Tgp chi Khoa hpc Trudng Dai hgc Cdn Tha Phdn B: Nong nghiep, ThOy sdn vd Cdng nghe Sinh hpc: 35 (2014): 65-73 I
Tap chl Khoa hoc Tru'dng Dai hgc Can ThcJ website: sj.ctu.edu.vn
PHAN LAP VA NHAN DIEN VI KHUAN VUNG RE
KICH THICH Sir SINH TRU'dNG (PGPR) TU" MOT SO LOAI RAU AN LA TRONG TAI TIIANH PHO CAN THO
Tran Thi Giang', Nguyen Thi Quyen^ va Cao Ngoc Diep^
' Viin Nghien cuu & Phdt trien Cdng nghi Sinh hoc, Truong Dgi hgc Cdn Tha
^ Hgc vien Cao hgc Sinh Thdi, Khoa Khoa hgc Tu nhiin, Trudng Dgi hgc Cdn Tha
Thdng tin chung:
Ngdy nhgn: 23/06/2014 Ngdy chdp nhgn: 30/12/2014 Title:
Isolation and identification of plant growth-promoting rhizobacteria on leaf-eating vegetables grown in Can Tho City
Tirkhda:
Co dfnh dgm, hda tan ldn, lAA, rau dn Id, siderophores, vi khudn viing
Keywords:
lAA, leaf-eating vegetables, nitrogen fixation, phosphate solubilization, rhizosphere bacteria, siderophores
A B S T R A C T
Seventy-six isdates were isolated from 25 rhizttsphere soil samples of 13 different kt^
eating vegetables species grown in 6 districts of Can Tho City. Among them, 48 isolates had good characlenstics as nitrogen fixation, phosphorus solubility and lAA synthesis.
Especially 5 isolates (NBT625. NPD721. NPD855, NOM131 mdNBT6l3) had relatively high abililies of nitrogen fixation, phosphorus solubilization (0.80-2.21 mg/L NHA 27.82- 50.63 mg'L POs) and 3 isolales (PBT622, POMl 12 and POM222) synthesized high lAA levels (782-8.25 mg/L) These eight isolales wei-e selected lo test the siderophm-e- producing ability and the results showed thai 7 isolales having light and color changes CAS medium. Six/seven isolates were chosen lo idenlifr with primers 27Fand 1492R. they were sequenced and compared with bacterial 16S rRNA genes in Genbank using BLAST N program. The results showed that NBT613 isolate wtis a 99% similarity with GQ181060 Agrobaclerium tumefaciens strain BLN4, NPD855 isolate had a 99% identity with KC934864 Ensifer adhaerens strain M27 and JQ322555 Sinorhizobium meliloli strain CHWIOB, PBT622 isolate was a 97% similarity with KF358257 Acinetobacter calcoaceticus strain L14, POM112 isolate had a 97% identity with JQ923444 Achromobacter xylosoxidans strain BL6, NBT625 isolate was a 98% similarity with KF870446 Rbtobium sp LS-079, and NPD721 isolate was a 99% similarity with KC833504 Burkholderia sp. TCP30, and 5 strains were selected lo evaluate their effects on leaf-eating vegetables in the pots and field experiments except POM! 12 strain.
T O M T A T
B^ muai sdu dong vi khudn dugc phdn lap tir 25 mdu ddt vung ricua 13 lodi rau dn Id trdng lgi 6 qugn-huyen cda Cdn Tha. Trong do, 48 ddng cd cd 3 dgc Hnh tdt nhu cd d/nh dgm. hoa tan ldn vd tong hop L4A; vdi 5 ddng (NBT625. NPD72I. NPD855, N0M13! vd NBT613) cd khd ndng cd dinh dgm, hoa lan ldn cao (0.80-2.21 mg/L NH4* 27 82-50 63 mgi P2O5) vd 3 ddng (PBT622. POMl 12 vd POM222) cd khd ndng ldng hgp lAA cao (7.82-8.25 mg/L). Kit qud khdo sdt khd ndng sdn xudt sideriyjhores. cd 7/8 ddng tgo dugc vong sang vdl^ doi mdu moi Intdng CAS Sdu dong 17 khudn ndy dugc chpn di nhdn
<£?n vat cap moi 27F vd 1492R vd gidi trinh lu gen 165 rRNA. so sdnh vdi gen 16S rRNA cm w khtdn trong GenBank bdng chuang trinh BLAST N. Kit qud cho thdy dong NBT613 tuang dong 99% vdi ddng GQ181060 Agrobaclerium tumefaciens BLN4 ddng NPD855 '^^99%vdi ddng KC934864 Ensifer adhaerens M27vdJQ322555Sinorhtobium mehloti CHWIOB. ddng PBT622 tuang ddng 97% vdi ddng KF358257 Acinetobacter calcoacencusU4. ddng POM112 tuang ddng 97% vdi ddng JQ923444 Achromobacter f^oxidcms BU. dong NBT625 tuang dong 98% vdi ddng KF870446 Rhizobium sp IS- 0J^'''"dmgNPD72!tuongadng99%vdiddngKC833504BuHdK,lderiaspTCP30.vd5 ajmg V, khuan dugc chpn ddnh gid hUu qud cOa chiing tren rau an Id trong chau vd ngoM ddngmi dong POMl 12 & • &
Tgp chi Khoa hpc Trudng Dgi hpc Cdn Tha 1 G i d l THIEU
Vi khuan viing re kich thich siito trudng d touc v^t (Plant Growto Promoting Rhizobacteria-PGPR) la vi Idiuin frong dit, sinh sing xung quanh hogc tren bi mdt re, true tiip hogc gidn tiip toam gia viec kich toich sinh trudng va phat triin cua touc vdt thdng qua san xuat va tiet ra nhiing chat hda hpc khac nhau d xung quanh viing ri. Ndi chung, PGPR tgo dieu kiin thudn lgi cho siito trudng thuc vgt bdi tryc tiip hogc hd frg viic tou thgp ngudn dudng chat (dgm, lan va cac khoang chit thiet yiu) ho§c diiu chinh hormone touc vat, hodc gidn tiip Idm gidm cac tac nhdn gdy b^nh khac nhau frc chi sinh tiirdng tiigc vat. Nhiiu nghiin cfru da ghi nhdn viic ling dyng PGPR frong frdng frpt Iam tang siic di khdng va ndng suat ciia cdc logi cay trdng khdc nhau frong cd diiu ki$n binh toudng va bit loi. Cdc vi khuin vdng ri toye vgt cd lgi cd toi Idm giam su phy toudc hoan todn vao ndng dugc nguy hgi gay bit on cho hi sirto thdi ndng nghiep. Cd toi ndi PGPR la luc Iupng tiin phong chi phdi viic tai su dung cac chat dinh dudng frong dat. Vi vay, chung rit quan frpng ddi vdi kha ndng phyc hdi ciia ddt.
VI thi, viec iing dung nhiing vi khuin vdng re cd lgi ndy frong san xuat phan vl sinh bdn cho cdy frdng ndi chung vd rau xanh ndi rieng, nham giam sfr dyng phdn bdn hda hgc, gdp phan bdo ve siic khde con ngudi va xdy dgng hi sinh thai ndng nghiep bin vihig.
Rau xanh dugc xera Id nguin touc phim dmh dudng quan frgng khdng toe toay toi (Ta Thu Ciic, 2005), frong rau cd dudng, dgm, vitamin, acid hihi CO va cac hgp chat khoang (sat, kali, canxi...) can toiit cho su duy fri va phai ttiin cda con ngudi, rau la v( thudc tg rtoien cd chda rdt nhiiu chit dugc - tinh. Hien nay, phim chat rau bi gidm siit do du Iugng hda chat dOc vd vi sinh vat gay hgi cho con ngudi vugt nhiiu so vdi ngudng qui dinh (Dd Thj Trudng 2009). Dac biit du Iugng nifrat ttong cac san phdm rau xanh gay nhieu h^u qud nghiem frgng, tii do phan bdn vi sinh vdt cho rau duoc nghien cfru va frng dung ngay cdng rpng rai nham han chi arto hudng tieu cgc cda phdn hda hpc.
Tdng diin tich ttdng rau cac loai ndm 2012 d tiidnh phi Cin Tho Id 6.845 ha, ndng suit 125,52 tg/ha, sdn Iugng 85.916 tan, frong do diin tich rau an Id 487,7 ha, nang suit 112,72 tg/ha, san Iugng 5497,2 tan (thing ke cua So Ndng nghiep va Phat ttiin Ndng tiidn Cdn Tho). Dya ttin diiu kiin tu nhiin va lgi thi dia Iy d nhftag vung ven sdng, cu lao, kenh rach bao quanh, Cin Tho da va dang xdy dyng cdc viing sdn xuit chuyen carto rau phuc vu
Phdn B: Ndng nghiip. Thity sdn va Cdng nghi Sinh hpc: 35 (2014): 65-73 nhu cdu sfr dung ciia todnh phd va cung cap cho cdc tinh ttong vung. Tuy nhien, frong khi rtou cau tiiu thu rau an todn ngay cang cao toi touc ti sdn xuit rau an todn d day cdn gdp nhieu khd khan.
Thuc te vdn chua cd nhiiu di tdi nghien cfru ve vai frd ciia vi sinh vat cd ich ttong dat trdng rau nin viic ling dyng chi phim vi sinh de gidm Iugng phdn bdn va thudc trfr sdu hda hpc fren dat frdng rau con hgn che.
Myc tiiu nghiin cfru la phdn Idp va n h ^ diin vi khuan viing ri kich thich su sinb tnrdng frin cay rau dn la frdng tai Cdn Tho.
2 PHU'ONG PHAP NGHIEN ClTU 2.1 Mau dat
Cdc mau dat viing re rau dn la (khoang 2 1 ^ dat bao gdm cd cdy rau) dugc tou to^p_tfrj;ac rudng rau tgi 6 quan-huyin (Cdi Rdng, Biito Thdy, Phong Diin, O Mdn, Co Dd, Thit Ndt) cua Cdn Tho. Dit viing ri (ldp dat bao quanli rl khoang 0,8-10 mm) cua timg mlu rau dugc di riing ttong tdi nilon sgch khuan (cd ghi nhdn va so toii tu), ttii ttong tii Ignh (4°C) khi chua sii dung. Gd Idp dat bao quarto re mdt each nhe nhdng, tdch riing, Ugn diu Iai di sii dung cho phan Idp vi khudn (md ta d phin sau).
2.2 Phdn Idp vi khudn vimg r i kich thich sinh trudng (PGGR)
Mau ddt viing re (I g) dugc cho vao binh tam gidc vdi 99 ml nude cit dd khii trung, lie 12 gid (200 vdng/phdt) cho cdc hat dit rdi ra va vi khuin phdn tdn deu frong nude. Djch vi khuan dugc de ling khodng 1 gid; lay 0,1 ml ttdi diu frin dia pefri chiia radi trudng Burk khdng dgm (phat hiin vi khuan cd dinh dam) (Park et al., 2005) vd mdi ttudng NBRIP (phalr^inCTgtkhudnHhdas (Nautiyal, 1999) 'da' du'gc'~chu^imsan, u d 30°C.
Sau 24-48 gid, cac khuan Igc mpc ttin be mgt mdi trudng dugc tiip tyc ciy chuyin sang mdi trudng mdi vdi lin din khi cdc khuin Igc xuat hiin trin dudng cay rdi nhau thi quan sat d^c diem khuan Igc (hinh dang, mau sac, dp ndi, dang bia, kich toudc).
Cac ddng vi khuan phan lap dugc kiem tta dp thudn bang phuang phdp gipt ep dudi kinh hien vi quang hpc d dd phdng dai 400 lin. Khi toiy vi khuan da touin nhit tiin hanh ciy chuyin sang dng nghiim chfra mii Irudng S^c tuang frng di trfr d 4°C va dugc xem nhu rapt ddng touan. Cac vi khuan phat ttiin dugc ttin mdi trudng Burk khdng dgm dugc ciy sang moi trudng NBRIP va ngugc Igi, chpn nhiing khuan Igc phat trien tten cd hai mdi trudng Burk khdng dam va NBRIP nhu Id cac ddng vi khuin cd khd nang co dinh dam va hda tan Idn.
Tgp chi Khoa hpc Tnamg Dgi hgc Cdn Tha Phdn B: Ndng nghiip. Thiiy sdn vd Cdng nghi Sink hpc: 35 (2014): 65-73 2.3 Dmh lupng dam, lan hda tan, lAA d
nhung ddng vi khuin phan ISp dugc Cdc ddng vi khuin phat triin fren timg logi mdi trudng dugc do khd ndng cd dinh dgm frong mdi tiirdng Burk khdng dam Idng bdng phuang phdp so mdu Indophenol Blue d budc sdng 636 nm (OD636nni); do khd ndng hda tan Idn frong mdi trudng NBRIP Idng bdng phuang phap so mau Blue-molypden d budc sdng 880 nm (ODssonm); do khd ndng ting hpp lAA frong mdi trudng phan ldp Idng bing phuang phap so mau Salkowsky d budc sdng 530 nm (ODssonm)-
2.4 Khao sat kha nang san xnat siderophores ciia cdc dong vi khuan da fuyen chpn
Vi khuin vung ri dugc tang sinh trong radi tiudng PS (peptone lOg/L, sucrose IOg/L)(Schwyn va Neilands, 1987), lie 200 vdng/phiit, d 30''C ttong 24 gid. Nhd gipt 50 pi dich vi k h u ^ Iin radi trudng chorome azurol S (CAS) agar vd u d 30°C trong 48 gid, sau dd quan sat kha ndng sdn xuit siderophores ciia tiing dong. Khi vi khudn sir dung sit trong mdi trudng toi mdu xanh ciia mdi ttudng CAS xung quanh khudn Igc khdng cdn niia, toay vdo dd vdng sang mau cam xudt hiin quanh khuan Igc dd la do cd su hifn dien ciia siderophores (Schwyn vaNeilands,1987).
2.5 Nhgn difn cic d6ng vi khuan bing ky thudt PCR-'l6S rRNA
- Tach chiet DNA vi khuan toeo md td cua Wilson (1997).
- Sau khi tdch chiit DNA tii cdc ddng vi khuan, phdn iing PCR gen 16S rRNA dugc tiin hdnh vdi cap mii 27F vd 1492R (Weisburg el ai, 1991) vdi ttinh hi nhu sau: 27F
(5'AGATTTGATCCTGGCTCAG3');1492R (5'GGTTACCTTGTTACGACTT3').
- Thanh phin phan ung PCR (25 pi): H2O 12 nl; buffer (chat dem) lOX 2,5 pl; MgCb 25 mM 2 pl; dNTP 4 \i.V, mdi 27F 1 pi; mdi I492R 1 pi;
BSA 0,25 pl; Taq polymerase 0,25 |il; DNA mSu 2 pi.
Phdn ling PCR: 95°C/5'; 30 chu ky:
(95''C/30", 53°C/30", 72°C/1'30"); 72°C/10".
- Sfr dyng dogn mdi 27F frong phdn iing PCR di nhgn diin vi khuin da md td d fren. San phim PCR dugc tinh sgch va giai trinh tu bing he todng may gidi frinh tu ttr ddng ciia Cdng ty MACROGEN (Hdn Quoc). Sfr dyng chuang frinh BLAST N di so sdnh frmh tu DNA cda cdc ddng vi khuin chpn loc vdi frinh tu DNA cda bd gen d cac Ioai vi khuin ttong GenBank va xdy dyng cdy phd hi gen giua cdc ddng vi khuan dugc giai ttinh ty bdng phin mira MEGA 6.05 (Tamura el al, 2011).
s i liiu dugc phdn tich thing ki bdng phin mim SPSS 16, Igp bdn ANOVA, sii dyng kiim dirto Duncan dk so sdnh su khac bi^t ciia tri trung binh cua tiing nghiira touc.
3 KET QUA VA THAO LU^N Bay muoi sau ddng vi khuan dugc phan ]|ip tii 25 mdu dat viing rl ciia 13 loai rau dn la khac nhau tou dupe d 6 quan-huyin cua Can Tho, frong do co 63 ddng phdt ttien frin cd hai logi mdi trudng Burk khdng dam va mdi trudng NBRIP; nhu vgy 63 ddng vi khuan nay vira cd kha ndng co djnh dam vira CO khd nang hda tan Ian (Hinh 1), da si chiing cd khuin lac frdn, trdr^ hogc vang, bia nguyin, dO noi md (Hinh 2); ti bao hinh que hay ovan, cd khd ndng chuyin dpng (Hmh 3).
Hinh 1: Khuan lac vi khuSn phan ldp it moi trudng Burk khdng N pbat triin tren mdi trufrng NBRIP sau 4S gid
Chu thich: A}=NCD534, A2=NOM131; A3=NBT614: A4=NOM232, A5^NPD725; A6=NCD565- B1=N0M222- B2=NTN422: B3=NOM214; B4^NPD721; B5=NBT625; B6^NBT622
Tgp chi Khoa hpc Trudng Dgi hpc Cdn Tha Phdn B: Ndng nghiip, Thuy sdn vd Cdng nghi Sinh hpc: 35 (2014): 65-73
Hinh 2: Dac diem khudn Igc (dang trdn; tring trong, dp nSi md) cua mpt so ddng (isolate) vi khuan phdn Igp
Hinh 3: Hinh dang mpt si d6ng vi khuan dudi kinh hiin vi diin tfr quet Chulhich: (A): DbngPBT622 (X8.000); (B): ddngPOMl}2 (XIO.OOO). (C) ddngNBT613 (X8.000)
Kit qud Bdng I va 2 cho toay kha nang cd dinh dam cua 63 ddng vi khuin sau 8 ngay u, ttong dd
Bdng 1: Kha nang co djnh dam ciia 38 ddng vi khuSn phan ISp trin mdi tririing Burk khdng dgm cd 11 ddng vi khudn ndi bdt todng qua ham Iupng NH4'^ tdng hgp kha cao (0,80-2,21 rag/L).
g^ «wug Lugng N H / vi khudn (mg/L) Ddng
Stt Ddng vi khuin
Lucmg N H /
Stt Dong Lupmg N H / vi khuan (mg/L) 1
2 3 4 5 6 7 8 9 10 11 12 13 14
DC NBT612 NBT613 NBT614 NBT622 NBT625 NBT626 NBT65I NCD534 NCD545 NCD546 NCD565 NCD574 NCR912
0,00 r 0,28 p 0,80 h 0,64 j 0,59 m 2,21a 0,01 r 0,75 k 0,74 k 0,78 h 0,01 r 0,63 j 0,94 e 0,03 r
15 16 17 18 19 20 21 22 23 24 25 26 27 28
NCR921 NCR933 NCR934 NCR936 N0M112 NOMfiS NOM131 NOM135 NOM143 NOM154 NOM155 NOM214 NOM22I NOM222
0,32 q 0,01 r 0,711 0,01 r . 0,01 r 0,38 p 1,53 c 0,01 r 1,00 d 0,52 n 0,01 r 0,01 r 0,01 r 0,701
29 30 31 32 33 34 35 36 37 38 39
NOM231 NOM232 NOM243 NPD721 NPD723 NPD855 NrN312 NTN411 NTN414 NTN421 NTN424 CV%
0,01 r 0,65 j 0,85 g 1,94 b 0,42 0 0,90 f 0,83 g 0,31 q 0,01 r 0,94 e 0,17 q 7,84
Nhung sd theo sau citng mgt cha cd su khdc biet khdng cd y nghia thong ki a mirc do 1%
Tgp chi Khoa hpc Trudng Dgi hpc Cdn Tha Phdn B: Nong nghiip, Thity sdn vd Cong nghi Sinh hpc: 35 (2014): 65-73 Bang 2: Kha ndng ci dinh dam ciia 25 ddng vi khudn phan lap tren mdi trirdng NBRIP
g Ddopvi LuomgNH/ _ Dongvi Lugng NH4*
khuan (mg/L) khuan (mg/L) Stt '^""^ ' Li'^^gNH^*
vi khuin (mg/L) 1
2 3 4 5 6 7 8 9 10
DC PBT622 PBT623 PBT651 PCD534 PCD537 PCD541 PCD546 PCD565 PCD572
0,00 n 0,27 b 0,09 j 0,24 c 0,89 a 0,18 g 0,10 i 0,07 m 0,17 gh 0,01 n
11 12 13 14 15 16 17 18 19 20
POMl 11 P0M112 POMl 14 POM215 POM217 POM222 POM224 POM243 POM244 POM247
0,20 f 0,17 gh 0,131 0,25 c 0,09 j 0,17 gh 0,22 e 0,28 b 0,10 i 0,01 n
21 22 23 24 25 26
PPD723 PPD734 PPD812 PTN311 PTN312 PTN427 CV(%)
0,16 h 0,01 n 0,111 0,23 d 0,141 0^4 d 7,18
NhSng so theo sau cimg nidi chit cd su khdc biil khdng cd y nghia thong ke d muc dp 1 Sau khi djnh Iugng dam, 26 ddng vi khuin
phdn ldp d radi trudng Burk khdng dgm vd 22 ddng vi khuan phan l|ip fren mdi trudng NBRIP dugc chpn di dinh lugng ldn hda tan va khd ndng tdng hgp indoI-3-acetic acid (lAA). Kit qud Bdng 3 va 4 cho thiy khd ndng hda tan lan ciia 48 ddng vi
khudn sau 20 ngay u va Iugng LAA ting hgp dugc sau 8 ngdy u. Trong dd, 11 ddng vi khuan cd hdm Iugng P3O5 cao tu 41,43 rag/L din-50,63 mg/L vi - 4 ddng vi khuin nii b^t thdng qua hdm lugng lAA khd cao (7,55-8,25 mg/L) Id cac ddng PBT622, PCD534, POMl 12 va POM222.
Bang 3: Kha nSng hda tan lan vd sinh tong hgp lAA cua 26 ddng vi khuin phan ISp trin mdi t r u ^ g Burk khdng dgm
STT 1 2 3 4 5 6 7 8 9 10 11 12
15 Dong vi DC NBT612 NBT613 NBT614 NBT622 NBT625 NBT65]
NCD534 NCD545 NCD565 NCD574 NCR921 NCR934 NOM123 NOM131
Lirong P2OS CmsflL) 0,00 n 23,28 h 27,82 fg 15,48Jm 26,32 g 29,05 e 28,33 ef 24,33 h 28,44 ef 13,52 m 27,60 fg 29,90 e 15,25 jm 20,65 i 36,60 d Nhung so Iheo sau citng moi chu co su
L u ^ g l A A (ms/L) 0,001 2,32 c 1,60 g 2,21 d 2,86 a 1,90 ef 1,59 g l,14h l,01h 2,14 d l,19h 2,35 c 1,47 g 2,44 b 1,81 f
16 17 18 19 20 21 22 23 24 25 26 27
khac biel khong co y ngh Dong vi
khuan NOM143 NOM154 NOM222 NOM232 NOM243 NPD721 NPD723 NPD855 NTN312 NTN411 NTN421 - NTN424 CV(%)
LlTQUg P2OS (ms/L) 48,11 ab
30,92 e 46,85 b 35,87 d 42,90 c 50,63 a 20,90 i 41,34 c 42,65 c 17,S8ii - ; T : g 3 0 , ' 9 4 ^
31,96 e 8,53
la Ihong ke a muc dQ 1%
Lin^glAA (me/L) 1,60 g 1,41 g l , l l h 1,44 g 1,77 f 1,58 g 1,92 ef 2,05 de 1,25 h 2,04 de
2,16 d 9,06
TQP chiKhoa hgc Trudng Dai hpc Cdn Tha Phdn B: Ndng nghiip, Ihuy sdn vd Cong nghi Sinh hpc: 35 (2014): 65-73 Bang 4: Kha n3ng hoa tan ldn va sinh tong hgp lAA ciia 22 dong vi kbuan phdn lap tren mdi tnrdng
NBRIP STT
1 2 3 4 5 6 7 8 9 10 11 12 13
Dong vi khuin DC PBT622 PBT623 PBT651 PCD534 PCD537 PCD541 PCD546 PCD565 POMl 11 POMl 12 POMl 14 POM215
Lu^ng PzOs (ms/L) 0,001 32,94 d 21,46 g 21,73 g 30,52 d 20,30 g 26,46 f 27,31 f 33,50 d
16,82 h 28,18 f 42,56 bc 20,87 g
LuornglAA ' (mg/L)
0,00 h 7,82 ab 4,48 d 5,10 c 7,55 b 3,88 e 2,48 g 3,93 e 3,27 f 3,44 f 8,19 a 3,45 f 3,64 ef
STT 14 15 16 17 18 19 20 21 22 23
D6ng vi khuSn POM217 POM222 POM224 POM243 POM244 PPD723 PPD812 PTN3H PTN312 PTN427 CV (%)
Lirong PiOs
• (mgfl.) 39,49 c 26,00 f 33,14 d 18,83 gh 28,10 f 36,79 c 45,79 b 49,66 a 48,02 a 42,93 bc 9,53
Lirong L\A ' (me/D
4,08 e 8,25 a 4,87 d 3,82 e 3,40 f 3,83 e 4,83 d 3,10 f 2,59 g 3,46 f 9,75
Nhang sd iheo sau ciing mpi chit cd sir khdc biit khong cdy nghia thdng kiamucdp 1%
Sau khi dmh Iugng dgm, Idn hda tan va lAA, 8 ddng vi khuan hfru hiiu nhdt (cd kha ndng c i dinh dam, hda lan Ian va long hgp lAA tdt vd i n dinh qua cdc ngay quan sat) dugc chpn di khao sat kha nang san xuat siderophores; do la cac ddng NBT6I3, NBT625, N0M131, NPD72I, NPD855, PBT622, POMl 12, POM222. Kit qud cho toiy 7
dong vi khuan cd khd nang ldm thay dii raau xanh ciia mdi trudng CAS todito mau cam xung quarto khudn lac, qua do cho tody chiing cd khd ndng sfr dung sit trong mdi trudng, gdp phin lic chi vi sinh vat gay benh cho cay frdng (ddng NPD855 khdng cd khd ndng sdn xuat siderophores)(Hinh 4).
Hinh 4: Cdc ddng vi khuin ISm xuit hiin vdng sdng mau cam sau 48 gifr phat trien tren mdi trudng CAS agar
Ddng todi, 8 ddng vi khudn nay dugc nhan diin bing kJ tougt PCR vdi cap mil 27F va 1492R. Kit qua cd 6 ddng cho band d vi fri khodng 1500 bp so vdi toang DNA chuin 100 bp plus (Hinh 5).
Cac ddng NBT613, NPD855, PBT622, POMl 12, NBT625, NPD721 dugc gidi tiinh tu dogn gen 16S rRNA vdi kit qud nhu sau: ddng NBT613 (1126 nu) cd ti li tuong ddng 99% vdi trinh tg DNA cua GQ181060 Agrobaclerium tumefaciens stiain BLN4 vd KFO10919 Agrobaclerium tumefaciens strain CSY-F4; ddng NPD855 (1285 nu) cd ti Ii hrang ddng 99% vdi trinh tu DNA ciia KC934864 Ensifer adhaerens 1500bp
Hinh 5: Phi difn di san phdm PCR dugc nhdn Ien tfr DNA ciia 6 ddng vi khuan vung rl Ghi chu: l=thangDNA chudn IOO bpplus. 2=NBT613, 3=NPD855. 4=PBT622. 5=POM112. 6=NBT625, 7-NPD721, 8=ddi chimg dm
Tgp chi Khoa hpc Trudng Dgi hpc Cdn Tha Phdn B: Nong nghiip, Thuy sdn vd Cdng nghi Sinh hpc: 35 (2014): 65-73 sfrain M27 va JQ322555 Sinorhizobium meliloti
strain CHWIOB; ddng PBT622 (1305 nu) cd ti Ii tuong ddng 97% vdi tiinh tu DNA cua KF358257 Acinelobacter calcoaceticus sttain L14 va FJ976525 Acinetobacter calcoaceticus sfrain LCR16. BGM3; ddng POMl 12 (1262 nu) cd ti Ie nrong ding 97% vcri ttmh pr DNA cua JQ923444 Achromobacter xylosoxidans strain BL6 va HQ676601 Achromobacter xylosoxidans sfrain M66; ddng NBT625 (1219 nu) cd d Ii tiiang ding di truyin 98% vdi frinh tu DNA cua KF870446
Rhizobium sp. LS-079 vaKF008229 Rhizobium sp.
BGM3; ddng NPD721 (1321 nu) cd ti If tuang ding di truyin 99% vdi frinh ty DNA cda KC833504 Burkholderia sp. TCP30 va JF772523 Burkholderia sp. bC28 (2011).
Nhu vay, trfr ddng POMl 12 ding hinh vdi ddng vi khuan cd tac ddng xau den mdi ttudng (nen logi ra), 5 ddng cdn Igi diu la vi khuin cd ich song a viing ri hay ndi sinh da dugc cdng bi d nhiiu qudc gia khdc nhau ttong ngan hdng du' liiu cua NCBI.
POMl 12
- JQ923444 Achromobacter xylosoxidans strain BL6 NPD855
KC934864 Enslfef adhaerens strain M27
•\
JQ322555 Sirarhizobium meliloti strain OIWIOB V N h a n h I - NBTC35
^—— FJ976525 Acinetobacter calcoaceticus strain LCR16
- KF358257 Acinetobacter calcoaceticus strain L14 JF77Z523 Burkhdderia sp. bC28[2011) - KC833504 Burkholderia sp. TCP30 99|KF870446RhizoWumsp. LS-079
IKF008229 Rhizobium sp. fiGM3
HQ676601 Achromobacter Kylosoxidans strain M66 KF010919 Agrobaclenum tumefaciens strain CSY-F4
NBTC13
381GQ181060 Agrobacterium tumefeciens strain BUW
> Nhdnh 2
Hinli 6: Cdy phd he (phylogenetic tree) trinh bay moi quan hf di truyin giua 6 dong vi khuin viing rl da dugc phSn ldp vd nhgn diin (theo Neighbor-joining)
Cdy phd hi dugc xdy dyng bdng phdn mim MEGA 6.05 Cay pha he (Hinh 6) cho toiy 6 ddng vi khuin phdn Idp nam tren 2 nhanh vdi nhdnh I gdra cac ddng NBT625, N0M131, NPD721, NPD855, PBT622, POMl 12, POM222, frong khi d nhanh 2 chi cd ddng NBT613.
Vi khuin ciing ri kich toich sy tdng trudng da dugc Kloepper va Schroto (1978) tira ra vd dat lin cho nhdra vi khuan sing d vung rl touc vdt nhung cd nhieu ich lgi cho touc vat nhu cung cip N sinh hpc, hda tan ldn khd tan, ting hgp lAA, tgo siderophore giup dii khdng lai nhdm vi sinh vat
gdy hgi cho touc viit (Basban vd de-Bashan, 2005).
Vi khuan Alcaligenes. Azoarcus. Azospirillum, Azolobacter, Bacillus, Klebsiella. Pantoe, Pseudomonas la nhfri^ PGPR cd khd nSng ci dinh N sinh hpc (Hurel et al., 1994; Baldani el al., 1997;
Riggs et al., 2001; Cakmakci et al.. 2008).
Gutierrez-Manero et al. (2001) tim toiy Bacillus pumulis va Bacillus licheniformis tgo nhiiu lAA sdng viing xl cdy xa lach vd Toro et al (1997) tim thiy Bacillus subtilis la nhung PGGR gia tdng Iugng lan hda tan sing d re cay hanh Id. Gin day (Farima et al., 2012) phdn lap va nh|in diin cdc
Tpp chi Khoa hoc Trudng Dgi hpc Cdn Tha Phdn B: Ndng nghiep, Thity sdn vd Cong nghi Sinh hpc. 35 (2014). 65-73 ddng vi khuin Agrobaclerium tumefasciens,
Burkhoderia, Enterobacter, Pseudomonas la nhirng PGGR cd kba ndng ting hgp lAA, c i dinh N sinh hgc, hda tan lan khd tan va tao siderophores frong re cay canola. Ngoai ra, vi khuan Achromobacter xylosoxidans dugc tim thdy frong dit, nuoc (McGuckin et al, 1982), Id vi khuin gram am hiiu khi, di dgng, hinh que, dugc md la lan dau vdo nam 1971 bdi Yabuuchi va Ohyama, hai dng phat hi^n chutig d nhimg benh nhdn viem tai giiia radn tinh vi vdy ddng ndy khdng dugc diing 6.h ung dung ttin cdy frdng mac du chung ciing cd rtoiing ddc tinh mong mudn. Nhttng kit qua dat dugc frong nghiin ciiu nay ciing phii hop vdi nhiing kit qua trudc ddy da bao cao ke ca ddng Achromobacter xylosoxidans, cd Ii ddng vi khudn la nhiing mdm b^nh tiim sinh frong dat nhung ciing cd nhiing ddc tirto nhu cac PGGR di chiing cd toi tin tgi trong diiu ki^n tu nhien khi khdng cd ky chu.
t KET LU^N VA D i XUAT
Bin ddng vi khuin (NBT625, NBT6I3, NPD72I, NPD855) cd khd ndng c i dinh dam, hda tan idn cao va ddng PBT622 cd kha nang tdng hgp lAA cao ddng todi cd khd nang uc che vi siito vat gdy b^nh cbo cay trdng, chiing dugc chpn de darto gia hiiu qud ciia chiing fren rau an Id frdng trong chau vd ngodi dong nham tien tdi san xudt phdn sinh hpc cho rau xanh.
L d i CAM TA
Cac tdc gid chdn toarto cam an Trudng Dai hpc Can Tho da hd frg kinh phi touc hiin di tdi.
TAI L i f u THAM KHAO
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