OIIMH TirVJH v i k h u A n m q i SIIMH TRONG c A TROIVG A TilMH B lv TRE V A LOIMG AIM

KHOA HOC C O N G N G H I X A C OIIMH TirVJH v i k h u A n m q i SIIMH TRONG c A TROIVG A TilMH B lv TRE V A LOIMG AIM y M IA Ng6 Hdng h1, Hoang Minh Tam2, Cao Ngpc Didp1 t 6 m t At Hai muoi tam d6ng vi khuin da dupe phan lap tu cac mau rk, than mia thu dugc tai cic tinh B n Tre vi Long An. Hiu heft cac dong vi khuin d u c6 hinh dang que ngin vi c6 khi ning chuyln dong. Khuin l?c c6 dang hinh trdn, miu ving, bia khuin lac nguydn ddu, khuin lac co dp noi mo cao, kich thuoc 1-3,4 mm. Bing phep thu sinh hoa da xic dinh dupe 15/28 ddng vi khuin co mang cic dac tinh tdt (co djnh dam, hoi tan lin vi tong hpp LAA). Tuy nhidn, chi nhin didn duoc mum ddng vi khuin npi sinh bing ky thuat PCR doan 16S rarn, nhung ddng vi khuin npi sinh niy dupe chpn de? giii trinh tu vi dupe so sinh vdi du lidu ngin hing gien cua NCBI bing phin mdm BLAST N. K t qui cho thiy trong tit ci 10 ddng phin lip dupe cd ti 1$tuong ddng 98-100%vdi cac loai thupc chi Bacillus. Phin tich cay di truydn dua trdn trinh ty doan 16S rarn bing phuong phip Neighbor-joining boi phin mdm MEGA 5.1 cho thiy mdi quan hd di truydn gin gui giua cic ddng vi ddng ; giua ddng vi ddng. Bdn ddng vi khuin ndi sinh Bacillus subtilis, Bacillus tequilensis, Bacillus cereus, Bacillus amyloliquefaciens cd d$c tinh tot, sd dupe su dung d sin xuit phin sinh hpc. Tu khoi: Bacillus, cd dinh dpm, hda tan lin, mia, tong hop IAA, vi khuin n$i sinh I.OATVANOt Dudng la san pham khong the? thidu trong bua in hang ngay cua con ngudi va ca trong chin nuoi gia sue. Song song vdi vide tang dan so cua the? gidi va su md rong chin nuoi vdi quy mo ldn, nhu cau vd dudng cung tang ldn rat nhanh. De? dip ung nhu ciu dd san lupng dudng trdn toan thd gidi cung tang theo. Tuy nhidn lupng dudng vln khdng dip ung du nhu cau tidu thu. Do vay, dd cd du lupng dudng cho tidu thu va phuc vu cho nhu cau chan nudi thi bdn canh vide tang didn tich trong mia ngudi ta cdn tim cach tang nang suat mia. Cu the?, de? cd 70 tan mia cay/ha ngudi ta phai bdn 60 kg N/ha. Thuc td con so nay ldn ddn 80 hay 100 kg/ha hay nhidu hon nua d nhung vung trong mia chuydn canh. Vdi tham vpng tang nang suat mia song song vdi vide giam lupng phan dam phai su dung, cac nha khoa hpc da vao cuoc vdi muc dich tim ra mot loai vi khuin song cong sinh vdi cay mia cd kha nang co dinh dam sinh hpc tu nito tu do trong khdng khi. Nam 1988 Cavalcante va Dobereiner da phan lap dupe loii vi khuin npi sinh trong cay mia va da dat tdn la Acetobacter nitrocaptans (hidn nay vi khuin cd tdn la Gluconacetobacter diazotrophicus) [3]. Kham pha 'Vi n Nghien cuu v i Phit trien Cong ngh? sinh hpc, D$u hpc Can The 2Trudng D?i hpc Saigon nay md dau cho cdng cuoc tim kidm cac vi khuin noi sinh trong cay mia trdn pham vi thd gidi. Hidn nay nhidu loai vi khuan noi sinh da dugc phan lap thanh cong d nhidu quoc gia vdi nhung dac tinh uu vidt nhu; co dinh dam sinh hpc, hda tan lan khd tan, tong hpp auxin. Md ra tridn vpng ldn cho su phat tridn cua nganh trong mia thd gidi. CT Vidt Nam da cd nhung nghidn cuu budc dau vd loai vi khuan huu ich nay. Tuy nhidn, vide nghidn cuu va hidu bidt vd chung vsn cdn nhidu han chd. Vi vay, vide phan lap, khao sat dac tinh va nhan didn cac ddng vi khuan nay dd san xuat phan bdn sinh hpc la mot vide lam cap bach va can thidt Muc tidu cua dd tai la phan lap, nhan didn va khao sat dac tinh cac ddng vi khuan noi sinh trong cay mia trong d tinh Bdn Tre va Long An. 2. VAT U 0 IV A PHUONG PHAP 2.1. vat lidu Hai muoi tam ddng vi khuan da dupe phan lap tnric day tir 10 mau tu than va rd mia trong dhai tinh Bdn Tre va Long An. 22. Phuong phip nghidn cuu 2.2.1. Phin l$p vi khuan Chpn cae khuin lac tu dia mdi trudng dang tru vi khuin, ciy chuyln nhidu lin sang mdi trudng LGI dac ddn khi cac khuan lac tach rdi ra. Tidn hanh kidm tra do rong (thuan nhit) cua cac ddng vi khuin. Quan sat bang kinh lup hinh thai cac d<mg cua khuin NONG NGHlfP VA PHAT TRI^N NONG TH6N - KY 2 - THANG 1/2014 41

KHOA HOC CONG N G H t lac nhir mau sic, do noi, hinh dang va dang bia; sir dung kinh hidn vi quang hoc va dien tir quan sat kha ning chuydn dong, kich thuoc va hinh dang cua cac td bao vi khuan. a. Khao sat kha ning co dinh dam RM dupe thidt kd boi Madhaiyan et al., 2004 >] voi trinh tunhu sau: i RB (i 5 AGAGTTT GATYMT C GCT CAG -3 i RM (i 5 GGACTACCAGGGTATCTAAT -3 xuoi) nguoc) Nuoi vi khuan trong moi trucmg Burk s khong dam long [ 12] tir 2 ddn 6 ngay va dinh luong amoni duoc tao ra bing phuong phap nitro- xyanhydrit natri hipocloril Mau duoc do o buoc song 640 nm. Cac san pham ADN sau khi duoc khudch dai, tidp tuc dien di trdn gel agaroza 1,2%, san pham PCR cho bang o vi tri khoang 900 bp. b. Khao sat hoat tinh nitrogenaza cua mot so ddng vi khuan tong hop amoni trien vong 2.2.3. Giai trinh tir va xay dung cay phat sinh loai (phylogenetic tree) Chon ra 5 dong vi khuan co kha nang tong hop amonium cao va dai dien cho noi thu mau, tidn hanh khao sat hoat tinh nitrogenaza bang phuong phap khu axetylen ( acetylene reduction assay - ARA) [4]. c. Khao sat kha nang hda tan lan kho tan Nuoi vi khuan trong moi trudng NBRIP long (8) tir 5 ddn 10 ngay va dinh luong lan hoa tan bang phuong phap so mau Oniani voi thuoc thu axit ascobic amonimolypdat kali antinomol tartrat o buoc song 880 nm. d. Khao sat kha ning tong hop indole-3-axetic axit (1AA) t so dong vi khuan hidn didn tot khi chay PCR bang cap moi RB-RM duoc tidn hanh giai trinh tu* Sau do, dung chuong trinh BLAST N d nhan didn vi khuan bang each so sanh voi tnnh tu gien cua cac dong vi khuan co trong ngan hang du lidu NCBI (National Center for Biotechnology Information). Dua vao khoang cach di truydn va ty Id phan tram tuong dong trinh tu nucleotit cua doan 16S rarn. Su dung chuong trinh MEGA 5.1, d<? xay dung cay phat sinh loai (hay cay pha hd). S. IET QIM VA H IM UMN Nuoi vi khuan trong moi truong LGI long khong bo sung tryptophan tir 2 ddn 6 ngay va dinh lupng LAA dupe tong hpp bang thuoc thu Salkowsky a buoc song 530 nm. 3.1. Phan l$p vi khuin npi sinh Hai muoi tam dong vi khuan da dupe phan lap tir cac mau than va rd mia thu dugc tai 3 huyen thuoc 2 tinh Bdn Tre va Long An. g do, 8/28 dong thupc 2.2.2. Tach chiet ADN va thuc hien phan ung huydn Bdn Luc, tinh Long An (28,57%), 7/28 dong PCR thuoc huydn Ham Luong, tinh Bdn Tre (25%), 13/28 ADN dupe tach chidt dua trdn qui tnnh chuan dong thuoc huydn Cay, tinh B^n Tre (46,43%). cua Neumann (1992) [9]. Sau do thuc hidn phan ling Nguon goc cac dong vi khuan nay dupe trinh bay PCR khudch dai doan 16S-rARN bang cap moi RB- trong bang 1. cua d c ddng vi khuin nqi sinh cay mia Stt VJ tri phan D$cdi m Kihi u Stt D$c di&n 1*P l p Ki hi u 1 Bdn Luc-Long An BL1 15 Ham Luong-B^n Tre HL6B 2 Bdn Luc-Long An 3 Bdn Luc-Long An 4 Bdn Luc-Long An BL2 16 Cay-B^n Tre R MCI 17 Cay-B^n Tre R BU 18 5 Bdn Luc-Long An 6 Bdn Luc-Long An 7 Bdn Luc-Long An R Rd BL5 Cay-Bdn Tre Cay-B^n Tre BL6 19 20 MC3 MC4 MC5 Rd 21 Cay-Bdn Tre Rk Rh Rl Rk 8 Bdn Luc-Long An 9 Ham Luong-Bdn Tre Rd BL8 22 MC7 HL1 23 Cay-B^n Tre Cay-B&i Tre Thin MC8 HL2 24 Cay-Bdn Tre HL3 25 Cay-BAi Tre M C10 10 Ham Luong-Bdn Tre 11 Ham Luong-Bdn Tre 42 Vi tri phan Cay-B&i Tre MC6 NONG NGHIDP VA PHAT TR( n N6NG TH6N - ICY 2 - THANG 1/2014

KHOA HOC C O N O N G H i 12 HAm Luong-Bdn Tre 13 Ham Luong-Bdn Tre 14 Ham Luong-Bdn Tre HL4 HL5 26 27 28 Cay-Bdn Tre Cay-Bdn Tre Cay-B^n Tre MC11 MC12 MC13 Kdt qui quan sat dac didm cua khudn lac va td bao vl khuin Nuoi vi khuan trdn moi trucmg LGI dac, u a 30 C trong 48 gia, kdt qua dupe trinh bay trong bang 2 BAng 2: B$c didm hinh thai cua cac d6ng vi khuin nfli sinh cay mia BAc didm ti bao Tt D6ng vi khuin 1 BL1 2 BL2 3 BO n6i Kich thu6c (mm) Nguyen 2,2 Tring trong Nguyen 1,5 VAng 1.4 Tr6n VAng 2,0 Tring due Lai 3,0 BL6 Tring trong 1.5 7 Que ngan 2,0 8 BL8 VAng 1,8 9 HL1 2,3 10 HL2 2,1 11 HL3 1.7 12 HL4 Khong d<ki VAng 2.0 13 HL5 M6 1.8 14 1.2 15 HL6B 1.2 16 MCI Tring due Lai 3,4 17 2.3 18 MC3 1.3 19 MC4 Tring trong 2.2 20 MC5 Tring trong 1.4 21 MC6 Tring due 1.8 22 MC7 2.0 23 MC8 2.0 24 M6 2.1 25 2.1 26 MC11 1.0 27 MC12 Trdn Mau hong 3.5 28 MC13 Tr6n 1.8 Hinh dang Chuyln d0ng B$c didm khuin lac Hinh d^ng MAu sic Que ngan 4 BL4 5 BL5 6 Chu thich: -: Vikhuin khdngchuyin dqng : Vi khuin chuyin ddngnhanh Dang bia : Vikhuin chuyin ddngch$m : Vi khuan chuyin dongrit nhanh N 6 N G NGHlfP V A PHAT TRll-N N 6 N G THdN - KY 2 - THANG 1/2014 43

KHOA HPC C6NO NOH j Da so cac dong vi khuan phan lap dupe d6u co khuan lac mau vang 20/28 (71,43%); 4/28 khuin lac co mau tring trong (14,26%); 3/28 khuan lac co mau tring due (10,71%); 1/28 khuan lac co mau hong (3,57%). V hinh dang khuan lac thi phin Ion cac dong vi khuan d u co dang tron 22/28 dong (78,57%), 6/28 dong con lai la dang khong d(ki (21,43%). Cac dong vi khuan nay deu tao khuan lac co dang bia nguydn tr n moi truong phan lap 28/28 dong ( 100%). V do noi cua khuan lac thi co 26/28 dong co khuan lac voi do ndi mo cao (92,86%); con lai la dang dai (7,14%). Duong kinh khuan lac cua cac dong vi khuan n&y dupe xic dinh nim trong khoang 1-3,4 mm. Quan sit hinh dang va khi nang chuyin dong cua nhung dong vi khuan bing phuong phap giot ep trong moi truong nuoc cat vo trung duoi kinh hien vi quang hpc o do phong dai 400 lin. Nhan thay 100% cic dong vi khuan d u co hinh dang que ngin va co khi ning chuyin dong trong do co 20/28 dong vi khuan co khi nang chuyen dong nhanh (71,43%), 8/28 dong vi khuan co khi nang chuyin dong cham (28,57%). Chitki dii cua nhung dong vi khuan nay bien thien trong khoing 0,769-2,578 pm. Bing 3: Khi ning t6ng hpp amonium (mg/ U cua D6ng vi khuin N giy 2 3,64b BL1 2,69" HL1 2,44( HL2 2,23 4,51* MC3 2,02h MC8 2,80d 3,10 2,73dc MC13 2,31g C.V(%) 1,97 Ng y 4 4,97b 2,53h 2,59*h 4,47d 7,56* 2,71* 3,14f 4,76c 3,55r 2,56h 2,26 N giy 6 Trung binh 3,08 0,62* 1,87 0,38h 2,09 l,24c 2,47 0,72" 0,25 0,62' 1,60" 0,87d 2,18* 0,52* 3,87 4,11 1,79 2,51 2,91 2,82 1,80 Nhung so theo sau cung mot chu (cung mot cdt) khdng khac biety nghia a muc 5%. b. Khao sat hoat tinh nitrogenaza Chpn ra 5/28 dong vi khuan co khi nang tong hpp amoni cao va dai di n cho noi thu mau d ki<?m tra hoat tinh nitrogenaza, va k t qui la 5 dong vi khuan BL1,,,, dupe chpn d u co khi nang khu axetylen thinh etylen. Dong vi khuan co khi nang khu axetylen nhidu nhat Vi vay, lupng nitrogenaza cua dong nay la cao nhat khoing 26,67 nanomol (Hinh 2). 26 67 e E 13 00 1 12 2 Hinh 1: Hinh'dang m$t s6 khuin lac (A) v i t^ bio ddng vi khuin (B) a. Kha ning co dinh dam Co 10/28 dong vi khuan co khi nang co dinh dam cao, do li cic dong: BL1, HL1, HL2,,, MC3, MC8,,, MC13 (Bang 3). Cac dong vi khuan niy cung co khi nang tao ra lupng amoni tuong doi cao b ngiy thu 2, lupng amoni tang dan d n ngiy thu 4 v i giim manh d ngay thu 6. Dac biet co dong co kha nang tong hpp amoni cao nhat CT ngiy thu 2 (4,51 mg/1) v i b ngiy thu 4 (7,56 mg/1), or ngiy thu 6 (0,25 mg/1) khic biet co y nghia so vdi cic dong khic. Lupng amoni trung binh do dong niy tao ra 4,11 mg/1 l i cao nhat 44 I* 1 40 I I B ll Hinh 2: Hoat tinh nitrogenaza (nanomol) cua 5 ASng vi khuin nqi sinh c&y mia c. Kha ning hoa tan lan 5/28 dong vi khuan co khi nang hoa tan lan cao do li:, HL2,,, (Bang 4). Cac dong vi khuan niy co khi nang hoa tan lan tang tu ngiy 5 den ngiy 10. g d6, dong co khi nang hoi tan lan cao nhat voi lupng lan hoa tan a ngiy thu 5 la 8,32 mg/1 va b ngiy thu 10 la 32,43 mg/1, khic bidt co y nghia so v6i cac dong vi khuin con lai. N6N G NGHIEP VA PHAT TRIEN N6NG TH6N - KY 2 - THANG 1/2014

KHOA HOC C6WO NGHfe Bing 4: K M ning hda tan lin (mg/1) cua 5 ddng vi khuin nqi sinh ciy mia Ddng vi khuin N giy 5 N giy 10 Trung binh 20,37 32,43 8,32a 17,72 3,99" 31,46a 10,31 6,47b 14,15d HL2 9,08 17,11 l,05d C.V(%) 0,22e 18,83b 4,95 3,45 9,52 Nhung so theo sau cung mot chu (cung mot cot) khdng khic bidty nghia a muc 5%. d. Kha ning tong hop indol-3-axetic axit (IAA) Kdt qua chpn dupe 5/28 dong vi khuin tong hop dugc lugng IAA trung binh cao nhat, do la die ddng BL2, BL4,,, MC11 (Bang 5). Hau hdt cic dong vi khuan ddu co xu hudmg tong hgp IAA dat dinh cao nhat la sau 4 ngay nuoi, sau d6 giam dan d ngay nuoi thu 6. Noi troi co dong MC11 tong hop lugng IAA cao nhit vao ngay thu 4 (4,12 mg/1), khic bidt co y nghia so voi cac dong vi khuan khic, lugng \ BLI INIC'2 M C3 IAA trung binh do dong vi khuan nay tao ra li 3,21 mg/1. Bing 5: Khi ning tdng hgp IAA (mg/1) cua 5 ddng vi khuin nqi sinh ciy mia Ddng vi khuin N giy 2 N giy 4 N giy 6 Trung binh 2,60b 3,24' 2,99 BL2 3,15a BL4 2,57b 3,39 2,87c 2,94 2,61b 3,45b 2,96b 3,01 2,74a 3,30 2,77d 2,94 2,59b 4,12a 2,93b 3,21 MC11 0,77 1,06 C.V(%) 1,29 Nhung so theo sau cung mot chu (cung mdt cdt) khdng khac biety nghia o muc 5%. 3.2. Nh n didn cic ddng vi khuin Kdt qui kiem tra trdn gel agaroza 1, 2% cho thay co 10/28 dong vi khuan cho bing (band) tuong ung 900 bp, phu hgp voi vi tri cua gien dugc thidt kd dua trdn trinh tu cua doan 16S rarn dung dd nhin didn vi khuan noi sinh song trdn ciy trong [13]. M C8 M 09 M O 10 M O I3 H L L 6A 900bp Giii trinh tu 10/11 dong vi khuan bing miy giai trinh tu tu dong ABI cho kdt qui dugc trinh biy trong bang 6. Bing 6: Kdt qui giii trinh tu Ddngvi khuin Sd nucleotit du>gc g iii (bp) BLI 723 775 MC3 MC8 753 748 749 750 Ddng vi khuin ddng hinh Curtobacterium citreum ddng CC-88221 Bacillus subtilis ddng TPL16 Bacillus subtilis dong LZHC10 Bacillus tequilensis ddng JF46 Bacillus subtilis dong ZN0871vll Bacillus amyloliquefaciens ddng h y ll Pseudomonasputida ddng GPSD-19 Pseudomonas monteilii ddng SB 3091 Acinetobacter calcoaceticus ddng MP9_2A Acinetobactercalcoaceticusd6ng MCMB881 Pseudomonas putida ddng BASUP87 Pseudomonas monteiliiddng PCWCW7 N6NG NGHI P VA PHAT TRIEN NONG TH6N - KY 2 - THANG 1/2014 Muc dd ddng hinh 100% 98% 98% 45

KHOA HQC CONG NGHfc 761 768 MC13 749 796 Bacillus tequilensis dong Ns9-18 Bacillus subtilis dong BS02 Bacillus cereus dong BDU5 Bacillus subtilis dong SU-10 Acinetobacter calcoaceticus ddng MP9_2A Acinetobacter calcoaceticus ddng MCMB881 Bacillus subtilis ddng ES179-1 Bacillus amyloliquefaciens ddng BVC48 98* lcm/*> 100% 99* Dua vao khoang cach di truydn va ty l phan trim tuong dong trinh tu nucleotit cua doan 16S rarn va su dung chuong trinh MEGA 5.1, da xay dung dupe cay phat sinh chung loai cua 10 dong vi khuan (Hinh 4).. JN105753_Becilus_subt]l*s_strawi_ZN0871v11 42 r G Q 28447e_Pseu<lom ones_n'k >r*e<l«_*tf»«i_pc W C W 7 ----- MM031974 Bacdlus mubt.la H u m BS-02 15 FN 39 3808 BsciRus subtilis s tr «n ES-179-1 rl su 9 9 1 BU. G U 19l92S_Ps«udom on«*_m ont.lh _»tr».n_sb _3091 --------- GU902972 B scilu s subtilis strain S U -10 - AY901 986_Curtobsct«n um _citr«um _s trai n CC-88221 18 ------------- KC l7201s_b scilus_taqi*tensis_strain_jf40 - JN644C21_Acinatobact«r_c k osc*t«cu f_strsm _W P9 _2A GU39C283_P*audomonas _put»ds_str»4n_basup87 1JN12O0Q1_Bacilus_amyloliquetecic<u_9train_hy 11 - JF083003_A cin «K>bactar_caicoac«icu*_stiB in_m C M B 88l - H Q 270550_P«audom ona«_pm ide_*tr»cn_g PSD -19 38 - G Q 8 e i4 0 9 _ B a c ilu s _ s u b th is _ «tra in _ L 2 H C 1 0 JQ 000631_B«cilus_arrylo<iqu«lacians_strain_BVC48 - E U 921468_A cm atotoactar_calco*crtcu s_f tr»n _H IR F A 3 0 EU373393_Bacilus_suM il>s_strbin_tpli0 15. MOO 25 9 8 1J)4)47S20_Bacdlus_car«us_strain_BDU5 MO - HQ 831414_B acilus_taqu I«nsi*_s*rain_Ns9-18 97,»K3 mai 13 ee BU BU 37 51 MQ st! Ma 0.5 Hinh 4: Ciy phi h di truydn (phylogenetic tree) trinh biy mdi quan hd di truydn giqa cic ddng vi n$i sinh phin lip vi nhin didn trdn mia tr6ng d tinh Bdn Tre v i Long An theo phuong phip neighborjoining. Cic gii trj hidn thj tai cic nhinh ciy li kdt qui phin tich boostrap vdi d0 l$p lai 1000 lin Dong vdi doan gien dai 775 bp cd moi quan hd di truydn gan gui vdi chi Bacillus va duoc nhan dien la Bacillus subtilis GQ861469.1 vdi ti Id tuong ddng nucleotit la, hai ddng vi khuan va cd quan hd di truydn gan gui vdi nhau va gan vdi chi Bacillus, cu thd la ddng doan gien dai 796 bp cd ti Id tuong ddng la vdi loai Bacillus amyloliquefaciens JQ660631.1; ddng cd doan gien dai 768 bp cd ti Id tuong dong la 100% vdi loai Bacillus cereus JX847620.1. Nhu vay ba ddng vi 46 khuan,, cd muc tuong ddng cao du chung dugc phit hidn d cac didm xa nhau va ca ba ddng vi khuan niy cung d nhinh ridng bidt vdi nhung ddng vi khuan cdn lai. Bay ddng vi khuan BLI,,, MC3, MC8,, MC13 cd quan hd di truydn gan gui vdi nhau v i gan vdi chi Bacillus. DSc bidt, ddng vdi chidu dai doan gien 761 bp dugc nhan didn li loai Bacillus tequilensis HQ831414.1 vdi ti Id tuong ddng la. Didu dd cho kdt luan ring du mia dugc trong d xa N 6NG NGHlfP VA PHAT TRIEN N6NG TH6N - KY 2 - THANG 1/2014

KHOA HOC C O N G N G H l nhau vd didu kidn dia ly nhung cac dong vi khuan noi sinh lai rat gan vd mat di truydn [1]. Ket qua co 10 dong vi khuan ndi sinh cay mia da dug«c nhan didn va xay dung trong cay pha hd (Hinh 5). 85 71 tequilensis HQ831414.1 cd ti Id ddng hinh la 98%; ddng MClO vdi ddng Bacillus cereus JX847620.1 (100%); ddng vdi ddng Bacillus amyloliquefaciens JQ660631.1 (), ddng vdi ddng Bacillus subtilis GQ861469.1 (). Bacillus tequilensis MCI Bacillus tequilana MC8 Bacillus tequilensismo Bacillus tequilensis MC 13 Bacillus tequilensis BLI Bacillm tequilensis Bacillus tequilensis Bacillus cerm MClO Bacillm subtilis B U Bacillus annloliquelaaens Hinh 5: Cky phi hd di truydn (phylogenetic tree) trinh bay mdi quan hd di truydn cua 10 ddng vi khuin nqi sinh phan lap va nh$n didn tr n mia trdng b tinh Bdn Tre va Long An theo phuong phap neighbor-joining. Cac gia tri hidn thi tai cac nhanh cay la kdt qua phan tich boostrap v6i d0 lap lai 1000 lin Hai muoi tam dong vi khuan phan lap dupe ddu co kha nang cd djnh dam, hoa tan lan va tong hop IAA; kdt qua nay phu hop vdi nghidn cuu cua Hernandez va dong tac gia [5], Mallaiah (2007) [7]. Theo kdt qua nghidn cuu truoc day thi phan Ion cac ddng vi khuan noi sinh sdng trdn cay mia thuoc cac chi nhu Pseudomonas, Azospirillum, Burkholderia. Hien nay kdt qua nghidn cuu nay cho thay vi khuan noi sinh sdng trdn cay mia cdn thuoc chi Bacillus [2], [10], [11]. 4. KET LUAN - Da phan lap duoc 28 ddng vi khuan tu cac mau rd, than cay mia; nhung ddng vi khuin nay cd dac diem que ngan, chuyen dong. Hau het cac khuan lac cd mau vang, bia nguydn, do ndi md, kich thudc 1-3,5 mm. - Cd 15/28 ddng vi khuin cd nhung cac d$c tinh tot trong dd ddng cd kha nang tong hgp amoni cao nhat, ddng cd kha ndng hoa tan lan cao nhit va ddng MC11 cd kha ndng tong hgp IAA cao nhit - Cay pha hd di truydn da xic dinh dugc mdi quan hd gin gui cua ddng vdi ddng Bacillus TAI L U THAM KHAO 1. Cao Ngoc Didp, Ai Chi (2009) Phan lap va dac tinh vi khuan noi sinh trong cay khom (.Ananas comosus L.) trdng trdn dat phen huydn Bdn Luc tinh Long An. Tuydn tap hoi nghi cdng nghd sinh h<?c phia Nam nam 2009, tai thanh pho Hd Chi Minh ngay 23-24 thang 10 nam 2009. 2. Cao Ngoc Didp, Van Mit (2007) Hidu qua phan vi khuan Gluconacetobacter diazotrophicus va vi khuan Pseudomonas stutzeri trdn nang suat va tru lugng dudng trong cay mia ( Saccharum offcinarum L.) trong trdn dat phu sa tinh Hau Giang. Tap chikhoa hoc, sd 7: 95-101. 3. Cavalcante V. A., Dobereiner J. (1988). A new acid-tolerant nitrogen-fixing bacterium associated with sugarcane. Plant Soil, 108: 23-31. 4. Dilworth M. J. (1966). Acetylene reduction by nitrogen fixing preparation from Clostridium pasteurianum. Biochem. Biophys., 127: 285-294. 5. Hernandez T. A., Cristales M. R B., Salgo T. J., Mellado J.C., Ramirez L E. F. (2000). Natural endophytic occurrence of Acetobacter diazotrophicus in pineapple plants. Microbiplogy Ecoli, 39:49-55. 6. Madhaiyan M., Saravannan V. S., Jovi D. S., Lee H., Thenmozhi R, Hari K., Sa T., 2004. Occurrence of Gluconacetobacter diazotrophicus in tropical and subtropical plants of Western Ghats, India. Microbiological research, 159:133-243. 7. Mallaiah K. V., Sridevi M. (2007). Production of indole-3-acetic acid by Rhizobium isolates fromsesbania species. African Journal of Microbiology, 1:125-128. 8. Nautiyal C. S. (1999). An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiology letters, 170: 265270. 9. Neumann P., Pospiech A., Schairrer H. U. (1992). Rapid isolation of genome DNA from Gram negative bacteria. Trends Genet8:332-333. NONG NGHI(P VA PHAT TRlfrl N 6N G TH6 n - id12 - THANG 1/2014 47

KHOA HOC CONG N G H I 10. Nguyen Huu Hi p, Tran Van Chieu, Dao h Hoang, Nguy n Khac Nlinh Loan (2005). Azospmllum w sinh vat co dmh dam voi cay khong thuoc ho dau. Tap chi Khoa hoc - Dai hoc Can Tha, so 2 (12): 4-6. 11. Nguyen Thi Phuong Tam (2011). Phan lap va khao sat dac tinh cua mot so dong vi khuan noi sinh trong cay mia. Luan van tot nghiep dai hoc chuyin nganh cong nghe sinh hoc. Trudng Dai hpc Can Tha. 12. Park A. E. (2005). Soil microorganisms and phosphorus availability. In: Pankhuurst C. E., Doube B. M., Grupta V. V. S. R, Grace P. R (eds). So. Biota, Management in Sustainable Farming >> stems. Melbourne, Australia: CSIRO: 50-62. 13. Zinniel K. D., Lambrecht P., Hans N. B., Feng Z., Kuczmarski D., Higley P., Ishimaru C.t Arunakumari A., Barletta G. R, Vidaver A. K., 2002. Isolation and characterization of endophytic colonizing bacteria from agronomic crops and praine plants. Appl. Environ. Microbiol., 59: 2198-2208. IDENTIFICATION AND CHARACTERIZATION OF ENDOPHYTIC BACTERIA IN SUGARCANE CULTIVATED IN BEN TRE AND LONG AN PROVINCE Ngo Hong h1, Hoang Minh Tam2, Cao Ngoc Diep1* 1BiotechnologyR&D Institute, CanTho University Summary 2 University o f Saigon, HoChiMinh city Twenty eight bacterial isolates were isolated from roots, stems of sugarcane cultivated in Ben Tre and Long An province. st of them have short rod-shape and motility. The majority of their colonies have roundshaped, yellow, emerge, whole cover, size about 1-3,4 mm. Among 15/28 bacterial isolates with several good characteristics were tested by the biochemical methods (such as IAA biosynthesis, phosphate solubilization and nitrogen fixation). However, ten isolates were identified as endophytic bacteria by PCR 16S-rRNA technique, these endophytes were chosen to sequence and their DNA sequencing were compared with GenBank database of NCBI by BLAST N software. The results showed that all of ten isolates were 98-100%of the identity with the species in Bacillus genus. Phylogenetic analysis based on partial 16S rrna the Neighbor^oining method by MEGA 5.1 software showed that relationships between isolate with MClO isolate closely; isolate and isolate closely. Four endophytes Bacillus subtilis, Bacillus tequilensis, Bacillus cereus MClO, Bacillus amyloliquefaciens have the best characteristics, they will be suggested for bio-fertilizer production. Key words: Bacillus, endophytic bacteria, IAA biosynthesis, nitrogen fixation, phosphate solubilization, sugarcane. Ngudi phan bi n: TS. Lft Nhu Ki u Ngiy nhfln bii: 10/8/2013 Ngiy thdng qua phin bi n: 9/9/2013 Ngiy duy t ding: 17/9/2013 48 NONG NGHlfP VA PHAT TRIEN N6NG TH6N - KY 2 - THANG 1/2014