhn ln dn
22

n s hn ch trong vic sn xut v s dng cc loi thuc tr su Bt [21], [16]. V mt qun l, nc ta cn rt th s v yu trong cng tc ging, cng tc kim nh, tng tr n nghin pht trin, thu thp lai to Mt khc, trong phn phi sn phm, nc ta li qu quan tm n qung b cho cc loi ho cht bo v thc vt ho hc, do vy vic qung b cho thng hiu Bt khng c quan tm ng mc. ng thi trong cng tc khuyn nng, nhng hn ch v tuyn truyn ngi nng dn nng cao hiu bit cn yu km, ngi nng dn ch mun c hiu qu tc thi v s dng thun tin, chnh v th m t quan tm n cc ch phm Bt. Tuy nhin, bc u cc chuyn gia nc ta thuc Vin cng ngh sinh hc (Vin Khoa hc v cng ngh) tin hnh nghin cu v sn sn xut thnh cng nhiu thuc tr su sinh hc Bt t hiu qu cao, ha hn mt tng lai tt p cho nng sn Vit Nam. Sau y l bng thng k cc nghin cu v mt s chng Bt chnh c ng dng trong sn xut ch phm Vit Nam( xem Bng 1.2 v Bng 1.3). Bng 1.2: Mt s chng Bt thng c ng dng trong sn xut ch phm STT 1 2 3 4 5 6 7 8 9 Tn gi Bacillus thuringiensis Bacillus thuringiensis subspecies aizawai Bacillus thuringiensis subspecies darmstadiensis Bacillus thuringiensis subspecies entomocidus Bacillus thuringiensis subspecies israelensis Bacillus thuringiensis subspecies kurstaki Bacillus thuringiensis subspecies konkukian Bacillus thuringiensis subspecies tenebrionis Bacillus thuringiensis subspecies galleriae K hiu Bt Bta Btd Bte Btk Btko Btt Btte Btg

23

Bng 1.3: Mt s nghin cu v vi khun Bt ( phn ny em tm m khng c nhiu, c c b sung cho em nh! Em cm n c!) STT Tn ti nghin cu 1 Nghin cu sn xut ch phm vi sinh t chng vi khun Bacillus thuringiensis subsp. israelensis serotype H14 dit lng qung mui Tc gi TS. H Th Hng Nhung v cs - Vin Pasteur TP.HCM (2009) Phn lp cc chng B. thuringiensis var. kurstaki Vit Bi Th Hng Nam v cs Vin sau thu hoch (2003) Phn loi B. thuringiensis bng phng php nh typ inh Duy huyt thanh ca Ohba v Aizawai Vit Nam KhngPhng Vi Sinh Phn t, Vin Cng ngh Sinh hc (2003) a dng phn t ca Bacillus thuringiensis cc tnh La Th Nga v bc b v bc trung b cs Vin Cng ngh Sinh hc (2003) Phn loi cc chng Bt theo typ huyt thanh Lecadet va cs (1999) Phn loi gen m ha ni c t Delta Bt v cn trng Hoft v ch tng ng Whiteley (1989) Phn loi gen m ha cho cc protein c t dit cn Hoft v trng Whiteley (1989)

5 6

24

CHNG 2
MC TIU I TNG - NI DUNG PHNG PHP NGHIN CU

2.1. Mc tiu nghin cu Mc tiu chung Phn lp c mt s chng Bt t cn trng b vi khun Bt k sinh v t. Trn c s chn lc c chng c hot lc cao trong dit su hi cy trng, lm tin cho nhng nghin cu to ch phm vi sinh trong bo v thc vt. Mc tiu c th + Phn lp c cc chng Bt t t, cn trng. + Xc nh c mt s c im ca cc chng Bt phn lp: hnh dng, mu sc khun lc, t bo, bo t, tinh th c. + Th hot lc dit su hi cy trng ca cc chng Bt phn lp. + To mt b su tp nh v vi khun Bt lu tr ti Trung tm ging & CNSH i hc Lm nghip. 2.2. i tng v gii hn nghin cu * i tng nghin cu Cc chng vi khun Bacillus thuringiensis tn ti trong: + 32 mu t thu thp khu vc Trng H Lm nghip - Xun Mai Chng M - H Ni. + 03 mu cn trng nghi b vi khun Bacillus thuringiensis xm nhim * Gii hn ca nghin cu Do iu kin th nghim, thi gian thc hin kha lun c hn, cng nh trnh bn thn cn hn hp. V vy, cha xc nh c chng vi khun Bt phn lp thuc loi, chi no v vic xem xt mt cch chnh xc s khc nhau gia cc chng Bt phn lp c s trng lp nhau khng ch l tng i. Kha lun ch tp trung vo phn lp c chng vi khun Bt c hot lc cao trong vic tiu dit cn trng gy hi cy trng.

25

2.3. Ni dung nghin cu - Nghin cu phn lp Bt t mu cn trng v t - Xc nh c im khun lc ca cc chng Bt phn lp. - Xc nh c im t bo, bo t, tinh th c. - Xc nh hot lc dit su da chut ca cc chng Bt phn lp. - Bo qun cc chng vi khun Bt phn lp. 2.4. Vt liu v thit b 2.4.1. Ngun vi sinh vt Ngun vi khun Bacillus thuringiensis (Bt) c phn lp t hai loi mu: - Mu cn trng cht c cc triu trng nghi l b cht bi vi khun Bt (xem Bng 2.1), ( xem Hnh 2.1). - Cc mu t thu thp cc a im khc nhau trong Trng i hc Lm nghip (xem Bng 2.2), ( xem Hnh 2.1).

B
Hnh 2.1 : Hnh nh cc ngun mu thu thp vi khun Bt 2.1-A : Hnh nh mu tm bnh 2.1-B : Hnh nh mu t

26

Bng 2.1: Ngun thu thp vi khun Bt t cn trng Loi su Su m Su keo Su tm Tnh trng cht cht cht K ch thc vt Cy m Lng Keo tai tng Cy Du a im thu thp Thi gian thu mu thp mu Ni Nut - HLN Ngy 21/04/2009 Ni Nut - HLN Ngy 02/05/2009 C s sn xut Ngy ging tm Mai 26/06/2009 Lnh - H Ni

TT 1 2

Bng 2.2: Ngun thu thp Bt t cc mu t khu vc Trng HLN TT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 K hiu mu t H T5 G6 MS1 MS2 MS3 MS4 MS5 MS6 MS7 MS8 MS9 MS10 MS11 MS12 MS13 MS14 MS15 MS16 a im thu thp mu (Trng HLN) Khu vc h Nh T5 Hi trng G6 Vn m Vn m Khu chi canh ni Nut Chn ni Nut Khu vn m ni Nut Khu KTX K13 Khu TH Tin Khu tp qun s ni Nut Khu ging ng G1 Khu chi canh ni Nut Chn ni Nut Khu tp qun s ni Nut nh ni Nut Khu vn m ni Nut nh ni Nut Khu vn m ni Nut
27

Loi hnh t t tht trung bnh t tht trung bnh t tht trung bnh t tht nh t tht trung bnh t tht trung bnh t tht trung bnh t tht trung bnh t tht trung bnh t tht trung bnh t tht nh t tht trung bnh t tht trung bnh t tht trung bnh t tht trung bnh t tht trung bnh t tht trung bnh t tht trung bnh t tht trung bnh

Thm thc vt Long no a l bng Re hng Su Cau Bng Sa D Cau Cm Lai V T Hp Keo L Trm Rng keo Long no Rng keo R cau Re hng Thng Hoa sa Rng tru Xoan ta

20 21 22 23 24 25 26 27 28 29 30 31 32

MS17 MS18 MS19 MS20 MS21 MS22 MS23 MS24 MS25 MS26 MS27 MS28 MS29

Khu vn m ni Nut Chn ni Nut Khu vn m ni Nut nh ni Nut Khu vn m ni Nut nh ni Nut Chn ni Nut Khu KTX K13 Khu B nc Khu Ngha trang Khu KTX K12 Khu KTX K11 Th vin c

t tht trung bnh t tht trung bnh

Lt hoa Thng

t tht trung bnh Khu vc m keo t tht trung bnh t tht trung bnh t tht trung bnh t tht trung bnh t tht trung bnh t tht nh t tht trung bnh t tht trung bnh t ct pha t tht trung bnh Thng Cm lai v Re hng Gi gc Nhi Tru Lim xt Keo L Trm Tru Tch

2.4.2. Su th nghim - Su n l da chut, da hu Spodoptera litura Fabricius h Noctuidae thuc b cnh vy (Lepidoptera) thu thp cc rung da ca b con nng dn khu vc Nam Phng Tin, Chng M, H Ni ( xem Hnh 2.2). - Su n l ci bp v su ho Plutella xylostella Linnaeus h Noctuidae thuc b cnh vy (Lepidoptera) thu thp ti cc rung rau ca b con nng dn khu vc Xun Mai, Chng M, H Ni ( xem Hnh 2.2).

A Hnh 2.2 : Hnh nh v su th hot tnh 2.2 - A :Hnh nh v su hi da chut, da hu 2.2 - B : Hnh nh v su hi ci bp v su ho.
28

2.4.3. Ha cht - Pepton - Tht b - Cao nm men - Glucose - Bt ng 2.4.4. Thit b - dng c * Thit b - Box cy v trng - T nui vi sinh vt hiu kh - T sy - T lnh - Ni hp kh trng * Dng c

- Bt u tng - Nc mm - Cn - Thuc nhum fuchsin - Agar

- Mt s cht khong nh: FeSO4, MgSO4, , ZnSO4....

- My n nhit - My lc nui vi sinh vt - Knh hin vi quang hc Olympus - Cn phn tch, cn in t - My o pH

- Cc dng c thu tinh nh ng nghim, a petri, bnh tam gic, pipet, ng ong, cc ong, n cn, que cy khun, que gt khun, lam knh, lamen...... 2.4.5. Cc loi mi trng s dng trong nghin cu * Mi trng phn lp - Mi trng phn lp c : Thnh phn Tht b Pepton: Axetat natri: Agar: Nc ct : pH g/l 100g 10 g 35 g 16 g 1000 ml 7

Ho cht sau khi pha, chun pH c em khi kh trng, ngui n khong 600C c rt vo cc a petri, bc giy parafilm, 24 gi
29

iu kin thng xem c nhim khng, nhng a thch khng b nhim mi s dng. - Mi trng phn lp lng : Thnh phn Tht b Pepton: Axetat natri: Nc ct : pH g/l 100g 10 g 35 g 1000 ml 7

Ho tan ho cht, chun pH, phn u vo cc bnh tam gic dung tch 100ml (mi bnh 30ml). Kh trng mi trng 121 0C trong 20 pht. sau 24h iu kin thng kim tra tit trng, khng b nhim mi ly ra s dng. * Mi trng nhn ging Thnh phn Tht b Pepton: CaCl2: KH2PO4.7H2O: ZnSO4.7H2O: Glucose: Nc ct: pH - Mi trng nhn ging cp 1 : (g/l) 100g 10 g 0,01 g 0,02 g 0,001 g 10 g 1000 ml 7

Ho tan ho cht, chun pH, phn u vo cc bnh tam gic dung tch 250ml (mi bnh 100ml). Kh trng mi trng 1210C trong 20 pht. sau 24h iu kin thng kim tra tit trng, khng b nhim mi ly ra s dng. - Mi trng nhn ging cp 2:

30

Lm theo cng thc ca mi trng nhn ging cp 1, ng trong cc cc bnh tam gic dung tch 250ml (mi bnh 100ml). * Mi trng gi ging Thnh phn Pepton: Glucose: Cao nm men: Agar: Nc ct: (g/l) 5g 10 g 3g 16 g 1000 ml

pH 7 Ha tan ha cht, chun pH, ng trong ng nghim ri em kh trng. Lm thch nghing, sau 24h iu kin thng kim tra tit trng, khng b nhim mi ly ra s dng. * Mi trng ln men Thnh phn Bt u tng: Glucose: Bt ng: ZnSO4: MgSO4.7H2O: FeSO4.7H2O: KH2PO4.7H2O: Nc ct: pH (g/l) 15 g 7g 10 g 0,01 g 0,3 g 0,01 g 0,05 g 1000 ml 7

Ho tan ho cht, chun pH, phn u vo cc bnh tam gic dung tch 250ml (mi bnh 100ml). Kh trng mi trng 121 0C trong 20 pht. sau 24h iu kin thng kim tra tit trng trc khi s dng.

31

2.5. Phng php nghin cu

2.5.1. Phng php lun

- Cc nhn t l ch tiu nghin cu: phi chia thnh cc cng thc th nghim khc nhau, phi c cng thc i chng. - Cc nhn t khng phi ch tiu nghin cu: phi bo m tnh ng nht gia cc cng thc th nghim. - S mu ca mi cng thc th nghim phi ln ( 30). - Th nghim c lp li 3 ln. 2.5.2. Phng php nghin cu c th 2.5.2.1. Phn lp Bacillus thuringiensis a) Phn lp t cc mu t - Phng php thu thp mu t Thu thp mu t t cc a im khc nhau khu vc Trng i hc Lm nghip. Cch thu thp mu t nh sau: Gt b lp t mt khong 2 3 cm, dng dng c sch thu ly 100 gam t. Mi im ly 3 ch sau trn u 3 mu ny trong ti nilon v trng. Ly 100 gam t cho vo ti v trng khc. Dn kn, bo qun nhit thp v kh ro. Trn mu t c ghi r a im thu mu, thm thc vt, ngy ly mu. - Phng php phn lp : Mu t ly v cn 1gam cho vo cc bnh tam gic cha mi trng lng. Nui trn my lc 280C trong 4 gi, sau ly ra t vo b n nhit 800C trong 15 pht. Ly 50l cy vo mi trng phn lp trn a petri, nui 28 0C trong 48 - 72 gi. La chn khun lc c hnh thi ging khun lc Bacillus thuringiensis nui tip trong 72 gi. Lm tiu bn nhum n vi fuchsin v kim tra di knh hin vi quang hc (vt knh x100). Cc mu c t bo sinh dng hnh que v c tinh th ni bo chnh l Bt. Thun khit li ging v bo qun ging. b) Phn lp t cn trng bnh Cn trng l i tng cm nhim ch yu ca Bt v l ngun vt liu quan trng phn lp loi khun ny.
32

- Phng php thu thp cn trng: Khi cn trng b cht do vi khun Bt, thn cn trng thng treo ngc trn phin l, qun vo trong l v ri xung t, xc cn trng c mu nu en. Cn trng mi cht, trn c th bm nhiu cc cht bn - l dch th do cn trng tit ra trong thi k nhim bnh hoc l cht bi tit ca n. Cn trng nhim khun cht l do c vi khun sinh sn trong nn vch c th rt mng v gin, khi thu thp mu t cn trng cn cn thn gp cn trng vo bnh nh v trng hoc ti nilon v trng. Trn ti nilon v trng ghi r a im ly mu, trng thi ca cn trng, loi thc vt b tn ph, thi gian thu thp mu. Kh trng: Mu cn trng c kh trng b mt. V trng phn gia ca cn trng trong ethanol 600 (2 pht) v NaOCl 5% (3 - 5 pht). Ra nhiu ln vi nc ct v trng. Phn lp: Trong iu kin v trng, dng ko ct m phn lng, c th c dch th chy ra. Dng ko n nh mt u cn trng p dch th chy ra. Phn lp theo phng php pha long trn a petri. t cc a petri trong t m 280C trong 48 - 72 gi. La chn khun lc c hnh thi ging khun lc Bacillus thuringiensis nui tip trong 72 gi. Lm tiu bn nhum n vi fuchsin v kim tra di knh hin vi quang hc (vt knh x100). Cc mu c t bo sinh dng hnh que v c tinh th ni bo chnh l Bt. Thun khit li ging v bo qun ging. 2.5.2.2. Quan st hnh dng t bo, bo t, tinh th c ca vi khun Bt Tin hnh lm vt bi sinh khi vi khun trn lam knh. Nhum vt bi vi thuc nhum fuchsin m c trong 1 pht. Ra nh bng ethanol 10%, sau ra vi nc ct v quan st tiu bn di knh hin vi vt knh x100. 2.5.2.3. Th kh nng dit su ca cc chng vi khun Bt trong phng th nghim xc nh c c lc ca cc chng vi khun Bt phn lp c i vi cc loi su n l cy da chut, da hu Spodoptera litura v su n l su ho, bp ci Plutella xylostella (u l nhng loi su l k ch c hiu ca vi khun Bt), ti ln lt thc hin cc th nghim sau:
33

* Th nghim 1: Nhn ging vi khun Bt Cy ging v nhn ging cp 1, cp 2 trong cc bnh tam gic cha 100ml mi trng nhn ging v trng chun b t trc. Tin hnh nhn ging mi cp 200 v/p, 28oC trong 24h. * Th nghim 2: Ln men to sinh khi vi khun Bt Ly 5ml ging cp 2 cy sang cc bnh tam gic 250ml cha 100ml mi trng ln men (t l tip ging 5%). Tin hnh ln men trn my lc vi tc 200 v/p 28oC trong 50 60h. * Th nghim 3: Xc nh s lng bo t/tinh th c ca vi khun Bt Do mi t bo ca cc chng Bt khi b ph v gii phng ra mt bo t v mt tinh th c. Cho nn bng cch m s lng bo t ta c th c tnh s lng bo t c trong dch ln men, cng nh theo di c tc sinh trng ca chng Bt ang s dng. m s lng bo t ta tin hnh x l mu 70 oC trong 10 pht ri pha long mu. Ly 0,1ml 3 nng pha long cui cy gt vo cc a thch v trng cha mi trng m s lng bo t (mi nng 2 a). vo t m 28oC trong 24h ri m s lng khun lc mc trn mi a. S lng bo t trong 1ml dch ln men s c tnh theo cng thc: X = n.10/a (bo t/ml) X: s bo t trong 1ml dch ln men. n: s khun lc trung bnh a: nng pha long. * Th nghim 4: Th kh nng dit su ca cc chng Bt trong phng th nghim th hot lc dit su ca cc chng vi khun Bt phn lp, ti tin hnh thu hi dch ln men tng ng vi tng chng thi im thu c nhiu bo t v tinh th c (50 - 60 gi). Ti tin hnh pha long 3 ln dch ln men ca tng chng Bt th hot tnh.

34

 c c s so snh v nh gi hiu qu dit su ca cc chng Bt phn lp, ti b tr thm 1cng thc i chng dng l ch phm Bt thng mi c sn xut bi mt cng ty sn xut thuc tr su sinh hc ca Trung Quc v 1 cng thc cng thc i chng m thc n c nhng vo mi trng ln men v trng khng c vi khun Bt. Ch phm c dng thuc bt, cha chng B. thuringiensis var kurstaki, c lu hnh v s dng Vit Nam. B tr th nghim: - Vi Su n l da chut, Da hu (Spodoptera litura Fabricius) c chia vo cc l th nghim b tr trong cc a petri v trng vi s lng su nh nhau (10 su/a) v tng i ng u v tui su. Thc n ca su (l da chut ti) sau khi ly v c ra sch di vi nc, kh t nhin. S dng panh v trng gp l da nhng vo dch ln men pha long tng ng vi tng chng Bt. Gp l da ra, ro bt dch sau cho vo cc a petri. Trn thnh a petri ghi r chng Bt, nng pha long, ngy th nghim... Th su vo cc l th nghim vi s lng 10 su/a. y cc a petri bng vi mn tha m bo mi trng trong a lun c thong kh. t cc a th nghim trong phng c nhit 250C. l i chng, thc n ca su c nhng vo mi trng ln men v trng chun b sn (khng c vi khun Bt) v tip cho su n vi lng thc n v lng su nh tin hnh vi cc l th nghim c dch Bt. Theo di v thng k s lng su sng/cht; tnh trng su sau 24h, 48h, 72h tt c cc l th nghim. Tnh t l su cht theo cng thc Abbott: (C - T) . 100 A= C Trong : A: % su cht C: S su sng st l i chng. T: S su sng st l th nghim. - Vi Su n l Ci bp v Su ho (Spodoptera exigua) th nghim c b tr tng t nh i vi su n l da chut v da hu.

35

2.5.2.4. Bo qun v lu gi cc chng vi khun Bt phn lp - Bo qun cc chng vi khun Bt phn lp s dng phng php ng lnh s dng glycerol 20%. Tin hnh nhn ging, thu sinh khi vi khun bng li tm, trn vi glycerol 20% bo qun bng ng eppendol iu kin - 850C, 200C . - Bo qun bng phng php cy truyn trn mi trng dinh dng trong ng nghim v bo qun iu kin 40C. S TH NGHIM
Mu t X l 800C Pha long mu Mu su Kh trng nh Pha long mu

Cy tri trn a petri Bacillus cereus + Bacillus thuringiensis

Nhum fuchsin Quan st di KHV Chn lc khun lc Bacillus thuringiensis Thun khit ging Gi ging

Bacillus thuringiensis

Nhn ging cp I, II
Tip ging 5%

Ln men
Bnh tam gic 250 ml c 100ml mi trng ln men

Pha long mu B tr th nghim xc nh hot lc dit su

36

CHNG 3 KT QU V PHN TCH KT QU

3.1. Kt qu phn lp Bacillus thuringiensis T 32 mu t v 3 mu cn trng thu thp quanh khu vc Trng i hc Lm nghip, chng ti thu nhn c 148 chng vi khun thuc nhm Bacillus cereus - Bacillus thuringiensis. Trong s c 66 chng c s sn sinh tinh th c (crystal) c nhn dng . Hnh nh trong qu trnh phn lp( xem Hnh 3.1). S phn b ca 51 chng Bt trong cc mu t v su:
S phn b ca 66 chng Bt trong cc mu t v su ( xem Bng 3.1 v Bng 3.2.).

Bng 3.1: Bt phn lp c t cc mu su TT 1 2 3 Ngun mu Su m Su keo Tm K ch thc vt Cy m lng Cy Keo Cy tm S lng 1 1 1 K hiu chng Bt BtK BtT

Bng 3.2: Bt phn lp c t cc mu t TT a im ly mu (Trng HLN) 1 2 3 4 5 6 7 Khu vc h Nh T5 Hi trng G6 Vn m Khu tp qun s ni Nut Khu ging ng G1 Khu chi canh ni Nut Thm thc vt Long no a l bng Re hng Cau Bng Rng keo Long no Rng keo 5 2 2 1 2 3 4 H1.1; H1.2; H1.3; H1.4 ; H1.5 T5.1; T5.2 G6.1; G6.2 MS2 MS8.1; MS8.2 MS9.1; MS9.2; MS9.3 MS10.2; MS10.3; MS10.4; MS10.5 S lng K hiu chng

37

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Chn ni Nut Khu tp qun s ni Nut nh ni Nut Khu vn m ni Nut nh ni Nut Khu vn m ni Nut Khu vn m ni Nut Chn ni Nut Khu vn m ni Nut nh ni Nut Khu vn m ni Nut nh ni Nut Chn ni Nut Khu KTX K13 Khu B nc Khu Ngha trang Khu KTX K12 Khu KTX K11 Th vin c

R cau Re hng Thng Hoa sa Rng tru Xoan ta Lt hoa Thng m Keo Thng Cm lai v Re hng Gi gc Nhi Tru Lim xt Keo L Trm Tru Tch

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

MS11.1; MS11.2 MS12.2; MS12.3 MS13.1; MS13.3 MS14.1; MS14.2; MS14.3; MS14.4 MS15.1; MS15.2; MS15.3 MS16.1; MS16.2; MS16.3; MS16.4 MS17.1; MS17.2 MS18.1; MS18.3 MS19.1; MS19.2 MS20 MS21.1; MS21.2 MS22.1; MS22.2 MS23 MS24.1 ;MS24.2 ; MS24.3 MS25.1 ; MS25.2 MS26.1 ; MS26.2 MS27.1 ; MS27.2 ; MS27.3 MS27.4 ; MS27.5 MS28 MS29.2 : MS29.1

Kt qu ch ra Bng 3.1 v Bng 3.2 cho thy: T 3 mu su cht chng ti phn lp c 3 chng Bt v t 32 mu t c 63 chng vi khun c nhn dng. Cc mu t thu thp mt s a im c kh nng phn lp c nhiu chng Bt gm: Khu vc h (5 chng), khu chi canh ni Lut (4 chng), khu vn m (4 chng), khu vc KTX K12 (5 chng). Kt qu nhn c cho
38

php khng nh rng t khu vc Trng HLN l mt a im tt thu thp cc chng Bt. iu c th c gii thch l do khu vc Trng HLN c h sinh thi a dng, tp trung nhiu loi thc vt rng vi s lng ln, l ni tr ng ca nhiu loi su hi cy lm nghip, cy bi... nn cng tim n mt qun th vi sinh vt i khng vi tn sut xut hin cao hn so vi nhng khu vc khc (Theo Ng nh Bnh v cng s, 2003, tn sut xut hin Bt trong t Vit Nam l kh cao so vi cc nc khc trong khu vc, khong 60% 90%). c im khun lc ca 66 chng Bt phn lp Trn cng loi mi trng phn lp trong cc a petri, cc chng Bt khc nhau s mc thnh cc khun lc c hnh thi v mu sc khc nhau ( xem Bng 3.3). Bng 3.3: Hnh dng, mu sc v ng knh khun lc ca cc chng Bt TT K hiu chng 1 2 3 4 5 BtK BtT Bt H1.1 H1.2 H1.3 H1.4 H1.5 T5.1 T5.2 G6.1 G6.2 MS2 Hnh sao, trng trong, b mt nhn. k 1- 2 mm Trn, trng sa, vin nhn, c tm dy vin mng. k 5 - 8 mm Hnh sao, trng trong, b mt nhn. k 1 - 2 mm Trn, trng c, b mt nhn. k 4 - 5 mm Trn, trng sa, b mt nhn, c tm dy vin mng. k 3 - 4 mm Hnh sao, trng trong, b mt nhn. k 4 - 5 mm Trn, trng trong, vin nhn, b mt g gh. k 2 - 3mm Trn, trng c, b mt nhn. k 2 - 3 mm Trn, trng sa, c tm dy vin mng. k 2 - 3 mm Trn, vng nht, b mt g gh. k 2 - 3 mm Hnh sao, trng sa, b mt g gh. k 3 - 4 mm Trn, trng c, b mt nhn. k 4 - 6 mm Trn, trng sa, b mt nhn. k 3 - 5 mm Hnh dng khun lc

6 7 8 9 10 11 12 13

39

14 15 16 17

MS8.1 MS8.2 MS9.1 MS9.2 MS9.3 MS10.2 MS10.3 MS10.4

Trn, trng c, c tm dy vin mng. k 4 - 6 mm Trn, trng trong, b mt nhn. k 5 - 6 mm Trn, trng sa, vin nhn, c tm dy vin mng. k 6 - 8 mm Trn, trng c, b mt g gh, c tm dy vin mng. k 3 - 5 mm Trn, trng c, c tm dy vin mng. k 3 - 5 mm Trn, trng sa, c tm dy vin mng. k 3 - 5 mm Hnh sao, trng trong, c tm dy vin mng. k 2 - 3 mm Trn, trng sa, vin c gh ni ln, gia khun lc lm. k 3 - 4 mm Hnh sao, trng trong, b mt g gh, c tm dy vin mng. k 8 10 mm Trn, trng sa, vin nhn, tm dy vin mng. k 9 11 mm Trn, trng c, c tm dy vin mng. k 7 9mm Hnh sao, trng trong, c tm dy vin mng, b mt g gh. k 4 7 mm Hnh sao, trng sa, b mt g gh, c vin dy. k 3 5 mm Trn, vin nhn, mu trng sa, c tm, b mt g gh. k 8 10 mm Trn, trng trong, c tm dy vin mng. k 2 4 mm Trn, trng sa, vin nhn dy. k 5 7 mm. Trn, trng trong. k 8 10 mm Trn, trng trong, b mt nhn, c tm dy vin mng. k 3 4 mm. Trn, trng c, c tm. k 3 4 mm Trn, trng trong, vin nhn, c tm dy vin mng. k 3 4 mm. Trn, trng trong, c vt lm tm b mt g gh, vin nhn. k 2 4 mm.

18 19 20 21

22

MS10.5 MS11.1 MS11.2 MS12.1 MS12.3 MS13.1 MS13.3 MS14.1 MS14.2 MS14.3 MS14.4 MS15.1

23 24 25

26 27

28 29 30 31

32 33

34

MS15.2

40

35

MS15.3

Trn, mu trng sa, c vt lm tm, b mt g gh. k 3 5 mm Hnh cu, trng sa, c tm dy vin mng, b mt g gh. k 3 5 mm Trn, trng sa, b mt g gh. k 2 4 mm Hnh sao, trng sa, b mt g gh. k 2 3 mm Hnh sao, trng trong, b mt g gh. k 3 5 mm Trn, trng trong, c tm dy vin mng, vin nhn. k 3 4 mm. Trn, trng vi, b mt g gh, vin nhn. k 4 - 6 mm Trn, trng trong, c tm lm, vin nhn. k 2 - 3 mm Trn, trng sa, c tm dy vin mng. k 7 - 10 mm Trn, trng sa, c tm dy vin mng. k 9 - 12 mm Hnh sao, trng trong, c tm, b mt g gh. k 7 - 10 mm Trn, trng sa, c tm dy, vin mng v nhn, b mt nhn. k 8 - 9 mm Trn, trng sa. b mt nhn, c tm dy, vin mng. k 2 - 5 mm Trn, trng vng, b mt nhn. k 2 - 4 mm Hnh sao, trng trong, c tm dy, vin mng. k 2 - 4 mm Trn, trng c, b mt nhn. k 8 - 10 mm Hnh sao, trng trong, b mt nhn. k 2 - 5 mm Trn, trng c, c tm dy vin mng. k 6 9mm Hnh sao, trng trong, b mt g gh. k 4 6 mm Hnh sao, trng sa, b mt g gh. k 3 4 mm Trn, vin nhn, mu trng sa, c tm. k 7 10 mm Trn, trng trong, c tm dy vin mng. k 4 6 mm Trn, trng sa, vin nhn dy. k 4 7 mm. Trn, trng trong, vin nhn dy. k 8 9 mm

36

MS16.1 MS16.2 MS16.3 MS16.4 MS17.1 MS17.2 MS18.1 MS18.3 MS19.1 MS19.2 MS20

37 38 39 40

41 42 43 44 45 46

47

MS21.1 MS21.2 MS22.1 MS22.2 MS23 MS24.1 MS24.2 MS24.3 MS25.1 MS25.2 MS26.1 MS26.2

48 49 50 51 52 53 54 55 56 57 58

41

59 60 61

MS27.1 MS27.2 MS27.3

Trn, trng trong, b mt nhn. k 3 5 mm. Trn, trng c, c tm, b mt nhn. k 3 4 mm Trn, trng trong, vin nhn, c tm dy vin mng. k 6 8 mm. Trn, trng trong, c vt lm tm b mt g gh, vin nhn. k

62 MS27.4 63 64 65 66 MS27.5 MS28 MS29.1 MS29.2

4 - 6 mm. Trn, mu trng sa, c vt lm tm. k 4 7 mm Hnh cu, trng sa, c tm dy vin mng. k 8 10 mm Trn, trng sa, b mt g gh. k 5 - 8 mm Hnh sao, trng sa, b mt g gh. k 3 - 5 mm

C
Hnh 3.1: Mt s hnh nh trong qu trnh phn lp Bt 3.1-A, 3.1- B: Vi khun Bt mc trn mi trng phn lp 3.1-C: Cc chng Bt thun khit trn mi trng gi ging

42

3.2. Kt qu quan st hnh dng t bo, bo t, tinh th c Bng phng php nhum n vi fuchsin, soi vt knh x100, chng ti nhn c cc thng tin v mt , hnh dng t bo, bo t v tinh th c ca 66 chng Bt (cc chng Bt c c im: tinh th ni bo bt mu sm; t bo sinh dng hnh que bt mu hng; bo t dng gn trn, c mp, khng bt mu( xem Bng 3.4 v xem Hnh 3.2). - Phng php ny ch c chnh xc tng i. Bng 3.4: Hnh dng t bo, bo t, tinh th c ca cc chng Bt TT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 K hiu chng BtK BtT Bt H1.1 H1.2 H1.3 H1.4 H1.5 T5.1 T5.2 G6.1 G6.2 MS2 MS8.1 MS8.2 MS9.1 Hnh dng t bo Hnh que ngn Hnh que ngn Hnh que ngn Hnh que ngn Hnh que ngn Hnh que ngn Hnh que ngn Hnh que di Hnh que di Hnh que ngn Hnh que di Hnh que ngn Hnh que ngn Hnh que ngn Hnh que ngn Hnh que ngn Hnh dng bo t Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng
43

Hnh dng tinh th c Qu trm Trn a gic Trn Kim a gic Qu trm Qu trm a gic Trn Trn, a gic Trn a gic Trn Kim Trn, a gic

17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

MS9.2 MS9.3 MS10.2 MS10.3 MS10.4 MS10.5 MS11.1 MS11.2 MS12.1 MS12.3 MS13.1 MS13.3 MS14.1 MS14.2 MS14.3 MS14.4 MS15.1 MS15.2 MS15.3 MS16.1 MS16.2 MS16.3 MS16.4

Hnh que ngn Hnh que di Hnh que ngn Hnh que ngn Hnh que ngn Hnh que ngn Hnh que ngn Hnh que di Hnh que di Hnh que ngn Hnh que ngn Hnh que di Hnh que di Hnh que ngn Hnh que ngn Hnh que ngn Hnh que di Hnh que ngn Hnh que ngn Hnh que ngn Hnh que ngn Hnh que ngn Hnh que ngn

Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng

a gic Trn a gic Kim a gic Trn Kim Trn, a gic Thoi Trn a gic Trn Trn, a gic a gic Kim Trn Trn Kim a gic Kim Trn, a gic Trn a gic

44

40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62

MS17.1 MS17.2 MS18.1 MS18.3 MS19.1 MS19.2 MS20 MS21.1 MS21.2 MS22.1 MS22.2 MS23 MS24.1 MS24.2 MS24.3 MS25.1 MS25.2 MS26.1 MS26.2 MS27.1 MS27.2 MS27.3 MS27.4

Hnh que ngn Hnh que ngn Hnh que ngn Hnh que di Hnh que di Hnh que ngn Hnh que di Hnh que ngn Hnh que di Hnh que ngn Hnh que ngn Hnh que ngn Hnh que ngn Hnh que ngn Hnh que ngn Hnh que di Hnh que di Hnh que ngn Hnh que ngn Hnh que di Hnh que di Hnh que ngn Hnh que ngn

Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng Hnh trng

Trn Trn a gic Trn Trn Trn, a gic Trn a gic Trn a gic Trn Qu trm Qu trm Trn a gic Trn Kim a gic Qu trm Qu trm a gic Trn Trn, a gic

45

63 64 65 66

MS27.5 MS28 MS29.1 MS29.2

Hnh que ngn Hnh que di Hnh que ngn Hnh que ngn

Hnh trng Hnh trng Hnh trng Hnh trng

Trn a gic Trn Kim

Kt qu xc nh s b s lng bo t/tinh th c ca 66 chng Bt phn lp di knh hin vi cho thy: c 20/66 chng c lng bo t/tinh th c cao hn so vi nhng chng cn li, gm cc chng: H1.2; MS8.1; MS15.3; MS16.2; MS16.3; MS17.1; MS21.1; BTK ; H1.5;T5.1;G6.1;MS27.4;MS27.1;MS23; T5.2 ; G6.1; MS26.1; BtT; MS11.1;MS19.2.

Hnh 3.2: Hnh dng t bo, bo t, tinh th c cc chng Bt 3.2-A: Hnh dng t bo, bo t, tinh th c ca chng H1.5 3.2-B: Hnh dng t bo, bo t, tinh th c ca chng MS21.1

46

3.3. Kt qu xc nh hot lc dit su 3.3.1. Kt qu nhn ging cp I, II v ln men

Sau khi xc nh c s b s lng bo t, tinh th c ca 66 chng Bt phn lp di knh hin vi, chng ti chn ra 20/66 chng sn sinh nhiu bo t v tinh th c hn so vi nhng chng cn li, gm cc chng: H1.2; MS8.1; MS15.3; MS16.2;

MS16.3; MS17.1; MS21.1; BTK ; H1.5;T5.1;G6.1;MS27.4;MS27.1;MS23; T5.2 ; G6.1; MS26.1; BtT; MS11.1;MS19.2 tin hnh th nghim tip theo.

Cc chng Bt sn sinh nhiu bo t, tinh th c la chn c tin hnh

nhn ging trn mi trng nhn ging cp I, cp II. Sau khi cy ging vo mi trng sau 3 gi 4 gi th vi khun pht trin v lm vn c mi trng, n 24 gi th mi trng c hon ton thi im ny, ti tin hnh cy chuyn sang mi trng nhn ging cp II. mi trng nhn ging cp II sau 24 gi khi vi khun pht trin mnh v lm vn c mi trng, ti tin hnh ln men vi t l tip ging 5% . Tin hnh ln men trn my lc 220 vng/pht 280C. Thu hi dch ln men thi im sau 50 gi - 60 gi thi im ny vi khun Bt phng thch nhiu bo t v tinh th c ra mi trng.

3.3.2. Kt qu xc nh s lng bo t Do mi t bo ca cc chng Bt khi b ph v gii phng ra mt bo t v mt tinh th c. Cho nn bng cch m s lng bo t ta c th c tnh s lng tinh th c c trong dch ln men, cng nh theo di c tc sinh trng ca chng Bt ang s dng. c c s cho pha long dch ln men ca 21 chng Bt khc nhau sao cho dch ln men ca cc chng sau pha long s c mt bo t v tinh th c tng i ng u nhau, chng ti tin hnh m s lng bo t ca cc chng Bt trong 1 ml dch ln men ( xem Bng 3.5). Bng 3.5: S lng bo t/ml dch ln men (thu hi sau 60h ln men) TT 1 2 3 K hiu chng BtK BtT H1.2
47

S lng bo t (x109)/ml dch ln men 8,1 6,03 3,07

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

H1.5 T5.1 T5.2 G6.1 MS8.1 G6.2 MS11.1 MS15.3 MS16.2 MS21.1 MS27.1 MS27.4 MS26.1 MS23 MS17.1 MS16.3 MS19.2

15,07 8,50 8,57 10,04 4,25 6,25 7,98 6,87 9,25 14,08 11,44 14,02 6,4 5,4 8,2 4,0 6,0

Kt qu m s lng bo t/ml dch ln men trnh by bng 07 cho thy s lng bo t ca 21 chng Bt nm trong khong t 3,07 x 109 bo t/ml n khong 15,07x 109 bo t/ml. 3.3.3. Kt qu kh nng dit su 3.3.3.1. Kt qu dit su n l da chut v da hu Su n l da chut, da hu (Spodoptera litura Fabricius) pht trin mnh vo v Thu ng v v Xun H, bm c chiu di thn khong 2025mm, si cnh rng t 35-45mm. Cnh trc mu nu vng, gia cnh c vn trng, cnh sau mu trng ng nh, trng c hnh bn cu, ng knh t 0,4 0,5mm.

48

Thi gian pht trin ca u trng ko di t 20-25 ngy, su c 5-6 tui tu thuc iu kin mi trng. Nu iu kin thun li su c th di t 35-53mm, hnh ng trn. Su tui nh c mu xanh lc, cng ln su chuyn dn thnh mu nu m. Trn c th c mt sc vng sng chy hai bn hng t t th nht n t th tm ca bng, mi t c mt chm en r nhng hai chm en t th nht to nht. Su cng ln, hai chm en t th nht cng to dn v gn nh giao nhau to thnh khoang en trn lng nn su n tp cn c gi l su khoang. Theo nhiu ti liu nghin cu trong v ngoi nc cng b [3], [6]. Trong s cc loi su n l thuc b cnh vy, th su n l da chut v da hu l mt trong nhng loi k ch rt c hiu ca vi khun Bt. Th nghim th hot lc dit su c b tr trong cc a petri, mi a 10 su (tng i ng u v kch thc). Nng bo t vi khun ca dch vi khun cho th nghim dit su khong 109 bo t/ml. Kt qu m s lng bo t mc 5.3 cho thy 21 chng Bt sn sinh s lng bo t trong khong 3,07 x 109 bo t/ml - 15,07x 109 bo t/ml. Do vy cn tin hnh pha long dch ln men m bo s lng bo t, tinh th c ca 21 chng Bt trong th nghim th hot lc dit su l tng i ng u. Cc chng Bt k hiu MS8.1; MS16.3; MS26; MS15.3; BtT; MS23; H1.2; MS19.2; G6.2 c s lng bo t/ml dch ln men 5 x 109 bo t/ml nn cn pha long 5 ln vi nc ct v trng t mt bo t trong khong 109 bo t/ml. Cc chng T5.1; T5.2; MS16.2; MS11.1; MS17.1; G6.1 ;BtK c s lng bo t/ml dch ln men 9 x 109 bo t/ml. Do vy chng ti dch ln men ca cc chng ny ra pha long 9 ln. Cc chng H1.5; MS21.1; MS27.4; MS27.1 c s lng bo t/ml dch ln men 13 x 109 bo t/ml. Chng ti pha long 13 ln i vi dch ln men ca cc chng ny. Cng thc i chng m l mi trng ln men v trng.

49

Cng thc i chng dng l ch phm thuc tr su Bt nhn hiu Anhuy do Trung quc sn xut. Ch phm ny cha chng Bt thuc th kurstaki sinh tinh th c hnh qu trm 8 mt, dng bt v c ho tan vi nc ct v trng to thnh dng lng, m bo s lng bo t cng khong 109 bo t/ml. Kt qu xc nh hot lc dit su cc thi im sau 24h, 48h v 72h (tnh theo cng thc Abbott), ( xem Bng 3.6 v xem Hnh 3.3). Bng 3.6: T l phn trm su n l da chut v da hu cht bi ch phm Bt TT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 K hiu chng C BtAn huy BtK BtT H1.2 H1.5 T5.1 T5.2 G6.1 G6.2 MS8.1 MS11.1 MS15.3 MS16.2 MS16.3 MS17.1 MS19.2 MS21.1 MS23 MS26.1 MS27.1 MS27.4 T l (%) su cht sau thi gian 24h 0 50 40 30 20 60 40 30 20 30 10 20 30 40 10 30 20 40 20 20 30 40 48h 0 100 50 40 30 80 60 40 40 50 30 30 60 60 30 50 50 90 50 30 50 70 72h 0 100 50 60 50 100 80 60 80 50 40 80 70 80 50 70 60 90 60 60 90 90

50

Xem kt qu bng trn ti thy: Cng thc i chng m (nhng l da vi mi trng ln men v trng) c t l su cht 0% sau 3 ngy th nghim. Cng thc i chng dng (BtAnhuy) s dng ch phm Bt thng mi ca Trung quc hiu lc dit su da chut v da hu rt cao. Sau 48h tt c cc su trong th nghim u cht (100%). Trong s 20 chng Bt sinh nhiu bo t/tinh th c phn lp, ng ch nht l hiu qu dit su n l da chut v l da hu ca cc chng sau: - Chng H1.5 c hiu qu dit su n l da chut v da hu cao tng ng hiu qu dit su n l da chut v da hu ca chng Bt thng mi ca Trung quc. Sau 24h (60%), sau 48h (80%) su cng thc th nghim ny cht, sau 72h (100%) su trong th nghim cht. - Chng MS21.1; MS27.4 ; MS27.1 c hiu qu dit su n l da chut v da hu cao gn bng hiu qu dit su n l da chut v da hu ca chng Bt thng mi ca Trung quc. Hiu qu dit su n l da chut v da hu ca 3 chng nh sau : Sau 24h - MS21.1 (40%), MS27.4 (40%), MS27.1( 30%); sau 48h MS21.1 (90%), MS27.4 (70%), MS27.1(50%); sau 48h - MS21.1 (90%), MS27.4 (90%), MS27.1( 90%) - 4 chng c hiu qu dit su n l da chut v da hu 80% sau 72h th nghim l T5.1, MS11.1, MS16.2, G6.1. Cc chng Bt cn li c hiu qu dit su thp hn. Khi xem xt kch c su v lng l da tiu th cc l th nghim chng ti nhn thy: Do su n l du hu v da chut trong mt ngy c th n ht mt lng l da ng k, tng ng vi lng l da su n, c th d dng quan st thy kch c su to ln sau tng ngy. Do vy, lng l da chng ti tip cho su n trc th nghim l y a petri nhng cng ch cho su n trong 24h , sau nu l th nghim no su n ht thc n th s phi nhng thm l da ti vi dch khun tng ng vi mi l th nghim tip thm cho su. l i chng m, lng l tiu th ht sch ch sau 1 ngy. Do vy, c sau khong 18h - 24h chng ti li thc hin nhng l da ti vi mi trng ln men v trng tip thm thc n cho su. Su sinh trng pht trin bnh thng nn c s ln ln v kch c v trng lng. cc cng thc th nghim b tr vi cc chng Bt c hiu qu dit su thp (nh MS23, MS26.1, MS19.3... ), chng ti cng phi tip thm thc n
51

nhng dch khun cho su, tuy nhin lng thc n cn tip thm khng nhiu nh l i chng m v kch c, trng lng ca nhng su cn sng st cng tng ln. L th nghim th hot lc dit su ca cc chng BtAnhuy, H1.5 , MS21.1, MS27.4 , MS27.1 , T5.1, MS11.1, MS16.2, G6.1. Sau 24h th nghim, lng thc n dnh cho su cng gim ng k, phi tip thm thc n cho su (tuy khng cn lng nhiu bng l i chng m v cc l th nghim khc). Tuy nhin, t 24h tr i, s lng su cn sng st khng nhiu, nhng su cn sng st th rt yu nn lng l da trong cc a hu nh khng c tiu th thm na. Su trong cc l th nghim ny hu nh khng tng ln v kch c v trng lng. Tng t, cc l th nghim khc c s tiu th lng l da nhiu t khc nhau tng ng vi hiu qu dit su ca tng chng Bt tng ng vi tng l th nghim. Hiu qu dit su thp, lng l da su tiu th nhiu. Hiu qu dit su cao hn th lng l da cn tip cho su n t hn.

Hnh 3.3: Hnh nh v hiu qu dit su n l da chut v da hu 3.3-A : Hnh nh v hiu qu dit su n l da chut v da hu ca chng H1.5 3.3-B: Hnh nh v mu i chng m 3.3.3.2. Su n l ci bp v su ho Su n l ci bp v su ho Plutella xylostella Linnaeus pht trin mnh vo v ng Xun. Bm di t 6 - 10 mm, si cnh rng t 10 - 15 mm. Cnh trc mu nu, gia lng c mt di gn sng, mu trng trn bm c v mu
52

vng trn bm ci, chy di n cui cnh. Bm c th sng n 2 tun v 200 trng. u trng mu xanh lc, mnh n to chnh gia, 2 u nhn, thn chia t r rng v c 3 cp chn gi t t bng th nm, ln sc mnh su di t 8 n 11mm. Su c 4 tui vi thi gian pht trin lu 7 - 10 ngy. Thi gian lm nhng lu 4 - 7 ngy. Khi mi hnh thnh nhng c mu xanh nht, khong 2 ngy sau thnh mu vng nht, chiu di nhng t 5 - 7mm, chung quanh nhng c kn bng t bao ph. Th nghim th hot lc dit su c b tr trong cc a Petri ging nh vi su n l da hu v da chut, mi a 10 su (tng i ng u v kch thc). Cng thc i chng m l mi trng ln men v trng. Cng thc i chng dng l ch phm thuc tr su Bt nhn hiu Anhuy do Trung quc sn xut. Kt qu xc nh hot lc dit su cc thi im sau 24h, 48h v 72h (tnh theo cng thc Abbott), (xem Bng 3.7 v xem Hnh 3.4).

Bng 3.7: T l phn trm su n l ci bp v su ho cht bi ch phm Bt TT 1 2 3 4 5 6 7 8 9 K hiu chng C BtAn huy BtK BtT H1.2 H1.5 T5.1 T5.2 G6.1 T l (%) su cht sau thi gian 24h 0 60 10 40 20 50 30 50 20
53

48h 0 100 50 40 20 90 60 60 40

72h 0 100 60 70 50 100 70 80 90

10 11 12 13 14 15 16 17 18 19 20 21 22

G6.2 MS8.1 MS11.1 MS15.3 MS16.2 MS16.3 MS17.1 MS19.2 MS21.1 MS23 MS26.1 MS27.1 MS27.4

20 20 40 30 40 20 20 20 40 20 20 40 50

30 30 60 60 50 30 50 50 80 60 40 70 90

30 50 80 40 90 70 60 50 90 60 50 90 100

Xem kt qu bng trn ti thy: Cng thc i chng m (nhng l da vi mi trng ln men v trng) c t l su cht 0% sau 3 ngy th nghim. Cng thc i chng dng (BtAnhuy) s dng ch phm Bt thng mi ca Trung quc hiu lc dit su n l Ci bp v Su ho rt cao. Sau 48h (100%) su trong th nghim u cht. Trong s 20 chng Bt sinh nhiu bo t/tinh th c phn lp, ng ch nht l hiu qu dit su n l ci bp v su ho ca cc chng sau: - Chng H1.5, MS27.4 c hiu qu dit su n l ci bp v su ho cao tng ng hiu qu dit su n l ci bp v su ho ca chng Bt thng mi ca Trung quc. Hiu qu dit su n l ci bp v su ho ca 3 chng nh sau: Sau 24h - H1.5(50%), MS27.4(50%); sau 48h - H1.5(90%), MS27.4(90%); sau 72h ( 100%) su trong th nghim u cht. Chng MS27.1, G6.1 , MS16.2, MS21.1 c hiu qu dit su n l ci bp v su ho cao gn bng chng Bt thng mi ca Trung quc v chng H 1.5 ,MS27.4 . Hiu qu dit su n l ci bp v su ho ca 4 chng nh sau: Sau 24h MS27.1(40%), G6.1 (20%), MS16.2 (40%), MS21.1(40%) ; sau 48h - MS27.1(70%), G6.1 (40%), MS16.2 (50%), MS21.1(80%); Sau 72h - MS27.1(90%), G6.1 (90%), MS16.2 (90%), MS21.1(90%). - 2 chng c hiu qu dit su 80% sau 72h th nghim l MS11.1, T5.2.
54

Cc chng Bt cn li c hiu qu dit su thp hn. Khi xem xt kch c su v lng l ci bp tiu th cc l th nghim chng ti nhn thy: Su n l ci bp va su ho trong mt ngy lng l su n gim i mt na, tng ng vi lng l ci bp su n, ta thy kch thc ca su tng chm . Do vy, lng l ti tip cho su n trc th nghim l y a petri cho su n trong 24h 36h , sau nu l th nghim no su n ht thc n th s phi nhng thm l ci bp ti vi dch khun tng ng vi mi l th nghim tip thm cho su. l i chng m, lng l tiu th ht sch ch sau 36h. Do vy, c sau khong 24h chng ti li thc hin nhng l ci bp ti vi mi trng ln men v trng tip thm thc n cho su. Su sinh trng pht trin bnh thng nn c s ln ln v kch c v trng lng. cc cng thc th nghim b tr vi cc chng Bt c hiu qu dit su thp (nh MS19.2, MS26.1, MS23...),chng ti cng phi tip thm thc n nhng dch khun cho su, tuy nhin lng thc n cn tip thm khng nhiu nh l i chng m v kch c, trng lng ca nhng su cn sng st cng tng ln. L th nghim th hot lc dit su ca cc chng BtAnhuy, H1.5 , MS27.4 , MS21.1 , MS27.1, G6.1 . Sau 24h th nghim, lng thc n dnh cho su cng gim ng k, phi tip thm thc n cho su (tuy khng cn lng nhiu bng l i chng m v cc l th nghim khc). Tuy nhin, t 24h tr i, s lng su cn sng st khng nhiu, nhng su cn sng st th rt yu nn lng l trong cc a hu nh khng c tiu th thm na. Su trong cc l th nghim ny hu nh khng tng ln v kch c v trng lng. Tng t, cc l th nghim khc c s tiu th lng l nhiu t khc nhau tng ng vi hiu qu dit su ca tng chng Bt tng ng vi tng l th nghim. Hiu qu dit su thp, lng l ci bp su tiu th nhiu. Hiu qu dit su cao hn th lng l cn tip cho su n t hn. T nhng s liu thng k phn trm hot lc dit su n l ci bp v su ho trn ta thy su n l hi ci bp v su ho cng l mt trong nhng loi k ch rt c hiu ca vi khun Bt. Kt qu th hot tnh vi hot lc dit su ci bp v su ho ca cc chng khng thay i nhiu so vi kt qu th hot tnh
55

i vi su n l da chut v da hu. iu ny chng t kt qu th hot tnh l tng i chnh xc, t ta c th thy cc chng vi khun Bt phn lp c hot lc cao chn c l cc chng tt cn lu gi v nghin cu su hn v loi, chi v hot lc ca chng i vi cc loi cn trng khc, cng nh tm cch nng cao hot lc ca cc chng ny.

Hnh 3.4 : Hnh nh v hiu qu dit su n l ci bp v su ho 3.4-A: Hnh nh dit su n l ci bp v su ho ca chng MS27.4 3.4-B: Hnh nh mu i chng m 3.3. Kt qu bo qun cc chng vi khun Bt phn lp Cc chng vi khun Bt phn lp c thun khit trong ng nghim t lnh 40C, sau khi tin hnh ln men th hot tnh tm ra cc chng c hot lc cao. Ti tin hnh bo qun cc chng bng phng php cy chuyn v ng lnh, nhm lu gi cho cc nghin cu lu di. i vi cc chng c th hot tnh nhng c hot lc thp, hot lc tng i v cc chng cha c th hot tnh( nh MS10.3, MS12.1, MS12.3...), ti tin hnh bo qun ng lnh bng glycerol - 850C. i vi cc chng c th hot tnh v c xc nh l c hot lc dit su 90% - 100% ( nh H1.5 , MS27.4 , MS21.1 , MS27.1, MS11.1,T5.2,G6.1 ), tin cho vic nghin cu ti tin hnh lu gi 3 iu kin - 850C, - 200C v 40C. Sau khi tin hnh bo qun 15 ngy, chng ti ly

56

cc chng c bo qun ra v em nui trn mi trng nhn ging thy cc chng ny pht trint bnh thng, chng t vic bo qun thnh cng. Kt qu bo qun cc chng vi khun Bt phn lp ti Trung tm Ging & CNSH Trng HLN (xem Bng 3.8) . Bng 3.8 : Kt qu bo qun cc chng vi khun Bt phn lp TT K hiu chng BtK BtT Bt H1.1 H1.2 H1.3 H1.4 H1.5 T5.1 T5.2 G6.1 G6.2 MS2 MS8.1 MS8.2 MS9.1 MS9.2 MS9.3 MS10.2 MS10.3 MS10.4 S lng mu - 850C 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

- 200C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

4 0C 0 0 0 0 0 0 0 2 2 2 2 0 0 0 0 0 0 0 0 0 0

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

MS10.5 MS11.1 MS11.2 MS12.1 MS12.3 MS13.1 MS13.3 MS14.1 MS14.2 MS14.3 MS14.4 MS15.1 MS15.2 MS15.3 MS16.1 MS16.2 MS16.3 MS16.4 MS17.1 MS17.2 MS18.1 MS18.3 MS19.1 MS19.2 MS20 MS21.1 MS21.2 MS22.1 MS22.2

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
58

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 2 0 0 0

51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

MS23 MS24.1 MS24.2 MS24.3 MS25.1 MS25.2 MS26.1 MS26.2 MS27.1 MS27.2 MS27.3 MS27.4 MS27.5 MS28 MS29.1 MS29.2

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 2 0 0 2 0 0 0 0

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KT LUN, TN TI V KIN NGH Kt lun Da vo kt qu thu c t cc th nghim, chng ti c th rt ra mt s kt lun nh sau: - phn lp c 66 chng vi khun Bt. Trong : + T 3 mu cn trng, phn lp c 3 chng vi khun Bt. + T 32 mu t, phn lp c 63 chng vi khun Bt. - xc nh c c im hnh thi, mu sc khun lc ca 66 chng vi khun Bt. - xc nh c hnh dng t bo, bo t v tinh th c ca 66 chng vi khun Bt. - xc nh c hot lc dit su ca cc chng vi khun Bt sn sinh nhiu bo t v tinh th c trong s 66 chng phn lp. + i vi su n l da chut v da hu, trong s 20 chng Bt xc nh c: * C 1/20 chng c hot lc dit su mnh, l cc chng H1.5 c hot lc dit su cao (t l su cht) 100% sau 72h. * C 7/20 chng c hot lc dit su t 80% - 90% sau 72h; l cc chng T5.1, MS16.2 , MS27.1, MS11.1, G6.1, MS21.1, MS27.4. + i vi su n l ci bp v su ho, trong s 20 chng Bt xc nh c : * C 2/20 chng c hot lc dit su mnh, l cc chng H1.5, MS27.4 c hot lc dit su cao (t l su cht) 100% sau 72h. * C 6/20 chng c hot lc dit su t 80% - 90% sau 72h; l cc chng MS16.2 , MS27.1, MS11.1,T5.2,G6.1, MS21.1, MS11.1. - bo qun c 66 chng vi khun Bt phn lp c ti Trung tm Ging & CNSH Trng HLN. Tn ti Do thi gian thc hin kho lun, trang thit b, s lng su th nghim c hn, cng nh trnh bn thn nn:

60

- Cha xc nh chnh xc c cc chng vi khun Bt phn lp thuc loi, chi no v chng c b trng lp hay khng. - Cha xc nh c chnh xc tim nng ca cc chng vi khun Bt phn lp v ti ch s dng mi trng nui cy chung cho vi khun Bt. - Cha xc nh c chnh xc hnh dng tinh th c ca cc chng vi khun Bt phn lp. - Cha th c hot lc dit su ca c 66 chng Bt phn lp. - Cha th c hot lc dit su vi mt s loi su hi cy nng - lm nghip khc. Kin ngh - Tip tc phn lp thm vi khun Bt cc a im khc quanh khu vc Trng HLN (y l mt a im c tim nng xut hin Bt vi tn sut cao v c hot lc dit su mnh). - Th hot lc dit mt s loi su khc (ngoi su da) ca tt c cc chng Bt phn lp. - Tip tc nghin cu xc nh cc chng vi khun Bt xc nh l c hot lc mnh ( nh H1.5, MS21.1,v MS27.4) thuc loi, chi no, chng c b trng lp hay khng v tm cch nng cao hot lc dit su ca chng. Tm ra mi trng nui cy ph hp nht i vi tng chng, chng c kh nng pht trin v sinh tinh th c tt nht.

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TI LIU THAM KHO Ti liu ting Vit 1. L Th Thu Hin, inh Duy Khng, L Trn Bnh, Nng Vn Hi( 1998), Gen khng cn trng v ng dng cng ngh chuyn gen thc vt . K yu vin cng ngh sinh hc. 2. Lu nh Thy (2005), Nghin cu sn xut thuc tr su sinh hc bacillus thruringiensis v ng dng trong phng tr su hi ( Kha lun tt nghip ). 3. Ng nh Bnh ( 2005), Thuc tr su vi sinh Nh xut bn i Hc Quc Gia H Ni, tr 10 43. 4. Nguyn Th Chnh, Nguyn nh Quyn(1998), Nghin cu sn xut thuc tr su Bt v ng dng trong phng tr su hi , Bo co nghim thu nhnh ti thuc d n hp tc Vit Nam CHLB c. VNM 9510-017. 5. Nguyn Ln Dng,( 1981). S dng vi sinh vt phng tr su hi cy trng, nh xut bn KHKT. 6. Ng nh Quang nh, Nguyn Qunh Chu, Nguyn Vn Thng, Nguyn nh Nguyt, Nguyn Hoi Trm, Trnh Th Cng( 2000), Nghin cu s phn b v a dng sinh hc ca Bacillus thurigiensis phn lp mt s tnh Vit Nam, Nhng vn nghin cu c bn trong cng ngh sinh hc, Nh xut bn i Hc Quc Gia H Ni, tr 484 488. 7. Nguyn Th Thanh Vn (1996), Nghin cu iu kin n gin nhn ging Bacillus thurigiensis, la chn phng php sn xut ging cung cp cho cc vng trng rau sch.( Lun vn thc s sinh hc). 8. Phm Th Thu (2004). Cng ngh sinh hc trong bo v thc vt, NXB i hc quc gia H Ni, tr 235 - 243. Ti liu ting Anh 9. Amos Navon(1993), Control of Lepidoteran pets with Bacillus thurigiensis ", Bacillus thurigiensis an environment biopestiside theory and practide, tr 126 146.

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10. Chilcot C.N and P. J Wigley (1994), Insecticidal activity of Bacillus thurigiensis crystal protein", Proceeding of the 2nd Canberra meeting on Bacillus thurigiensis, tr 43 52. 11. Choma C . T . W. K. surewicz, P. R. Carey, M. Pozsgay, T. Raynor and H. Kaplan (1990), Unusual proteolysis of the protoxin and toxin Bacillus thurigiensis, Eur.J. Biochem189: tr 523 527. 12. Deluca A.J, Simonson J.G and larson A. D(1981), Bacillus thurigiensis distribution on soil of the United States, Microbiol, 27: tr 865 870. 13. Hofte. H and H. R. Whitelog (1989) Insecticidal crystal protein of Bacillus thurigiensis", Microbiol. Rev. 53: tr 242 255. 14. Iizuka T(1999), Historical review on Bacillus thurigiensis", Biotechnology of Bacillus thurigiensis, Eds : Yuzinin, Sun Ming and Liu Ziduo, Vol 3: tr 35. 15. Laurent P. H, Ripauteau H, Dumamoir V.C, Frachon E, Lecadet M-M(1996), A micromethod for serotyring Bacillus thurigiensis, Microbiol.22: tr 259-261. 16. Pries F . G(1992), Biological control of Mosquitoes and other bitting flies by Bacillus thurigiensis and B sphaericus, Journal of Applied

Bacteriology,72: tr 357- 369 17. Powel, C.A. Charlton and T. Yanamoto(1994), Recent advences in structure and fuction reseach on Bacillus thurigiensis crystal protein, Bacillus thurigiensis biotechnology and environmental benefits, Vol 1: tr 1-20. 18. Sakhi. V. F. P. Parenti, G.M.Hanazet, B.Giordara, P . Lluthy(1986), Bacillus thurigiensis toxin inhibits K+ - gradient dependent amino acid transport across the brush border membrane of pieris brassicae midgut cell, Microbiol Rev.53: tr 213 218. 19. Thiery and E. Frachon( 1997), Indentification, isolation culture and preservation enbromo pathogenic Biologocal techniques manual of technology in Insect pathology, A academicpress, tr 55 56.

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Cc trang web 20. http\\:www.agbiotech.com.vn 21. http\\:www.ebook.edu.vn 22. http\\:www.thiennhien.net

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