KHOA HQC C O N G NGHfi
PHAM L A P V A M H A M DlEIXl VI KHUAIXI CO iCH [CO OflVH OAIVI, HfJA TAM LAM, P H A M GIAI KALI]
TRQMIi DAT VLJMii R I M G O T R O M G a HLiYEM TRAMG BDIU, TIMH DOMG MAI
Ddng Thi Ngpc Thanh', Cao Ngpc DiOp^
T6MTAT
Cay ngo {Zea mays L) la cay luong thuc quan trpng sau c&y liia. Ayxiyc trong nhi^u rr vung dat mi^n Dong Nam bp, tuy nhien c&y ngo can nhieu phfin bon dd c6 nang sudt can. vi v$y ngo thirenig clm.rc nong dan canh tac tren vixng ddt co do phi kha. Ben canh ngudn dinh duong tir phftn bdn hoa hoc, trong ddt co nhung nhom vi khuan c6 kha nang co d|nh dam, hoa tan lan va phan giai kah co the dap img phdn nao nhu cau dinh duong cho cay ngo. Mac tieu cua nghien cuu la phAn lap va nh^n dicn nhom vi khuan cd ich nay song trong dat vung r^ ngo. Cd 28 chiing vi kliuan cd kha nang co dinh dain. hoa tan lan. phan giai kah tren moi trudng Burk vo dam, NBRIP va kaobmt, trong do c6 10 chung vi khuan cd dp huu hi^u cao va cluing duoc nhan di^n bang ky thuat PCR vdi cap moi tong (SF va 1492R), san ph£m PCR diroc giai trinh tu doan gien 16S ArRN va so sanh trinh tu gien trong ngan hang dir lieu NCBI vdi phan m^m BLAST N va xay dung cay pha he vdi phanm^m MIiC.A 5.05 cho thay chiing la nhom vi khuan gram am (trir chung DN 181a vi khuan gram duong) thupc nhdm vi khuan cd dinh dam sdng tu do hay ndi sinh ho5c vi khuan not re
TCr khda: .\gd, da't Ming r^. PCR-16SArRN, phin lap, \i khudn co ich.
LDATVANDE
Ngd {Zea mays L.) Id cay luong thuc quan trpng rong ndn kinh te todn cdu, dung thu 2 chi sau cay lia vdl dien tich 1.086,8 ngan ha, nang sudt 40.8 ta/
la dat san luong 4.431,8 ngdn tan vao nam 2009 Thdng ke Viet Nam. 2010). Theo Trung tam Khdo ighidm Gidng Cay ti'dng Trung uong (1995), Ddng
•^am bp dupe xem la vung ngd hang hda cd tiem lang nhat trong tdm vung smh thai trdng ngd ciia cd lude, trong dd Ddng Nai la tinh cd dien tich frong igd Idn nhdt trong viing (54,4 ngan ha), vdi nang iuat trung binh 5.8 tan/ha, dat san luong 315,3 ngan :an. Dd't den cd dien tich 99.100 ha, chiem 4,23% tong hen tich tii nhien vimg Ddng Nam bd, phan bd tap ning d nhimg vung dat cd nhidu hpng nui lua nhu Dinh Qudn. Tan Phu. Thdng Nhdt, Long Khdnh (tinh Ddng Nai). Chau Thanh, Xuyen Mdc (tinh Bd Ria - /ung Tdu). Dat nay cd dp phi nhieu cao, cdc chat hnh dudng rat can ddi ngoai trir kali, cdu tnie dodn ap toi xdp. Tuy nhien tang ddt nay thudng rat mdng, dn nhieu dd va ket von, nhieu da Id dau. Diem ndi sat cua loai ddt nay la lan tong so, nhat la tren mieng nii lua cd hdm lupng PaOs dat tdi 0,9%, gdp hdng
Khoa Su ph^m Khoa hpc Tir nhien, Dai hpc Sai Gon V[?n NC&PT Cong ngh? Sinh hpc, E)?i hpc Can Tho
chuc lan cdc loai dat khac; giau canxi va rat ngheo kali. Dat tu it chua den trung ti'nh, rat gidu cation trao ddl. CEC cao va dp no baza rat cao. Dat den frdn mieng niii lua cd ham luong lan tong sd rat giau nhung lan dd tieu trung binh.
Theo Nguydn Nhu Hd (2006) cay ngd cdn den 269 kg N, 111 kg P P s vd 269 kg KaO/ha de cho nang suat cao, vi vay dien tich trong ngd, nhat la giong ngd lai. hi han che neu nhu khdng cd ngudn cung cap phan bdn. nhat la phan khoang so vdi nhung cay frdng khdc; tuy nhien bdn qud nhieu phan dam hda hoc se dan dd'n chi phi cao, khdng nhung lam d nhidm mdi trudng md cdn gay ton hai den siic khde va anh huong tidu cue ddn hd smli thai. Hien nay, cdc nha khoa hpc tap trung nghien cuu va su dung cdc chung vi khuan cd dinh dam sinh hoc. Do dd, nghien cuu va tuyen chpn cde ehiing vi khuan cd kha nang cd djnh dam hull hieu bon cho cay ngd Id gdp phan nghien cuu ngudn dam sinh hpc vd cdn cho su phat frien ndng nghidp b^n vung nhu d Thai Lan; thi nghiem dupe tidn hanh fren cay ngd (1984-1985) vd sdn luong tang 15-35% do chung AzospirUlum lam giam hay tiet kiem dupe lupng phan hda hpc tii 1/3-1/2 (Vasuvat etal., 1986'', dong thdi rd cay ngd cd chung vi khuan AzospirUlum thi cd ham lupng lAA. IBA rat cao so voi ddi chung khong chung vi khuan (Fallik et al.,
vJONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 7/2012 49
KHOA HOC C O N G NGHfi
1989); vi khuan Pseudomonas sp. hda tan ldn, phAn lap b dat vung rd cay so dua (Cao Ngpc Dicp et al, 2009). tren gidng ngd lai DK888 cho tliay hidu qua tuong duong vdi cay ngd bdn 60 kg I'jOr,/ha vd chic^u cao cay. dudng kinh bdp nhung giiip hgt ngd to hon, nhieu hat/bdp ndn nang siKit ngd lai cao hon cfty ngd bdn 60 kg PjO^ha frdng frdn dat phii sn huydn Tdn Hiep. Kidn Giang (Nguydn Van DIRR- vd Cao Ngpc Diep. 2004). Phan ^mli hpc. bao gdm nhCnig chiing vi khuan cd ich, khdng lam tliay ddi sinh thai va an toan mdi truimg vd thdng qua phdn sinh hpc cdc vi sinh vat xam nhidm vdo viing rd tdc ilnng cung cdp chdt dinh dudng cho cay Irdnj; de tang nang suat (Rodriguez (V,-i/.19<>9).
Muc dich cua ngliidn cuu Id phan l;i]i nhung nhdm vi smh vat cd ich, bao gdm cd dpih dam, hda tan lan, phan giai kali, trong dat vung rd ngd va tuyen clion nhCmg ddng vi khuan tdt d6 cd the sir dung nhu la ngudn phan vi sinh dc thay tlic hay tidt kidm mpt luong phan khoang NPK trong nhu cau dinh dudng cua cay ngd
L NGUYEN UEU VA PHUONG PflAP 1. Mau dat
Cac mdu ddt viing rd ngd duoc thu Uiap tu cdc rupng ngd tai 3 diem (dp) cua xa S<ing Trdu. huy^n Trang Bom, tinh Ddng Nai cd thdnh phan ly hda tinh duoc tiinh bdy frong bang 1 cho tliay dat d dp Sudi Tidn cd pH thap nhung N tong sd. K frao ddi vd chat hiru co cao so vdi dp 2 vd ap 3.
Bang 1. Thdnh phdn ly hda tinh cua ddt (den) trdng ngdcrxS
Dia diem ApSuol
Tien Ap2 Ap3
Songi pH 4,43 5,63 5.71
rrau, hu N long so
«)
1,02 0.21 0,23
iin Trang B P de ueu (mgP.Os/
lOOgdSt) 59,88 116,86 87,60
om, tinhD K trao doi (mg K.O/kg)
582,42 339,56 323,21
6ngNai Ciia'l hiju coOi)
9,26 4,82 4,68 2. Phan ldp vi khudn c6 d}nh dam, hda tan 1 ^ vd phan giai kali
Dat vimg rd ngd sau khi de khd o nhiet dp phdng. diing cdi sir nghian min. Sau dd. can 1 g mau dat cho vao binh tam gidc voi 99 ml nudc cat da dupe khii tiling, ldc 12 gid- vdi toe dd 200 vdng/phut cho cac hat ddt rdi ra vd vi khuan phan tan deu frong nudc. De ldng khoang 3 gid, lay 0,1 ml dung djch nudc (phan frong) frai deu fren dia petii chua mdi
trudng Burk vd d^im (d^ phdt hidn vi khudn co dinh dam) (Park et al., 2005), mdi tiirdng NBRIP (phat hi^n vi khudn hda tan lan) (Nautiyal. 1999) va moi frudng kaolinit (phdt hidn v'i khudn phan giai kali)(Lin Qi-mei etal.. 2002) da dupe chuan bi san.de khd. Ll d 30'C. Sau 24 - 48 gid. cdc khudn lac mpc tr^n b^ mjt ludi trudng du<;c ti^p tuc cay chuydn sang mdi frudng mdi vdi ldn den khi cdc khuan lac xuai hi^n frdn dudng cdy rin nhau va quan sat dflc di^m khuiin lac (hinh dang, mdu sdc, dp ndi, dang bia, kich tiiudc) (Hinh 1). Ki6m fra dd thudn bdng cdch quan sdt dudi kinh hidn vi bdng phuong phdp gipt ep. Khi thdy VI khuan da thudn nhat thi cdy chuydn sang ong nghi<'m chua mdi trudng dac tuong iing d^ tru a 4*C vd du(/c xem nhu mpt chiing (isolate) thudn. Cacvi khuan mpc dupe trdn mdi trucmg Burk vd dam dupe cdy sang mdi trudng NBRIP va ngupc l?i; chpn nhijng khuan lac phat tnen ti-dn ca hai mdi truang Burk vd dam vd NBI^IP nhu Id cac chung vi khuan c6 kha nang cd dinh dam va hda tan lan; cac chiing vi khuan nay d.myc cay sang dIa mdi trudng kaolinit de xdc dmh khd nang phan giai kah ciia chiing (Hinh 2}
3. Ddnh gid khd nang cd dinh dam, hod tan 1^, phan gidi kali ciia c^c ehung vi khudn
r
tik,-
ir--' • •
1 ; t ,>,T11 E l -
1
• 1 ^
''• '^
Chung OMO
Hinh 1. Cdc khudn l?c rdi r^c dupe cdy chuydn vdo dng nghidm d^ trtr l^nh vd xem nhu la 1 chung
(isolate)
Cac chiing vi khuan phdt tii^n frdn tiing loai moi tindng dupe do kha nang cd djnh dam bdng each u frong mdi tmdng Burk vd dam long bang phuong phap so mdu vdi thuoc thu phenol nifro-xyanhydiat, frong mdi truong NBRIP long (glucoza, 10 %^
(NH4)2S04 0.1 g/l; MgS04.7H20 0,25 g/l; MgCl^S g/l; KCl 0.2 g/l; Ca3(POj2 5 g/l; bromothymolxanh 5 ml/1; pH=7.2-8 chinh bang KOH 0,1M) de do kha nang hda tan ldn khd tan bang phuong phdp so nu"
vdi thudc thu xanh-Molybden (Oniani) [Phong ^i nghidm chuydn sdu, D?ii hpc Cdn Tho]; sau do dinh gia kha nang phan giai kali bang cdch sang HW"
50 NONG NGHlfP VA PHAT TRIEN NONG THON - KY 2 - THANG 7/2012
KHOA HOC C O N G N G H |
ti-udng kaolinit Idinh tinh] vd each danh gia theo dudng eay (hinh 2) sau 2 ngay ii Id + (phdt tiien binh thudng). ++ (phdt frien tdt), +++ (phat trien rat tdt).
4. Nhdn di^n cdc chting vi khudn bdng ky thudt PCR-16SArRN
Tdch chiet ADN vi khudn theo md ta ciia Neumann etal. (1992).
- Sau khi tdch chiet ADN tir cac chung vi khuan.
phan irng PCR gien 16S ArDN dupe tidn hdnh vdi cap ' mdi 8F vd 1492R a u m e r et al., 1999).
8F 5'-AGA GTTTGATCC TGG CTC AG-3' 20 (moi xudi)
• 1492R 5'-TACGGTTACCTTGTTACGACTT-3' 22 (mdi ngupc)
- T h a n h p h d n p h d n i m g P C R (25 p i ) : H . O 12 pi;
buffer ( c h a t d e m ) lOX 2,5 pi; M g C l . 2 5 m M 2 pi;
d N T P 4 pi; m o i 8 F 1 pi; m o i 1492R l p i ; B S A 0.25 pi;
. T a q - p o l y m e r a z a 0,25 pl; A D N m a u 2 pi.
P h d n i m g PCR: 9 5 C / 5 ' ; 3 0 e h u ky: ( 9 5 C / 3 0 " , 5 5 C / 3 0 " , 7 2 C / 1 ' 3 0 " ) ; 7 2 C / 1 0 ' .
Stt d u n g d o a n m d i x u d i frong p h a n i m g P C R d^
n h a n d i e n vi k h u a n d a m d t a o- p h a n frdn. S a n p h a m PCR d u p e t i n h s a e h t h e o k i t I n v i t r o g e n v a gidi t r i n h t u b a n g h e t h d n g m a y giai t r i n h t u t u d p n g c u a C d n g ty M A C R O G E N (Hdn Q u d e ) . S u d u n g c h u o n g tiinh BLAST N d e s o s d n h t r i n h tu A D N ciia c d c c h i i n g vi k h u a n c h p n l p c v d i t r i n h t u A D N ciia b d g i e n d c a c loai vi k h u a n frong G e n B a n k vd x a y d u n g c a y p h a hd g i e n giira c d c d d n g vi k h u a n d u p e giai t r i n h t u b a n g phdn m e m M E G A 5.05 ( T a m u r a etal., 2011).
•.KET QUA VA THAO LUAN l.K^tqua
Cd 28 chiing vi khuan phan lap va phat tri^n frdn 2 ca hai loai mdi tnrdng Burk vd dam vd mdi tnrdng NBRIP; nhu vay ca 28 chiing vi khuan nay vira cd kha nang cd dinh dam vira cd kha nang hda tan lan (hinh 2), da sd khuan lac cd dp noi md. hinh cau, mau , frdng frong suot hay vdng dam; da sd chiing cd dang : que ngdn, chuyen dpng nhanh, tuy nhidn chi cd chiing vi khuan cd khd nSng phan giai kali (hinh 3).
K^t qua til bdng 2 cho thay da sd cac chiing vi khuan phdn lap phdt trien rat tdt frdn mdi trudng 1 NBRIP, dieu nay eho thay chung ed kha nang hda ,tan lan tdt; the nhung chi eo mot sd chiing vi khuan
phdt tii^n tdt frdn mdi trudng Burk vo dam, di^u nay
cho thay chung cd kha nang cd dinh dam 6 miic dd trung binh. Tuy nhien chi cd 2 chiing vi khuan khdng phat frien fren mdi trudng kaolinit. cdn lai deu phdt tri^n trdn mdi trudng nay; tdng hpp kdt qua cho thay da sd chung vi khuan cd kha nang cd djnh dam, hda tan lan va phan giai kah khd nhung khdng dong ddu.
(A) mdi trudng Burk vd dam (B) mdi trudng NBRIP
(C) mdi trudng vd dam va P khd tan Hinh 2. Vi khudn phat tri^n frdn mdi tnrdng Burk vd d?jn (A), frdn mdi hirdng NBRIP (B) vd cd frdn mdi
trudng Burk vd dam vd P khd tan
Hinh 3. Vi khudn phdt tri& frdn mdi trudng Burk vd d^m vd ch^ia P khd tan ( ^ , tii day cdy chuydn sang mdi tru6ti£ kaolinit (phan giai kali) (B), dang que b
kinh hi^n vi di^n hi (C)
I N O N G NGHIEP V A PHAT TRIEN N O N G THON - KY 2 - THANG 7/2012
KHOA HQC CdNONCHi
TT
ni
02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Bdng 2. Dd Tdn chting
vi khuan DNl DN2 DN3 DN4 DNS DN6 DN7 DN8 DN9 DNIO DNll DN12 D N B DN14 DN15 DN16 DN17 DN18 DN19 DN2D DN21 DN22 DN23 DN24 DN25 DN26 DN27 DN28
: tinh vi khudn phdn IdP dupe frong ddt viing r§ frdng ngd b xA Sdng Trdu, huvOn Trdne Bom. tinh Ddng Nai
Burk vd dam
+ + + +++
4-M- + + t +
++
+++
+++
+++
++
•H- +++
++
+++
+++
+++
+++
++
+ + +++
+ ++
+ + +++
NBRIP + + ++
+ +++
+++
+++
+++
. . . j t ± L _ +++
+++
+++
+++
-H-+
+++
+++
•M-+
+++
+-H- +++
•H- ++
+++
+ +++
+ ++
+++
Kaolinit
+ ++
-*•++
+++
+++
++
++
++
+++
++
+ + +++
+++
++
++
+++
++
++
+ + ++
++
+++
+ +++
Dja didm thu mdu dp 2 dp 2
i p 3
dp Sudi Tidn
;i|) Sudi Tidn dp 3 jiP 2 .ip2 Ap3 dp 3 dp 3 dp 2 dp 2 dp Sudi Tidn
dp 2 dp 3 dp 3 dp Sudi Tidn
dp 3 dp 3 dp 2 dp 2 dp Sudi Tien dp Sudi Tidn
dp 2 dp 3 dp 3 dp Sudi Tidn
Hinh d ^ g vi khudn Que ngdn, chuydn dpng Que ngdn, ehuydn dpng Que ngdn, chuydn ddng Que ngdn, chuydn dpng Que ngdn, chuydn ddng Que ngdn. chuydn ddng Que ngdn, chuyen ddng Que ngdn^chuydn ddng Que ngdn, chuyen dpng Que ngdn, chuyen ddng Que ngdn^chuydn ddng Que ngdn, chuyen ddng Que ngdn, chuyen ddng Que ddl. chuyen ddng Que ngdn, chuyen ddng Que ngdn, chuyen ddng Que ngdn, chuyen ddng Que ngdn, chuyen ddng Que ngdn, chuyen ddng Que ngdn. chuyen ddng Que ngdn. chuydn ddng Que ngdn. ehuyen ddng Que ddi, chuyen ddng Que ngdn. chuyen ddng Que ngdn. chuyen ddng Que nedn. chuven dpng Que ngdn. chuyen dong Que ddi. chuyen ddng
I-khuan Ifc phat tritl + khuan lac phat trien it sau 2 ngiy, ++ khuiin l^c phit tnen vira sau 2 ngiy, i
nhieu sau 2 ngiy
Kit qua tir bang 3 cho thay kha nang co djnh dam (thong qua smh tong hpp NH4* trong dung dich nuoi cay) va kha nfing hoa tan lan (thong qua ham luong P2O5 hinh thanh trong moi truang nuoi cay v6i lan apatit) vao ngay thii 5 sau khi ij, c6 11 chung vi khuan ndi bat thong qua ham luting NH4* va P2O5 kha cao dong thoi cac chung vi khuan nay cung c6 kha nang phan giai kali k h i tdt, dd la cac chiing DN4, DNS, DNIO, D N l l , DN14, DN15, DN16, DN17, DN18, DN20, DN28.
11 chung vi khuan du(7C chpn dd tien hanh cac phan ling PCR va phd di^n di ciia phan ung PCR ciia AND ciia 11 chiing vi khuan nay d^u b band 1500 bp so vdi thang c h u i i 100 bp (Hinh 4). Tuy nhidn chi 10
chiing vi khuan dupe giai trinh ttr doan gien 16S Tmng Qudc) vdi miic dd 98%; chting DN15 "
ArRN vdi kdt qua chiing DN4 ddng hinh vdi chiing hinh vdi chiing EF054875 GQ169811 Agrobacterium tunietaciens strain B228 (ddng vi khuan npi sinh trong r^ liia d Tnmg Quoc) voi miic dp 9896; chiing DN5 ddng hinh vdi chiiiig JQ659867 Burkholderia anthina strain R7-112 (vi khudn npi sinh d cay Jatropha dupe Sinhgapore rtn?
bd) cd miic dd 99%; chiing DNIO ddng hinli vS chiing FJ436055 Burkholderia viebiamlensis strain SYe*586 (vi khuin cd djnh dam d vimg xh c4y ngo * MEXICO) voi miic dO 99%; chiing D N l l ddng hii*
vdi chiing HM462444 KlebsieUa sp. VITPGPSM («
khuan kich thich tang tnrdng thuc vat d virng A ^ trdng d An Dp) d miic dp 97«; chiing DN14 ^ hinh vdl chung GQ169811 Agrobactof tume&iciens strain B228 (vi khuan npi sinh cSy W'"
52 NONG NGHIEP VA PHAT TRIEN N 6 N G THON - KY 2 - THANG 7/2012
KHOA Hpc C 6 N G N G H |
tumefaciens sfrain ORS 3405 (vi khuan not sdn b cay keo {Acacia) b Senegal, chau Phi) cd muc dp 98%;
chung DN17 ddng hinh vdi chung GU290320 Klebsiella sp. 200913 (vi khuan cd dinh dam sdng viing rk thuc vat 6 Trung Qudc) vdi muc dd 98%;
chung DN18 ddng hinh voi ehung GQ284325 Arthrobacter oxydans sfrain TSWCSN38 (vi khudn mdi trudng d An Dp) a muc dp 99% vd chiing DN28 ddng hinh vdi chung HM037179 Klebsiella sp. SHN-1 Bdng 3. Khd nang ed dinh d?un vd hda tan lan cua 28 vdd^m
(vi khudn cd dinh dam sdng d vimg rd thuc vat 6 Tmng Qudc) d muc dd Id 99%.
Nhu vay, trir chung DN18 dong hinh vdi chung vi khudn mdi trudng. cdn 9 ehiing ddu la vi khuan ed ich sdng vimg r^ hay nOi sinh da dupe edng bd o nhidu qudc gia khdc nhau frong ngdn hang dir lidu ciia NCBI. frong dd chiing DNIO Id chiing Burkholderia vietnamiensis rdt ndi tidng frdn the gidi, la chung vi khudn cd ich.
ehung vi khudn sau 5 ngdy chiing vdo mdi trudng Burk vd NBRIP
Chung vi khuan Ddi chiinff DNl DN2 DN3 DN4 DNS DN6 DN7 DN8 DN9 DNIO D N l l DN12 D N B DN14
Lupng NHj*
(mK/1) 0,005 f 1,072 c 1,269 cd 1,287 c 1,344 b 1,446 a 1,277 cd 1,329 c 1,224 d 1,287 c 1,387 b 1,411a 1,773 e 1,200 e 1,317 c
Lupng PjOs (mg/l) 0,020 g 5,351 be 3,001 e 5,239 be 4,381 cd 6,183 a 5,848 ab 4,750 c 3,653 e 5,338 be 6,040 a 5,208 be 4,493 ed 3,552 e 5,352 be
Chiing vi khuan DN15 DN16 DN17 DN18 DN19 DN20 DN21 DN22 DN23 DN24 DN25 DN26 DN27 DN28 CV (%)
Lupng NH4*
(mg/l) 1,317 e 1,288 e 1,355 b 1,261 ed 1,372 b 1,298 e 1,112 e 1,187 e 1.281 e 1,161 e 1,291 c 1,199 e 1,261 cd 1.332 b
2,78%
Lupng PjOs (mg/l) 4,566 c 3,333 e 6,453 a 4,233 d 5,416 b 4,163 d 3,012 e 4,021 d 3,431 e 2,981 ef 2,457 f 2,149 f 3,113 e 5,327 be
5,14%
* Nhimg sd theo sau cung rndt chd khdng khac biet v nghia thdng kd dmucdd 1%
1 2 3 4 5 6 7 8 9 1 0 1 1 12 13 1500 bp
Hinh 4. Phd di$n di cua san phdra PCR dupe nhSn ldn txi ADN cua 11 chiing vi khudn frfin gel agaroza
1,2%
Ghi chii: 1: tiiang chuan 100 bp, 2-DN4, 3=DN5, 4=DN10, 5=DN11. 6=DN14, 7=DN15, 8=DN16, 9=DN17, 10=DN18, 1UDN20. 12=DN28, 13=Dd chung am
So sanh miic dp quan hd cua chiing thdng qua eay pha hd di truydn dua fren trinh t\r gien 16S ArRN cho thay cay pha hd chia thanh 2 cum (cluster) vdi chung DN18 b cum 2 (hinh 5a).
Oil^W
* « « . n m « . » .wtBw.iBjFWiiKm
jaaB_Mm«.i«n.B».!l-it:jP«WX.J>™WjSKi«Hl IJom.UNBn.inn.in.STttaSjMLCDi
aempioMffiimMtjxf^vmji,
|Oif||.» J
cl-ciuig^nig
( • O M I W
~ i i l ( ! ] i B i i.ifUnii.iMlDn.nii.eztjOICMrTt.KEiaH) K1 craWS.'fitMgui.UWn.in.aG.M;)jUn.niilii|!(ltSil|
Oaum
t - ^ _J Cum 2 Hinh 5a. C;ay pha h^ (phylogenetic free) trinh bdy mdi quan hd ciia 10 chiing vi khudn vdi cdc chiing ddng hmh trong ngdn hdng gien (NCBI) (theo
Neiborgh-jommg free vdi 1000 bootstr^) NONG NGHIEP VA PHAT TRIEN NONG THON • KY 2 - THANG 7/2012 53
KHOA H p c C 6 N G N G H t
Cum 1 chia ra 2 nhdnh vdi cum la, bao gdm cac chung vi khudn D N l l , DN28 vd DN17 ddng hinh vdi chiing Klebsiella sp. vd quan h^ mat thidt vdi chiing DN16 la vi khudn ddng hinh vdi chiing Enterobacter cloacae va chiing DNS, DN4 vd DNIO ddng hinh vdi chung Burkholderia sp. frong cum lb; Irong cum 2 gdm cd 2 chiing DNH vd DN15 ddng hinh vdi chiing Agrobacterium tumeiaciens. Phan tich 10 cluing vi khuan ndy frong cdy phd hd cho thdy chiing chia ldm 2 cum rd ret vdi cpm 1 eung bao gdm cdc chiing Klebsiella sp.. Enterobacter cloacae, Burkholderia sp. va Agrobactenum tumefaciens (Hinh 5b.)
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Hinh 5b. Cay pha h§ (phylogenetic free) trinh bdy mdi quan h$ ciia 10 chung vi khudn vdi nhau dya frdn trinh tir gien 16S ArRN (theo Neiborgh-joining
free vdi 1000 bootstrap) 2. Thao lu^n
De cung cap day du dinh dudng cho cay frdng can su dung phdn hda hpc, nhdt la dam, Ian, kali...;
tuy nhidn cac l o ^ phdn hda hpc thuang khdng dupe cdy frdng su dung het ma con tich hi 6 cde tang m?it ciia ddt noi cd rd cay, frr dd gay d nhiem tdng ddt mat.
ngodi ra du lupng phdn bdn cdn lam d nhidm thitc an va anh hudng ddn siic Wide ngudi tidu dimg va lam mat can bdng h$ sinh thdi ndng nghidp. Dd han ehd tinh frang frdn can gidm lupng phan hda hpc diing frong h^ thdng ndng nghidp bdng cdch dp dyng cdng nghd sinh hpc vdo san xudt. frong dd su nghidn eim thanh cdng phan sinh hpc dd thay thd ddn phan hda hpc vi phan sinh hpe khdng lam thay ddi smh thdi vd an toan mdi trudng.
Vi khuan Burkholderia sp. nhdt la Burkholderia vietnamiensis, da dupe Tran Van Van et al. (2000) phan lap frong dat o ddng bdng sdng Ciiu Long; kd ddn la Ngd Thanh Phong va CTV (2010, 2011) phdn
ldp vd nhdn didn frong ddt vimg rd liia d- Kien Gianj vd Vinh Long vd vi khudn Burkholderia tiopicavi L tropicalis dupe xdc djnh Id vi khudn npi sinh troni cSy khdm (dua) frdng b viing ddt phen cdc tinh naj (Cao Ngpc Didp vd Nguydn ^ Chi, 2010; Cao Ngpi Didp vd Nguydn Thdnh Dung. 2010; Cao Ngpc Di^
vd Phan Thj Nha, 2011) vd Trdn Thanh Phong vd Cac Ngpc Di^p (2011a). Cdc tdc gia da phan lap va nhar didn cdc chiing vi khudn Burkholderia tropica, B.
tivpicalis, Enterobacter sp. vd Klebsiella sp. ]k vi khudn npi sinh frong cay khdm (diia) frdng d viing dat phdn huy^n Tan Phudc, tinh Tidn Giang. Trong dd khd nang cd djnh dam. hda tan iSn va sdn xuitck kich thich td thue vdt eiia vi khudn ndy dang dugc quan tdm vd nghidn cuu dd sdn xudt phdn sinh hpc hay phan huu co vi sinh bdn cho cdy khdm rat hi^
qud CTrdn Thanh Phong vd Cao Ngpc Di^p, 2011c va 2011c); ddng thdi Nguydn Thj Ngpc Tnie va CTV (2010) eung phan ldp va nhdn di^n vi khuin Burkholderia tropica vd Enterobacter cloacae trong dat frdng rau b Tidn Giang. Ngodi ra, Zhao et d (2008) nhan didn cdc ddng vi khuan hda tan kali frong dat ndng nghidp la Bacillus mucdaginosus va Agrobacterium tumefasciens. Vi vay vide phat hiSn cac chiing vi khuan cd kha nang cd djnh dam, hoa tan lan va phan giai kali frong dat frdng ngo b huyen Trdng Bom. tinh Ddng Nai md' ra trien vong ung dung cdc chung vi khudn cd ich ndy cho cay frdng de hdt ki?m dupe phan nao lupng phan hda hpc NPK cdn bdn cho cdy frdng ndi chung va cay ngd noi ridng.
rv. KET LUAN
Vi khudn cd ich (vi khuan cd djnh dam, hda tan ldn, phan giai kah) sdng frong dat vimg rd ngd trong frdn ddt cr huy^n Trang Bom, tinh Ddng Nai phong phii va da dang chiing loai, tir nhdm vi khuan cd dinh d^m sdng tir do, npi smh den vi khuan ndt san;
chung gdp phdn tang cudng vd eai thidn dp phi ciia ddt frdng ngd.
TAI U i u THAM K H A Q
1. Cao Ngpc Di^p, Trdn Thanh Phong, TrdnlTiJ Giang, 2009. Phin I$p vi tiiydn chgn cic chung ri khuan Pseudomonas cd kha ning hoa tanliavasish tdng h(>p auxin cao. T?p chi Nong nghidp va Phat fridn Ndng thdn, 9,32-36.
2. Cao Ngoc Didp vd Nguydn Ai Chi, 2009. B^
lip vi die tinh cUa vi khudn ndi sinh trongdy^^^
54 N 6 N G NGHlfP VA PHAT TRIEN N 6 N G THON - KY 2 - THANG 7/2012
KHOA H9C C O N O NOHJ
trdng trdn dat phen huydn Bdn Luc, tinb Long An, Viet Nam. Tuyen tap Hpi nghi Cong ngh& Sinh hpc toan qupc nim 20091» chiic tai tai thanh php Ho Chi Minh, 23-24, thang 10, 2009 tr:69-73.
3. Cao Ngpc DiSp va Nguyin Thtoh Dung, 2010 Dac tinh vi khuan n6i sinh phan lap trong c^y kh6m trong tren dat phen Vinh Thuan, tinh Ki^n Giang.
Tap chi Khoa hpc Tniong Dai hpe Can Thp 15a, 54- 63,
4. Cap Ngpc DiSp vi Phan Thj Nha, 2011. Phin lip vi xic dmh die tinh vi khuiin ndi sinh trong ciy khdm (Ananas comosus L.) trdng tren dat pben thf xi VI Thanh, tinh H$u Giang. Tap ehi Cong ngh^
Sinh hoe. Vi^n Khoa hpc v^ Cong ngh0 Vi^t Nam 9 (2), 243-250,
5. Fallik, E., Y. Okon, E. Epstein, A. Goldman and M. Fischer, 1989, Identilication and quallUcation of lAA and IBA in Azosphilluni brasilense- inoculation maize roots. Soil Biol. Biochem., 21,147- 153.
6. Un Qi-mei, Rao Zheng-Hung, Sun Yan-Xing, Yao Jun and Xing Li-Jun, 2002. Identilication and practical apphcation of silicate-dissolvnig bacteria Agric. Sci. China, 1:81-85.
7. NauUyal, C. S. (1999). An efScient microbiological growth medium for screenmg phosphate soIubiUzing microorganisms. FEMS Microbiology Letters, 170, 265-270.
8. Neumann, B., A. Pospiech and H. U.
Schairrer, 1992. Rapid isolation of genomic DNA tom Gram-negative bacteria Trends Genet. 8,332- 333.
9. Ngo Thanh Phong, Nguyfc Thj Phuomg Thu va Cao Ngpc DiSp, 2010. Phin lap va nhin dien vi khuan cddinh dam vung rl Ida ti-dng ti-in datphii sa tinh Kien Giang Tap chi Cong nghd Sinh hpe. Vi$n Khoa hpc va Cong nghe Viet Nam 8 (3A), 1015-1020.
10. Ngo Thanh Phong, NguySn Thi Phuong Thao vi Cao Ngpc Di4p, 2011. Phin lip vi nhin diin
vi khuan cd dinh dam tiong dat vung re lua tiong tien dat phu sa tinh Vinh Long. Tap chi Cong ngh4 Sinh hoe. Vifn Khoa hpc va Cong nghf Vi6t Nam 9(4), 521-528.
11. NguySn Thi Ngpc Tnie, L4 Thi Thu Hong vi Cao Ngpc Di«p, 2010. Khio sit khi ning nhin sinh khdi vi sin smh axit huu ca cua cic ddng vi khuan
Burkholderia tropica vi Enterobacter cloacae du^
phan lip tir dat tidng rau tai Tidn Giang. Ky ydu boi ngbi khoa hoc "Phit tri^n nong nghifp bin vung thieh ung v6i sir bi^n doi khi hau". Dai hpe Can Tha, 2010. NhJ xuSit ban N6ng nghiep, H4 Npi, Vift Nam.
12. Nguyfn Van Dupe va Cao Ngpc Diep, 2004.
Hieu qui phin sinh h(?c trdn ciy diu nanh vi bip lai trdng tivn ddt phd sa huydn Tin Hiep, tinb Kidn Giang Tap chi Khoa hpe - Dai hpe Cin Tho 1,98-104.
13. Nguyin Nhu Hi, 2006. Giio tiinh phin bdn cho ciy tidng. NXB N6ng nghiep. Ha Npi.
14. Park, M., C. Kim, J. Yang, H. Lee, W. Shin, S.
Kim and T. Sa, 2005. Isolation and characterization of diazotiophic growth promoting bacteria from rbizosphere of agricultural crops of Korea Microbiological Research, 160,127-133.
15. Rodriguez, H. and R. Fraga, 1999. Phosphate solubizing bacteria and their role in plant growth promotion. Biotechnology Advances, 17,319-339.
16 Tong cue Thdng ke Viet Nam, 2010.
littp://www.gso.gov.vn/
17. Trdn Thanh Phong va Cao Ngpc Diep, 2011a.
Phin lap vi die tinh vi khuan ndi sinh tiong ciy khdm (Ananas comosus L) tidng tien dat phen huyen Tin Phudc, tinh Tien Giang. Tap chi Ceng nghe Sinh hpc. Vien Khoa hoc va Cong nghe Viet Nam 9(1), 125-132.
18. Tran Thanh Phong va Cao Ngpc Diep, 2011b.
Anh hudng cua vi khuan cd dinh dam Burkholderia tiopicalis & B. tiopica, phin dam vd co, phin bdn li ddn ning sudt vi chat luong tiii khdm (Ananas comosus L) tiong tidn dat phen huydn Tan Phudc, tinh Tidn Giang. Tap chi Nong nghiep va Phat trien Nong thon, BO Nong nghifp va Phat trien Nong thon 12,23-28.
19. Tran Thanh Phong va Cao Ngpc Difp, 2011c.
Hieu qui cua phin hdu co- - vi sinh bdn cho ciy khdm ti-dng tidn dat phen huyen Tin Phudc, tinh Tidn Giang. Tap chi Khoa hoc cua truang Dai hgc CanThpl9b,179-186.
20. Tran Van Van., Serge 0 „ Ngo Ke S., Balandreau J., Heulin T„ 2000, Repeated beneScal effects of rice inoculation with a stiain of Burkholderia vietnamiensis on early and late yield components in low fertility sulphate acid of Vietnam.
Plant Soil 218,273-284.
N 6 N G NGHIEP V A PHAT TRIEN N O N G THON - KY 2 - THANG 7/2012 55
KHOA HOC C 6 N G N G H £
21. Trung tam Khao nghidm Gidng Cdy frdng small subnit rRNA sequence analysis. J. of Trung uong, 1995. Ky tiiuit tiong cic gidng ciy Eukaryotic Microbiology 46:327-338.
trong mdi ning sui't cao.'^hd.xyii'ih^n(n\zngh\^\X 24. Zhao. F., X. Sheng, Z. Huang and L. He, Hd Npi, 285 frang. 2008. Isolation of muieral potassiums
22. Tumura. K., D. Peterson, N. Peterson, G. bacteria stiains tom agricultural sods in Shandong Stecher, M. Nei and S. Kumar. 2011. MEGA5: /^oraice. Biodeversity Science, 16(6), 593-600.
Molecular Evolutionary Genetics Analysis using Maximum Ukelihood, Evolutionary Distance and Maxmmni Parsmiony Methods. Moi. Biol, Evol.
28:2731-2739.
23. Turner, S , K. M. pryer, V. P. W. Miao and J.
D. Palmer, 1999. Investigating deep phylogenetic relationships among cyanobacteria and plastids by
25. Vasuvat. Y. B. Fangcham, S. Siripin, D, Fusang and P. Chanaram, 1986. Yield maximizatioii of feed grain by associate N^fbdng bacteria, k:
Proceeding of International seminar on yield maxhiiization of feed grain through. Soil and Fertilizer Management, 12-16.
ISOLATION AND IDENTIFICATION OF BENEFICIAL BACTERIA ON SOIL RHIZOSPHERE OF CORN CULTIVATED ON TRANG BOM DISTRICT, DONG NAI PROVINCE
Dang Thi Ngoc Thanh, Cao Ngpc Diep Summary
Com {Zea mays L.) is an important crop after nee which has been cultivated on soils of Eastern of South Vietnam, however com plant needs to be used a lot of chemical fertilizers to ensure high yield therefore com has often been planted on high fertility of soil. Besides that, nutrients fi-om inorganic ferbUzers, there are nitrogen-fixing bacteria, phosphate-solubilizing bacteria and potassium-solubihzing bacteria in soils which can response nutrient requirement for com growth and the aims of this study were (i) isolation and (ii) identification of these beneficial bacteria in soil rhizosphere of com. Twenty-eight bacterial isolates having ability of nitrogen-fixing, phosphate and potassium solubilization were isolated on Burk without N.
NBRIP and kaobnite media among ten isolates had high effectiveness and they were sequenced with 16S rRNA gene universal primers (8F and 149210 and compared with Genbank database of NCBI by BLAST N software and MEGA 5.05 software was apphed to analyse the phylogenetic free. The results showed that 9 isolates are gram-negative bacteria (except isolate DN18 is gram-positive bacteria) which were identified as nitrogen-fixing bacteria and they are legume rhizobia. endophytes and PGPR Iplant growth promoting rhizobactena].
Keywords: BeneScial bacteria, com, isolation, PCR-16S-rRNA, soil rbizosphere.
Ngudi phdn bi^n: PGS.TS. Ph^un Van Todn Ngdy nhdn bdi: 12/6/2012
Ngdy Ihdng qua phan bi^n: 11/7/2012 Ngdy duy^t ddng: 16/7/2012
5^ N 6 N G NGHlfP VA PHAT TRI^N N 6 N G T H 6 N - KY 2 - THANG 7/2012
