NGHIEN ClTU U’NG DUNG VIKHUAN NOl SINH Bacillus velezensis VY03 TRONG PHONG CHONG BENH BAC LA LUA Nguyen Thi Hieu Thu1, Nguyen Duy Tbi1, Lai Tien Dung1 2, Nguyen Kim Nfl Thao3, Dinh Thuy Hang1 TOM TAT[.]
NGHIEN ClTU U’NG DUNG VIKHUAN NOl SINH Bacillus velezensis VY03 TRONG PHONG CHONG BENH BAC LA LUA Nguyen Thi Hieu Thu1, Nguyen Duy Tbi1, Lai Tien Dung12, Nguyen Kim Nfl Thao3, Dinh Thuy Hang1 TOM TAT Chung vi khuan noi sinh lua Bacillius velezensis VY03 co hoat tinh doi khang cao vbi vi khuan Xanthomonas oryzae pv oryzae (Xoo) gay benh bac la lua Dieu kien nuoi cay toi du de chung VY03 sinh trdbng va sinh hoat chat doi khang Xoo la moi triidng co pH 7, NaCl 10 g/L, sucrose la nguon carbon va nang ldpng, cao thit la nguon nitcf, nhiet nuoi cay la 37°C, thbi gian nuoi cay 40 gib Thi nghiem nha liibi danh gia hieu qua phong chong benh bac la tren giong lua Bac Thcfm so cho thay hieu qua kiem soat benh cao (71,6%) sif dung dich nuoi chung VY03 theo che phdng-chong (phun tnibc va sau nhiem Xoo) Biibc dau danh gia hieu qua kiem soat benh bac la cung tren giong lua Bac Thom so dieu kien td nhien cho thay, chung VY03 co hieu qua kiem soat benh tot diioc ap dung ket hop xd ly dat licfm ma va phun xuat hien benh, hieu qua dat 75 - 85% Ben canh do, sb dung chung VY03 lam tang nang suat lua ~12% so vbi doi chbng khong xb ly Ket qua thu dbOc mb tiem nang bng dung vi khuan noi sinh kiem soat sinh hoc doi vbi benh bac la, dong thbi kich thich tang trbbng cua cay, gop phan giam thieu sb dung thuoc hoa hoc canh tac lua Tb khoa: Benh bac la lua (Xanthomonas oryzae pv oryzae), vi khuan noi sinh lua (Bacillius velezensis), kiem soat sinh hoc I DAT VAN DE dinh dbbng va bi heo kho (Huang et a l, 1997) Xoo Benh bac la dope phat hien lan dau tien tai vung Fukuoko, Kyushu b Nhat Ban nam 1884 (Mizukami and Wakimoto, 1969) Benh da xuat hien vao nhbng nam 1960 b Dong Nam A, hien day cung la noi benh dien bien nhat (Goto, 2012) Tren toan cau, benh bac la co the lam thiet hai tbi 50% nang suat, gay anh hdbng lbn den nen kinh te cua cac quoc gia lua (Mew, 1992) co the lay lan nhanh qua gib, mba va ndbc tbbi, cung nhb qua hat giong va dat (Murty & Devadath, Vi khuan Xanthomonas oryzae pv oryzae (Xoo) la nguyen nhan gay benh bac la lua Vi khuan xam nhap vao la thong qua cac vet thbong hay 16 khong, thuy khong, sinh trbbng gay tac cac mach dan lam cho la lua khong nhan dbpc chat 1984) B enh bac la thbbng phat sinh vbi cac dau hieu dien hinh la la heo ua va chay kho tb ngon xuong, bat dau b giai doan cay de nhanh, dinh diem b thbi ky hoa, co the keo qua thbi ky tro hat va chin (Mew et al., 1993) Khi hau am va mba la dieu kien thuan lpi de benh bung phat va lan rong (Adhikari & Basnyat, 1999) O Viet Nam, theo bao cao cua Cue Bao ve thbc vat, nhbng nam gan day dien tich lua bi benh bac la tang len moi nam, dac biet dien tich benh nang dan den mat trang tang tren 10 nghin ha, phan anh mbc nghiem cua 1Vien Vi sinh vat va Cong nghe sinh hoc, Dai hoc Quoc gia Ha Noi 2Vien Bao ve there vat, Vien Khoa hoc Nong nghiep Viet Nam 3Truong Dai hoc Khoa hoc Tu nhien, Dai hoc Quoc gia Ha Noi 63 Tap chi Khoa hoc va Cong nghe Nong nghiep Viet Nam - So 06(127)/2021 benh Thong thiidng benh diipc tri bang cac thuoc hoa hoc nhif thuoc diet vi khuan Bismerthiazol, thuoc diet nam Zineb Bui 80WP (Zhu et al., 2013) Tuy nhien, viec sd dung thiidng xuyen va lau dai cac hoa chat bao ve thiic vat la nguyen nhan dan den thoai hoa dat va gay nhiem moi triidng Kiem soat cac benh tren cay sd dung cac tac nhan sinh hoc la hiidng di diipc quan tam, nham giam thieu sd dung hoa chat canh tac nong nghiep (O’Brien, 2017) Vi khuan noi sinh thvfc vat la nguon gen co tiem nang dng dung cao kiem soat sinh hoc, doi khang vdi nhieu loai vi sinh vat gay benh thiic vat tren cac loai cay ky chu khac Hien nay, co khoang hon 200 chi vi khuan noi sinh da diipc cong bo, chu yeu thuoc nganh Actinobacteria, Proteobacteria va Firmicutes (Afzal et al, 2019) Vi khuan noi sinh dc che vi sinh vat gay benh theo nhieu cP che, nhif (i) sinh cac hoat chat khang khuan, (ii) canh tranh ve dinh diidng va khong gian sinh triidng, (iii) tang ciidng sdc khoe va kha nang mien dich cua cay (Afzal et al., 2019) He vi khuan noi sinh d cay lua co mdc da dang cao va bao gom nhieu chung mang dac tinh sinh hoc co lpi cho cay chu (Bertani et al., 2016) Nghien cdu ve he vi khuan noi sinh lua cung nhii mot so loai thpc vat khac da chi rang cac loai Bacillus nhii B amyloliquefaciens, B subtilis, B velezensis, B pumilus co hoat tinh doi khang nhieu loai vi sinh vat gay benh thpc vat, vay co tiem nang dng dung kiem soat sinh hoc (Bertani et al., 2016; Chung et al., 2015) MT256303) phan lap td re cay lua Bac Thdm so vu Xuan He 2018 tai Thai Binh co kha nang doi khang tot vdi vi khuan gay benh bac la Xoo Chung diipc liiu tai Trung tam nguon gen Vi sinh vat Quoc gia vdi ma so VTCC60016 Chung Xanthomonas oryzeae pv oryzeae XR5 diipc phan lap td lua bi benh bac la d Viet Nam, diipc liiu gid tai phong Vi sinh Nong nghiep, Vien Vi sinh vat va Cong nghe Sinh hoc, Dai hoc Quoc gia Ha Noi 2 P h iiP n g p h a p n g h ie n c d u 2.2.1 D anh g id dnh httdng cua cac dieu kien nuoi cay den h o q t ttnh khdng X oo cu a chung VY03 Chung VY03 diipc nuoi cay tren moi triidng Landy dich the: glucose 20 g; KH2P g; M gS04.7H20 0,5 g; F eS 04.6H20 0.0012 g; CuS0 4.7H20 0.0016 g; M n S04 0.0004 g; L-glutamic acid g; cao nam men 10 g; niidc cat L; pH 7,5 (Wu et al., 2015) Cac dieu kien nuoi cay diipc danh gia gom co: nhiet (25, 30, 37 va 42°C), nong muoi (NaCl 0, 5, 10, 15 va 20 g/L), pH (td den 9), nguon carbon (glucose, sucrose, glycerol, methanol, tinh bot, ri diidng) va nguon nito (cao nam men, cao thit, peptone, triptone, casein) Trong cac thi nghiem, vi khuan diipc nuoi d dieu kien lac 160 vong/phut 40 gid 2.1 Vat lieu nghien cvtu Dich canh triidng (1 mL) cua vi khuan nuoi d cac dieu kien khac diipc tach chiet bang dung moi ethyl acetate (ty le 1: ve the tich), lam bay hdi chan khong Can chiet tho sau diipc hoa tan 50 pL DMSO va sd dung de danh gia hoat tinh khang Xoo bang phiidng phap khuech tan tren dia thach (Magaldi et al., 2004), tom tat cac biidc thpc hien nhii sau: cac dia petri chda moi triidng PSA (khoai tay 300 g; sucrose 15 g; Na2H P 4.12H20 g; C a(N 03)2 0,5 g; peptone g) diipc bo sung vi khuan Xoo d ty le 1% (v/v) Sd dung ong hut vo trung due cac gieng thach (diidng kinh mm) tren dia, sau nho 50 pL hoat chat tho cua chung VY03 hoa tan DMSO vao cac gieng va u dia d 30°C 24 h Nhii vay, liipng mau thd moi gieng tiicfng diidng mL dich nuoi cay Hoat tinh doi khang Xoo diipc danh gia qua kich thiidc vong khang khuan AD = D - d, D la diidng kinh vong khang va d la diidng kinh 16 thach Chung vi khuan noi sinh lua Bacillus velezensis VY03 (trinh 16S rDNA co ma GenBank la 2.2.2 Nghien cvtu kh d ndngphdn g chong benh bqc Id cu a chung VY03 dieu kien n htdi Chung vi khuan noi sinh Bacillus velezensis VY03 diipc phan lap td re cay lua Bac Thom, co hoat tinh doi khang cao vcfi vi khuan Xoo gay benh bac la Trong nghien cdu nay, anh hiidng cua cac dieu kien sinh triidng tdi hoat tinh khang Xoo cua chung VY03 diipc danh gia chi tiet in vitro de phuc vu viec dng dung kiem soat sinh hoc Hieu qua phong chong benh bac la cua chung VY03 cung diipc danh gia dieu kien nha liidi va ngoai dong ruong, theo hiidng dng dung chung vi khuan noi sinh phong trd benh Xoo gay diipc ban luan II VAT L IE U VA PH LfO N G P H A P N G H IE N C U tJ 64 Tap chiKhoa hoc va Cong nghe Nong nghiep Viet Nam - So 06(127)/2021 Thi nghiem dope thpc hien tren giong lua Bac Thom so (BT7), ditpc trong dieu kien nha litbi tai vien Bao ve thpc vat va cham soc theo quy trinh thiibng quy Thi nghiem gom cac bribe nhii sau: Cac dat thi nghiem co kich thiibc m2 (1 m x m) difqc lam sach co, biia va bon lot theo quy trinh thiibng quy Hat giong lua BT7 dripc u ma - 10 ngay, cay vao cac dat theo ty le 30 khom/m2, khoang each cay each cay x cm; hang cac hang 20 x 20 cm Duy tri mpe niibc mat cm va thpc hien cham soc theo quy trinh thiibng quy Chung vi khuan Xoo XR5 dripc nuoi moi trribng TSB 1/2 (tryptone 17 g; soya peptone g; NaCl g; K2H P 2,5 g; glucose 2,5 g; pH 7,3; lripng sh dung cac phan giam xh ) b 30°C, lac 160 v/p 24 gib Dich nuoi sau dripc pha loang bang moi triibng TSB 1/2 vo trimg tefi O D ^ = 0,2 Cay lua tuan tuoi dope nhiem Xoo bang each dung keo vo trung nhung vao dich vi khuan Xoo chung XR5 va cat dau ngon la lua khoang - cm Chung VY03 dope nuoi moi trribng Landy b dieu kien toi liu Dich nuoi dope pha loang vbi mlbc theo ty le 5% va phun Cac cong thhc thi nghiem gom: Cong thhc T phun dich nuoi vi khuan VY03 triibc nhiem Xoo va lien tuc sau nhiem Xoo Cong thhc 2: phun dich nuoi vi khuan VY03 lien tuc sau nhiem Xoo Doi cluing A: phun Bismerthiazol vbi lieu liipng thiibng dung (0,025 g pha 50 mL node phun cho m2 thi nghiem) theo che phong chong benh, the la phun trObc nhiem Xoo va lien tuc sau nhiem Xoo Doi chiing B: phun Bismerthiazol vbi lieu lopng thobng dung theo che chong benh, the la phun lien tuc sau nhiem Xoo Doi cluing C: nhiem Xoo, khong xb ly Cac cong thhc thi nghiem dope lap lai lan (3 dat cho moi cong thiic) Ty le benh bac la cac cong thOc thi nghiem dope danh gia bang each lpa chon ngau nhien 10 danh lua b gifla cac dat thi nghiem (bo qua cac danh lua b bia ngoai), miic ton thoong cua la tai cac thbi diem trObc phun va sau phun 3, 5, 7, 14, 21 va 28 Hieu lpc phong trii dope danh gia theo cong thOc Abbott (Abbott, 1987) nhO sau: H (%) = - ^ t T x 100 Trong do: CvaT Ian lUdt la chi so benh tren cay d cong thiic doi chiing va cong thiic thi nghiem, dUOc tinh bang chieu dai ton thiidng (mm) 2.2.3 Nghieti ctiu Jcha tian gphdn g chong ben h b ac Id cua chung VY03 th i nghiem don g ruong Thi nghiem dong ruong dope thpc hien tren giong lua Bac Thom so 7, gom cac bobc nhO sau: Chung VY03 dope nuoi moi trObng Landy dich the b dieu kien toi Ou Te bao vi khuan dope thu bang ly tam va hoa dung dich NaCl 0,9% tbi gia tri ODfifl0 = 0,5 Dich te bao chung VY03 dope tObi vao dat u ma trObc gieo hat (ty le 100 mL/m2, tron deu 10 cm dat mat) Cay ma sinh trObng tren dat co bo sung te bao cua chung VY03 15 trObc dOa ruong cay Dien tich lua thi nghiem la luong lap lai, moi luong 30 m2 (dai x rong la m x 10 m) Trong moi luong, lua dope cay tifng danh vbi mat 42 danh/m2, khoang each cay each cay la 15 x 15 cm, hang each hang la 16 x 16 cm Cay lua dope cham soc theo quy trinh thobng quy Trong giai doan cay lua lam dong den tro (70 75 tuoi), la thbi diem d i nhiem benh bac la, b cong thhc thi nghiem (CTTN) tien hanh phun dich nuoi chung VY03 pha loang (OD600 = 0,2), tan suat lan (tranh mOa), tong cong lan Doi chhng gom co (i) ruong khong phun thuoc (DC) va (ii) ruong phun thuoc hoa hoc Kasumil 2SL vbi lieu lOpng thobng dung (40 mL thuoc pha binh 16 L phun cho 300 - 400 m2) (DCks) Theo doi tien trien benh b cac ruong thi nghiem va doi chhng, danh gia hieu lOc phong trb benh bac la lan th phun thuoc cho den thu hoach Cu the, moi cong thhc danh dau dieu tra 10 diem, moi diem dieu tra 10 danh ngau nhien, thpc hien dem so la bi benh va phan cap benh Tinh toan chi so benh va ty le benh theo QCVN 01-166:2014/ BNNPTNT (PhOcfng phap dieu tra phat hien dich hai lua) bang cac cong thhc sau: Chi so benh (CSB): CBS (%) = (n 1+3n3+5nl t Z n7+9n9) _ x ioo NxK Trong do: nl, n3, n9 Ian lUdt la so la bi benh d cac cap benh; N la tong so la dieu tra; K la cap benh cao nhdt quan sat dtidc 65 Tap chiKhoa hoc va Cong nghe Nong nghiep Viet Nam - So 06(127)/2021 Ty le benh (%) = So la bi benh — — - x 100 So la dieu tra Cac yeu to cau nang suat va nang suat lua diidc danh gia theo QCVN 01-55:2011/BNNPTNT gom: so bong/m2, so hat chac, khoi liidng 1.000 hat, nang suat ly thuyet va nang suat thdc thu (so lieu diidc tlnh toan n = 30 mau) Mau diidc thu thap theo TCVN 5451:2008 Hai Quang, huyen Hai Hau, tinh Nam Dinh vu Xuan He nam 2021 III K E T Q U A VA T H A O LUAN A n h h iid n g c u a c a c d ie u k ie n n u o i c a y d en h o a t tin h d o i k h a n g Xoo c u a ch u n g V Y Nhiet nuoi cay da khao sat khoang 25 - 42°C khong anh hiidng dang ke den hoat tinh doi Nang suat ly thuyet (ta/ha) = (So bong/m2 x So hat chac/bong x M 1.000 hat)/10.000 khang Xoo cua chimg VY03 (Hinh 1A, ID ) Mac Nang suat thdc te (ta/ha) = (nang suat 6/30 m2) x 10.000 m2 cua chung dat mdc cao nhat nhiet 25°C - 30°C 2.2.4 Xii It so lieu giam nhe nhiet 37 - 42°C Do pH khoang So lieu thi nghiem dupe xd ly bang phan mem Excel va IRRISTAT 5.0 du vay, co the nhan thay hoat tinh doi khang Xoo (diicfng kinh vong khang khuan la 17,5 mm), va - thich hpp vdi chung VY03, pH chung co hoat tinh doi khang Xoo cao nhat (diibng kinh vong khang khuan 19 mm) (Hinh IB, IE) T hdi g ia n v a d ia d ie m n g h ie n ctiu Thi nghiem nha liidi diipc thiic hien tai Vien Bao ve thiic vat vu Xuan He nam 2020 Thi nghiem dong ruong diipc tien hanh tai xa Hoat tinh doi khang Xoo cua chiing VY03 khong thay doi dang ke moi triidng co nong muoi khoang - g/L (Hinh 1C, IF), mdc hoat tinh cao nhat la nong muoi 10 g/L Hinh Anh hiidng cua cac dieu kien nuoi cay tefi hoat tinh doi khang Xoo cua chung VY03 Ghi chu: A, D - Anh hiidng cua nhiet do; B, E - Anh hiidng cua pH; C, F - Anh hiidng cua nong muoi Khac vdi cac dieu kien nhiet do, pH hay nong sd dung lam cd chat va cho hoat tinh khang Xoo cao muoi, nguon carbon hdu cd s£f dung moi vdi diidng kinh vong khang dat 22 mm Dieu tnlimg nuoi cay co anh hiidng ro ret tdi hoat tinh co y nghia thpc tien quan cho viec giam gia doi khang Xoo cua chung VY03 (Hinh 2) Chung cua moi trddng nuoi cay de len men thu sinh YY03 the hien hoat tinh doi khang Xoo cao nhat khoi chung VY03 Acetate va methanol la cac cd dupe nuoi moi triidng co glucose hoac chat it phu hdp nhat doi vdi chung VY03 Doi chdng su crose, dudng kinh vong khang dat mdc 26 va 26,5 thi nghiem la moi triidng TSB 1/2 co ham m m , D an g chu v, ri dudng cd the diipc chiing VY03 liidng glucose thap (1,25 g/L), dan den chung VY03 Tap chiKhoa hoc va Cong nghe Ndng nghiep Viet Nam - So 06(127)/2021 nuoi tren moi trddng chi dat hoat tinh doi khang Xoo b mdc 58,49% so vdi nuoi moi triibng co sucrose (20 g/L) (Hinh 2A, 2C) Nhd vay, nguon carbon hdu cd co anh hiicfng ldn den hoat tinh doi khang Xoo cua chung VY03, ca ve ban chat hoa hoc va lieu liidng sd dung Hinh Anh hiicfng cua cac nguon carbon va nitcf tcfi hoat tinh doi khang Xoo cua chung VY03 Khac vbi nguon carbon hflu cd, nguon nitd khong anh hiidng den hoat tinh doi khang Xoo cua chung VY03 Cu the, chung VY03 diidc nuoi moi triidng sii dung cac nguon nitd khac nhii peptone, casein, cao thit hay cao nam men deu dat diidng kinh vong doi khang Xoo mdc 26 - 27 mm (Hinh 2B, 2D) Doi chifng la moi trddng TSB co nguon nitd la tryptone d mdc cao tiidng diidng vdi dieu kien thii nghiem, nhien ham liidng carbon thap, dan den hoat tinh doi khang Xoo thap hdn (Hinh 2B, D) Ket qua khang dinh tarn quan cua nguon carbon quyet dinh hoat tinh doi khang Xoo & chung VY03 So vdi moi tnidng TSB, moi triidng Landy co cd chat la sucrose (20 g/L) va nguon nitd la cao nam men (10 g/L), la moi triidng phu hdp cho chung VY03 de dat hoat tinh doi khang Xoo cao 3.2 D a n h g ia h ieu q u a k ie m s o a t b e n h b a c la c u a ch u n g VY03 tr o n g d ieu k ie n n h a lifefi Thi nghiem nha liicfi diidc theo doi 28 va tien trien cua benh bac la diidc danh gia diia tren cac bieu hien dac tning cua benh nhii ua vang, heo kho d dau la (dinh tinh) va chieu dai mdc ton thiidng (dinh liidng) Ket qua chd thay hieu qua kiem soat benh cao nhat la of cac cong thdc xii ly theo che phong va chong benh (phun triidc va sau nhiem Xoo), gom cong thdc phun dich te bao chung VY03 va doi chdng A phun thuoc Bismethiazol Hieu qua kiem soat benh tren cd sd chieu dai mdc ton thddng va xd ly thong ke (Hinh 3) cung khang dinh xd ly theo che phong chong co hieu qua cao hdn Chung VY03 co hieu qua rat dang chu y kiem soat benh bac la dddc ap dung theo phddng thdc phong va chong benh, tdc la phun lien tuc triidc va sau nhiem benh, hieu qua dat 71,6%, gan tiidng diidng vdi cong thdc doi chdng A sd dung thuoc hoa hoc Bismerthiazol cung theo phddng thdc phong va chong benh (hieu qua dat 75,7%) 100 80 £ g> 60 *3 40 cr 20 Cdng thtrc thf ngh^m Hinh Hieu lUc kiem soat benh bac la thi nghiem d dieu kien nha liidi Tuy nhien, cong thdc sd dung chung VY03 theo phddng thdc chong benh, tdc la chi phun sau 67 Tap chi Khoa hoc va Cong nghe Ndng nghiep Viet Nam - So 06(127)/2021 nhiem Xoo thi hieu qua kiem soat thap hdn han, chi dat ~20% Trong do, doi chdng B sif dung thuoc hoa hoc Bismerthiazol theo cung dieu kien vln dat hieu qua cao la 69,4% Theo nghien cdu cua nhom tac gia Chung va cong tac vien (2015), hai chdng vi khuan Bacillius sp YC7007 va YC7010T co the kiem soat benh bac la lua d mdc 61,2 - 70,8% Ro rang, viec sd dung chung VY03 theo phiidng thdc phong va chong dat hieu qua kiem soat benh bac la cao tiidng diidng vdi cac nghien cdu da cong bo Ket qua gdi md hitdng sd dung cac tac nhan kiem soat sinh hoc theo phiidng thdc phong chong, tdc la tao dieu kien cho cay lua tiep xuc sdm vdi vi sinh vat doi khang triidc bi nhiem benh, tham chi d giai doan hat mam hoac cay ma va ket hop phun tri benh cay co bieu hien mac benh xam nhap vao cay td giai doan dau cua qua trinh sinh triidng Ket qua dieu tra mdc nhiem benh bac la giai doan cay lua lam dong den trd (70 - 75 tuoi) cho thay benh bac la xuat hien d tat ca cac ruong thi nghiem vdi ty le benh tang dan qua cac ky dieu tra (Hinh 4) Tuy nhien, co the nhan thay sU chenh lech ro ret gida cong thdc thi nghiem (lua diipc tiep xuc sdm vdi chung VY03) va cac ruong doi chdng Cf CTTN, tai thdi diem tro bong ty le benh bac la la 19,83 - 20,05% va chi so benh la 9,21 - 9,38%, sai khac d mdc co y nghia thong ke so vdi doi chdng la 48,72 % va 26.25% (Hinh 4A, B) Sau sp gia tang cua benh bac la cac ruong thi nghiem diipc xac dinh, dich te bao cua chdng VY03 diipc phun d CTNN va so sanh vdi doi chdng la ruong khong xd ly benh (DC) va rupng 3 D a n h g ia h ie u q u a k ie m s o a t b e n h b a c la c u a cua cac ho dan xung quanh phun thuoc Kasumin 2SL (DCrs) Ket qua cho thay CTTN chdng ch u n g V Y tr o n g d ieu k ien d o n g ru o n g VY03 lam giam ty le benh va chi so benh mot Khac vdi thi nghiem nha liidi, thi nghiem each ro ret, d mdc tiidng diiong vdi thuoc hoa hoc dong ruong te bao chung VY03 diipc difa vao dat Kasumin 2SL (Hinh 4) gieo hat va itom ma de tao dieu kien cho vi khuan A 80 100 I CTTM I BCk, 70 „ 60 ^ sz5 & 90 Is 80 40 70 SB- 30 ^ 20 50 10 40 24-05 Ngay di&u tra Ngay diki tra 01-06 07-06 Ng8y ffi^u tra Hinh Hieu qua kiem soat benh bac la dieu kien thiic te ngoai dong ruong Cu the, ket qua danh gia d thdi diem lua chin sap, ruong diipc phun dich te bao chdng VY03 (CTTN) co ty le benh va chi so benh tiidng dng la 12,96 va 6,63%, tiidng diidng vdi ruong phun thuoc Kasumin 2SL (DCKS) la 10,45 va 4,12% Ruong doi chdng khong xd ly (DC) co ty le benh va chi so benh tiep tuc tang nhanh qua cac ky dieu tra va dat cao nhat d giai doan la 67,35 va 44,75% (Hinh 4A, B) Hieu qua kiem soat benh bac la d ruong xd ly bang chung vi khuan VY03 va ruong phun thuoc Kasumin 2SL tiidng dUdng suot qua trinh dieu tra, dat mdc 75 - 85% 68 (Hinh 4C) Hieu qua kiem soat benh cao hdn so vdi thi nghiem dieu kien nha liidi, phan anh tac dung cua viec cho cay tiep xuc sdm vdi chung vi khuan noi sinh VY03 bang xd ly dat lidm ma vdi chdng Ben canh do, hieu qua cua chung VY03 dc che benh bac la Xoo diipc danh gia thong qua cac yeu to cau nang suat va nang suat lua Quan sat hinh thdc bong lua co the thay d CTTN bong lua co mau sac hat vang sang, it hat lep, d doi chdng (DC) bong lua co nhieu hat lep, mau nau toi (Hinh 5) Tap chiKhoa hoc va Cong nghe Nong nghiep Viet Nam - So 06(127)/2021 cao hdn DC (mdc sai khac co y nghia) Khoi ldpng 1.000 hat CNTN cung cao hdn DC (mdc sai khac co y nghia) Nang suat ly thuyet va nang suat thiic te cua CTTN dao dong td 64,7 - 70,4 ta/ha, cao hdn cong thdc ddi chdng d mdc sd sanh co y nghia td 12 - 13% Hinh Bong lua d ruong doi chdng (DC) va CTTN Ket qua danh gia chi tiet (Bang 1) cho thay, so bong/m2 cua CTTN khoang 268,8 - 289,8, so hat chac/bong giao dong td 104,22 - 118,12 hat, Tong hdp cac ket qua phan tich thd nghiem dong ruong cho thay chdng VY03 co anh hitdng tich cUc tdi sd phat trien cua cay lua, gom (i) kiem soat tot benh bac la va (ii) tang nang suat lua Mac du co tinh man cam cao vdi nhieu benh hai, dac biet la benh bac la Xoo gay ra, giong lua Bac Ihdm so diidc bien nhat d cac tinh dong bang song Hong nhieu nam bdi co chat lddng gao tot Chung VY03 nghien cdu the hien tac dung nhii mot probiotic cho cay lua, co tiem nang dng dung cao canh tac lua hdu cd, giup giam thieu sd dung hoa chat trdng nong nghiep Bang Yeu to cau nang suat va nang suat lua cua cac cong thdc thi nghiem NSLT (ta/ha) Nang suat Tang(%) NSTT (ta/ha) 20,56 64,7 12 59,2 19,25 53,9 - 48,6 Cong thdc Bong/m2 So hat chac/bong M 1.000 hat (g) CTTN 285,6 110,25 DC 268,8 104,22a Ghi chu: M: Trong lUdng; NSLT: Nang suat ly thuyet; NSTT: Nang suat thutc te IV K E T LU A N VA D E N G H I K e tlu a n Chung vi khuan noi sinh lua Bacillus velezensis VY03 co hoat tinh doi khang vi khuan Xoo cao the hien cac thi nghiem in vitro Hoat tinh on dinh cac dieu kien nhiet do, pH, nong NaCl, va nguon nito, chi phu thuoc vao nguon carbon sd dung moi trddng nuoi cay, glucose va sucrose la phu hdp nhat Thd nghiem chung VY03 kiem soat benh bac la lua d dieu kien nha lddi cho thay sd dung theo phddng thdc phong va chong benh (phun triidc va sau nhiem Xoo ) cho hieu qua kiem soat 71.6%, tddng diiOng vdi thuoc hoa hoc Bismerthiazol ap dung cung dieu kien Trong do, neu sd dung theo phiicfng thdc chong benh (phun sau nhilm Xoo ) thi hieu qua kiem soat chi dat mdc 20.4% Thd nghiem d dieu kien dong ruong theo phiiong thdc phong va chong, ket hdp xd ly dat liOm ma bang chdng VY03 vdi phun phong benh lua d giai doan lam dong va phun tri benh phat hien benh bac la da cho hieu qua kiem soat benh tot (75 - 85%), tddng dddng thuoc hoa hoc Kasumin 2SL, dieu kien benh lay nhiem tvi nhien Qua trinh xd ly benh bang chung VY03 cung lam tang nang suat lua -12% so vdi doi chdng khong xd ly D e n g h i Nghien cdu phat trien che pham sinh hoc td chung Bacillus velezensis VY03 va tiep tuc thd nghiem d dieu kien dong ruong de danh gia hieu qua, lam cd sd difa che pham vao phuc vu cho san xuat lua hdu cd LCtt CAM ON Nghien cdu dddc tai trd td de tai NDT.34.ITA/17 Bo Khoa hoc va Cong nghe tai trd TA I L IE U T H A M K H A O Abbott, W.S., 1987 A method of computing the effectiveness of an insecticide Journal of the American Mosquito Control Association, (2): 302-303 69 Tap chi Khoa hoc va Cdng nghe Nong nghiep Viet Nam - So 06(127)/2021 Adhikari, T.B., Basnyat, R.C., & Mew, T.W., 1999 Virulence of Xanthomonas oryzae pv oryzae on rice lines containing single resistance genes and gene combinations Plant Disease, 83 (1): 46-50 Afzal, I., Shinwari, Z.K., Sikandar, S., & Shahzad, 5., 2019 Plant beneficial endophytic bacteria: Mechanisms, diversity, host range and genetic determinants Microbiological Research, 221: 36-49 Bertani, I., Abbruscato, P., Piffanelli, P., Subramoni, 5., & Venturi, V., 2016 Rice bacterial endophytes: isolation of a collection, identification of beneficial strains and microbiome analysis Environmental Microbiology Reports, (3): 388-398 Chung, E.J., Hossain, M.T., Khan, A., Kim, K.H., Jeon, C.O., & Chung, Y.R., 2015 Bacillus oryzicola sp nov., an endophytic bacterium isolated from the roots of rice with antimicrobial, plant growth promoting, and systemic resistance inducing activities in rice The Plant Pathology Journal, 31 (2): 152 Goto, M., 2012 Fundamentals of bacterial plantpathology Academic Press Huang, N., Angeles, E.R., Domingo, J., Magpantay, G., Singh, S., Zhang, G., & Khush, G.S., 1997 Pyramiding of bacterial blight resistance genes in rice: marker-assisted selection using RFLP and PCR Theoretical and Applied Genetics, 95 (3): 313-320 Magaldi, S., Mata-Essayag, S., De Capriles, C.H., Perez, C., Colella, M.T., Olaizola, C & Ontiveros, Y., 2004 Well diffusion for antifungal susceptibility testing International Journal o f Infectious Diseases, 8: 39-45 Mew, T.W., 1992 Compendium of rice diseases American Phytopathological Society St Paul, MN: 62 Mew, T.W., Alvarez, A.M., Leach, J.E., & Swings, J., 1993 Focus on bacterial blight of rice Plant Disease, 77 (1): 5-12 Mizukami, T., & Wakimoto, S., 1969 Epidemiology and control of bacterial leaf blight of rice Annual Review of Phytopathology, (1): 51-72 Murty Ih ri, V.S., & Devadath, S., 1984 Role of seed in survival and transmission of Xanthomonas campestris pv oryzae causing bacterial blight of rice Journal of Phytopathology, 110 (1): 15-19 O’Brien P.A., 2017 Biological control of plant diseases Australasian Plant Patholology, 44:1-12 Zhu, X.F., Xu, Y., Peng, D., Zhang, Y., Huang, T.T., Wang, J.X., & Zhou, M.G., 2013 Detection and characterization of bismerthiazol-resistance of Xanthomonas oryzae pv oryzae Crop Protection, 47: 24-29 Wu, L., Wu, H., Chen, L., Yu, X., Borriss, R and Gao, X., 2015 Difficidin and bacilysin from Bacillus amyloliquefaciens FZB42 have antibacterial activity against Xanthomonas oryzae rice pathogens Scientific Reports, : 12975 doi:10.1038/srep12975 Study on application of endophytic bacterium Bacillus velezensis VY03 in controlling rice bacterial blight disease Nguyen Thi Hieu Thu, Nguyen Duy Toi, Lai Tien Dung, Nguyen Kim Nu Thao, Dinh Thuy Hang Abstract The rice endophytic bacterium strain Bacillius velezensis VY03 has high antagonistic activity against the phytopathogen Xanthomonas oryzae pv oryzae (Xoo) causing rice bacterial blight disease The optimal culture conditions for the strain to grow and have the highest antagonistic activity against Xoo were pH 7, NaCl 10 g/L, sucrose as the carbon and energy sources, meat extract as nitrogen source, temmperature 37°C, and culturing time 40 h The experiment in net house condition using Bacthom N7 cultivar revealed that culture broth of strain VY03 had a high protective effect (71.6%) against the bacterial blight disease when applied at the protective-fighting mode, i.e before and after the Xoo infection Preliminary results of the field test using the same rice cultivar showed that a protective effect against the bacterial blight disease was obtained as high as 75 - 85% if the strain was used as an additive to soil for the seedlings in combination with spraying when the disease began in the field In addition, using the strain VY03 also increased the rice yield to -12% compared to non-treatment control The study showed significant potential for application of endophytic bacteria in controling the bacterial blight disease, while stimulating plant growth, thus reducing the use of agrochemical in rice cultivation Keywords: Bacterial blight disease (Xanthomonas oryzae pv oryzae), rice endophytic bacteria (Bacillius velezencis), biocontrol Ngay nhan bai: 14/5/2021 Ngay phan bien: 02/6/2021 Ngifdi phan bien: PGS.TS Nguyen Van Viet Ngay duyet dang: 29/6/2021 ... Chung vi khuan noi sinh lua Bacillus velezensis VY03 (trinh 16S rDNA co ma GenBank la 2.2.2 Nghien cvtu kh d ndngphdn g chong benh bqc Id cu a chung VY03 dieu kien n htdi Chung vi khuan noi sinh Bacillus. .. pv oryzeae XR5 diipc phan lap td lua bi benh bac la d Viet Nam, diipc liiu gid tai phong Vi sinh Nong nghiep, Vien Vi sinh vat va Cong nghe Sinh hoc, Dai hoc Quoc gia Ha Noi 2 P h iiP n g p h... M: Trong lUdng; NSLT: Nang suat ly thuyet; NSTT: Nang suat thutc te IV K E T LU A N VA D E N G H I K e tlu a n Chung vi khuan noi sinh lua Bacillus velezensis VY03 co hoat tinh doi khang vi