IJIVIG DLJiXIG KY
T H U A TPCR TROIMG VIEC X A C DirUH V I KHUAIXI Leifsania jcyli s u b s p . jcylh GAY BEIXIH
cAlV MIIA GOC (RSD) Q VLJIXIG DOIVIG iXIAIVI BO
Hd Dinh TudnS Ld Dinh D6n^ Biii Cdch Tuyln^
T6MTAT
Ung dung ky thuat PCR dl xac dinh Leifsonia A7//subsp. xy//trong djch chilt true tilp tir cSy mia va qua nuoi cay nh^n tao da dupe thuc hifn trong nien vu 2009-2010. Cap moi Lxl (5'- CATTGACATrGGTGCAGAGC-S') va Lx2 (S'-CATCCACCGTrTGCTdTAG-S') dupe tiiilt kl cho vifc khuech dai viing 16-23S thuoc rRNA cua Leifsonia xyli subsp. xyli, san phdm PCR co kich thuoc 363 bp.
Chu trinh nhift cho phan img PCR vol chu ky ddu 95"C (3 phuO. 40 chu ky Ulp theo [QSX (30 giay), 55"C (30 giay) va 72"C (1 phuOl va ket thuc b 72"C (10 phuO- Trdn ca so so s^nh trinh tu nucleotit cua cac mau thu thap cho thdy co su tuong dong cao voi ca sa dCr lifu tren GenBank, ket luan da chung to vi khuan Leifsonia Ay/r'subsp. Ay^'cung la t^ic nh^n gSy bfnh cdn mia gdc a viing Dong Nam bf.
Tirkh6a: Nudi cdy nhin tao. dich chi A, PCR. binh cin mia gdc (RSD).
L DAT VAN D^
Bfnh cdn mia gdc (ratoon stunting disease-RSD) da dupe phdt hifn tir rat lau trdn till gidi. Vi khuan cu tni trong hf thdng mach dan ciia cay mia, gay tdc mach hoAc gidm kha nAng van chuyin nudc vd chat dinh dudng lam cho cay mia bi cdi epe Qglesia, 2003). Vdi dAc diim gdy hgi ciia bfnh, thdng kd d nhilu nude trdn t h i gidi cho thay nd thudng xuydn lam tiiat thu nAng suat 5 60%, cd bift cd nhilu nAm da gay thift hgi ldn den trdn 30% san lupng mia
(Davis vd Bailey, 2000).
Nghidn ciiu trdn t h i gidi cho thay vi khuan gay bfnh cdn mia gdc rat dd lay lan ngodi tu nhidn, nhung khd nudi cay trdn moi trudng nhan tgo. Chinh vi vay, vifc xac dinh tdc nhan gay bfnh gAp nhilu khd khAn vd ddi hdi nhilu thdi gian. Trong khi phuang phdp kinh hiin vi vd huylt thanh doi khi dgt hifu qua khong cao nlu mat dp vi khuan trong dich chilt cua cay mia thap vd phu thupc vdo ngudn khang thi ban ddu. Vifc ting dung ky thugt sinh hpc phan tu d l chan dodn bfnh da dupe cdng bd ddu tidn bdi Pan vd ctv (1998), Fegan vd ctv (1998); qua dd cho thay uu diem v l miic dp chuan xdc trong thdi gian ngan, ngay cd khi dich chilt ehiia lupng vi khuan thdp. Chinh vi vgy, vifc xac dinh nhanh vd chinh xdc vi khuan gay bdnh cdn mia gdc rat cdn tiiilt cho qua trinh kiim dich gidng mdi, gdp phdn vdo vile san xuat gidng sgch bfnh cung cap cho cdc vimg mia nguydn lifu.
L VAT UfU VA PHU01UG PHAP NGHIEN CUU
Bdng 1. Ky hifu cdc mdu vi khudn thu thdp cho nghidn ciru
TT 1 2 3 4 5 6 7 8 9 10 11 12
13
Ky hi^u mSu Comus_BD
K84_BD QD23_BD VN85 BD
S7 BD VN84 BD
F156_DN My55_DN R0C16_TN
R570 TN VN85_TN K84_TN_SC
VN84 BD _S8
Giong mia Comus K84-200
QD23 VN85-1427 Suphanbury7
VN84-4137 F156 My55-14
R0C16 R570 VN85-1427
K84-200
VN84-4137
Cach thu mau Chiet true ti^p
11
M 11 11 11 11
II II
II tl
Nuoi c^y trfen moi truong SC Nhan sinh khoi trong moi trudng S8
Dia diem thu thap Thu Dau Mot,
Binh Duong Ben Cat, Binh Duong
\\
»
11 11
Dinh Quan, Dong Nai
11
Duong Minh Chau, Tay
Ninh
II
Tan Chau, Tay Ninh
II
Ben Cat, Binh Duong
' Trung tam Nghien cim va Phat trien Mia ducmg
^ Trucmg D?i hpc Ndng LSm Tp. HCM
^ Bp Tai nguyen va Moi trucmg
Tiln hanh thu thap mau vi khuan tir 3 khu vuc khde nhau thupc viing mia nguydn lifu Dong Nam bd (Ddng Nai, Binh Duong vd Tdy Ninh) trong nidn vu 2009-2010 nhu sau. (Bang 1):
1. Xdc dinh bfnh cdn mia gdc di^ trin trifu chiing bfnh vd mft sd d$c diim hinh thdi chinh ciia vi khudn
Ap dung theo quy trinh Koch d l tao trifu chiing bfnh diln hinh ciia bfnh cdn mia gdc bdn trong cay.
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 11/2011 41
Thi nghifm dupe tiln hdnh trong dilu kifn nhd kinh, tuang hJ phuang phap cua Davis vd ctv (1980).
Xdc dinh mpt sd dgc diim hinh tiidi chinh cua vi khuan: Vi khudn dupe thu ti-uc tilp tir dich ho$c dem nudi cdy ti-dn moi tiirdng SC (Davis vd ctv, 1980). Cd dinh mau trong dung dich glutaraldehyt 2,5%, chup anh bdng kinh hiin vi difn tir d dp phdng dgi 2.000 din 12.000 ldn.
2. Xdc djnh tdc nhAn gAy bfnh cdn mia gdc vd phdn tich tuong ddng dva trdn vimg 16-23S rRNA ciia vi khudn
Mdi rupng chpn 5 cdy/gidng cd bilu hifn bfnh cdn mia gdc, mdi gidng chpn ngdu nhidn tir 3 rupng trong ciing khu vue (tdng cpng 15 cdy/gidng/khu vue). Tiln hanh chilt dich tir ldng tiiii 3 vd 4 (tinh tir gdc) cua cAy mia bdng cdch sir dyng bom chdn khong hoAc ly tSm 5.000 vdng/phiit ti-ong 10 phiit.
Trong trudng hpp khong su dung ngay, dich mia dupe bdo qudn d -20°C vd rA dong d 37"C trude khi tiln hdnh chilt xudt ADN. Phuang phdp CTAB dupe dp dung dl chilt xuat ADN tdng sd cua vi khuan trong dich chilt, phuang phdp phenol/clorofom thuc hifn cho vi khuan nudi cay nhan tgo.
Bang 2. Trinh tif cdc cdp mdi dupe sir dyng trong nghidn Cliu
khudn lgc hinh trdn trdn mAt thgch bdt dau xudt hifn rai rdc khodng 1 tudn sau khi cdy, hdu hit dudng kinh ciia khudn lgc ndy cd kich thude nhd hon 0,3 mm; khdng mdu din trdng nhgt vd nhd lln khdi m$t mdi trudng. Khudn lgc tAng ddng kl b tudn thir 2 sau khi cdy truyin vl mgt sd lupng nhung kich thudc dudng kinh cdc khudn lgc khdng tAng ldn (hinh lb).
Bdn cgnh dd, sir dyng vi khudn tir mdi trudng nudi cdy chung ldn cdy mia sgch bfnh dupe trdng tiong dilu kifn nhd kinh cung cho trifu chiing diln hinh ciia b|nh cdn mia gdc. Trong dd, cdc ddm cd dgng hinh ddu phdy, hinh trdn vdi mdu sdc tir vdng nhgt din dd sdm xudt hifn or vi tri dai sdp khi che dpc cdy mia (hinh la).
•.J»- .^ii^A'f^^m-
\
i . ^ ^ i i . . . . . i...i..A
(a) Tric'u chirng xuat hivn tiong cay sau khi chunp brnh
0:)) Kluian lac trdn moi truOTig SC
4a
Mdi
Cxxl Cxx2
Lxl Lx2
Trinh tu mdi
5'-CCGAAGTGAGCAGATTGACC-3' S'-ACCCTGTGTTGTTTF CAACG-3' 5'-CATTGACATTGGTGCAGAGC-3' 5'-CATCCACCGTTTGCTCT TAG-3'
Chu trinh nhift cho phan img PCR vdi chu ky ddu 95°C (3 phut), 40 chu ky tilp tiieo [ 9 5 ^ (30 giay), 55''C (30 gidy) vd 72°C (1 phut)] va k i t tiiiic a 7 2 ^ (10 phiit). San phdm PCR dupe difn di tiong gel 1% agaroza vd 0,5 jig/ml ethidium pha trong dung dich TAE Ix. Gel dupe ehgy trong b l difn di cd TAE vdi hifu difn t h i lOOV ti-ong 40 phut. Kiim ti-a vd chup anh ban gel dudi tia tii ngogi. Giii san phdm PCR d l giai trinh tu tgi Cdng ty Macrogen (Hdn Qudc). K i t qua giai trinh tu nucleotit dupe phdn ti'ch, so sdnh bdng chuong trinh BLAST trdn c a s a dii U|u NCBI vd phan m i m ClutalW 2.1.
BLKFT QUA VATHAO LUAN
1. Xdc dinh bfnh cdn mia gdc dua trdn trifu chiing bfnh vd mft sd d$c diim hinh thdi chinh ciia vi khudn
Chilt dich tir cay cd trifu chirng hi bfnh cdn mia gdc, cay trdn mdi trudng SC dl or 28±0,5°C, cac ddm
(c) Huih dgng vi kliuaii (d) Huili dang vi ti-ong moi trudng SC khuan trong dich chilt Hinh 1. Hinh dgng vi khudn gdy bfnh cdn mia gdc vd
trifu chung trong bfnh
Ddng tiidi, quan sat dudi kinh hiin vi difn tii cho thd'y vi khudn cd hinh que hogc hinh chiiy vdi kich tiiudc chilu rfng khodng 0,37 0,48 um, chilu dai khoang 1,1 1,8 ]im (hinh lc & Id), kit qua phii hpp vdi nghiin ciiu vl dgc diim Lei^nia xylysuhsp. xyli ciia Davis vd ctv (1980, 1984), Comsti)ck (2002), Brumbley vd ctv (2006), Gao va ctv (2008).
2. Xdc dinh tdc nhdn gdy bfnh cdn mia gdc dya ti-ln vimg 16-238 rRNA ciia vi khudn bdng ky tiiuat PCR
Dl xdc dinh vi khuan gay bfnh cdn mia gdc d viing Ddng Nam bd, cAp mdi Cxxl/Cxx2 (Pan va ctv, 1998) Cling da dupe dp dung cho ra san phdm PCR sau khi difn di ti-ln gel 1% agaroza cd kich tiiudc khoang 438 bp or cac mau vi khuan chilt true tilp tir
42 NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 11/2011
cdc gidng mia trdng dgi tra Qiinh 2a), kit qua tuang trudng S8 vd khi chilt xuat. MAc dii vay, qua kit qua tu vdi nghidn ciru trude day cua Pan vd ctv (1998), nay cd thi so bp kit luan bfnh cAn mia gdc d viing Iglesia va ctv (2005), Gao vd ctv (2008). Bdn cgnh dd, Ddng Nam bd do Leifsonia xyli subsp. xyli gay ra.
tiep tuc tiiuc hifn vdi cAp mdi Lxl/Lx2 dupe tiiilt kl Tuy nhidn, miic dp tuang ddng vd su gan gQi giiia mdi dl xac dinh trinh tu vimg 16-23S rRNA cua cdc
mdu vi khuan thu thdp tir dich chilt true tilp vd qua nuoi cay nhan tao.
E15f? WW""." '•^''•"^^JW^^I^^
f-
ii«
Sir dung primer Cxxl/Cxx2; Ladder lOObp
Sir dung primer Lxl/Lx2; Ladder 50bp
cdc mau vi khudn cdn phu thupc vdo so sdnh giiia cac trinh tu nucleotit thu dupe vdi GenBank (bang 3 vd hinh 3).
3. PhAn tich tuong ddng giiia cdc mdu vi khudn thu th$p vd nghidn ciiu vl Leikonia xyli subsp. xyli da cdng bd trdn GenBank
MAC dil vi khudn gay b|nh cdn mia gdc dupe biit din tir rdt lAu, nhilu nghidn ciiu dA tiln hdnh khd cong phu. Tuy nhidn, kit qud nghidn ciiu d miic dp phan tii dupe cdng bd khong nhilu, thdng kd trdn GenBank cho thay cd 7 trinh tu nucleotit cua vi khuan Leifsonia xyh suhsp. xyli, bao gdm nghidn ciru tgi Brazil (AE016822), My (AF056003), Trung Qudc (EU723209) vd Uc (DQ232616, AF034641, BZ715457, BZ715458).
Sau khi khulch dgi vimg trinh tu ADN cua cdc Hinh 2. Sdn phdm PCR khulch dgi vimg 16-23S
rRNA ciia vi khudn gAy bfnh cdn mia gdc dr vimg Dong Nam bp bdng primer Cxxl/Cxx2 (a) vd primer
Lxl/Lx2 (b)
Qua dd, kit qua difn di cho tiidy san pham PCR mdu vi khudn tiiu dupe bdng primer Lxl/Lx2, kit qua ciia vi khuan tir 2 ngudn nay tirong duang nhau vd gidi tiinh tu san pham PCR cho tiiay hdu hit cdc mau khoang 363 bp (hinh 2b), kich tiiudc ndy phii hpp vdi vi khuan cd muc dp tirong ddng rat cao so vdi tiinh tir tinh todn ly tiiuylt khi tiiilt kl mdi. Tuy nhidn, band nucleotit da cong bd ti-dn GenBank vdi mA sd
(dai) san pham difn di mdu K84_TN_S8 doi khi DQ232616, AE016822, AF056003 d ty If 100% ti-dn cdc khong xuat hifn hoAc mdr, cd till chat lupng ADN mau, kl tilp ma sd AF034641 cd ty tirong ddng dr 96%
chua dam bao do qud ti-inh nhdn sinh khdi bdng moi so vdi mau My55_DN va 97% so vdi cdc mau cdn Igi.
Bang 3. T^ If tuong ddng (%) giiia trinh h^ ADN ciia vi khudn gdy bfnh cdn mia gdc dr Ddng Nam bf so vdi
TT 1 2 3 4 5 6 7
k i t qud nghi Mau thu thgp Comus BD K84 TN SC My55_DN R0C16 TN VN84 BD VN84 BD S8 VN85_BD
dn Cliu v l LeiK xmiaxUy subs Gen DQ232616
100 89 100 100 100 100 100
AE016822 100
99 100 100 100 100 100
p. xilyd& cdng bd ti-dn GenB ank Bank Accession No.
AF056003 100
89 100 100 100 100 100
AF034641 97 89 96 97 97 97 97
EU723209 81 89 83 83 69 76 83
eu733ao»
* Ghi chd- Tyli tuong ddng (%) duoc tinh ddc vf tii nucleotit thay ddi (khdng bao gdm thim hoic mdt nucleotit) sd dling chuong trinh BLAST trin NCBI
Bin cgnh dd, su tuong ddng dgt thap hon khi so sdnh vdi mA sd EU723209 vdi ty If tir 69%
(VN84_BD) din 83% ti-dn mdu My55_DN, R0C16_TN vd mau VN85_BD O^dng 3). Ddng tiidi, mdu K84_TN_SC tirong ddng rat cao (99%) vdi AE016822 (bp gien ciia Leifsonia xyU subsp. xyU) Hinh 3. Khoang cdch di truyin dya trdn trinh fa; nhung dgt 89% vdi cdc mA sd cdn Igi. Trong khi dd, nucleotit eiia Leifsonia j^/f subsp. xyUtrtn cdc gidng tiinh tir nucleotit ciia mA sd BZ715457 vd BZ715458
mia phd biin 6 vCing Ddng Nam bd so vdi GenBank da dupe nghiin cim ngodi viing 16-23S rRNA ciia
\/(>4B.« B ^ s a
- x a ^ "Tv^ mo
NONG NGHIEP VA PHAT TRIEN
N 6 N GTHON KY 1 - THANG 11/2011 43
Leifsonia xily subsp. xily da khong chi ra sir tuang ddng vdi cdc mau vi khuan thu thap d viing Ddng Nam bd (khong bao cdo sd lifu). Dilu ndy phii hpjj vdi mo phdng ly tiiuylt khi thidt kl primer Lxl/Lx2.
Dua tidn trinh ti; nucleotit san phdm PCR cua cac mau vi khuan dupe tiiu tiidp trdn nhirng gidng phd bien d viing mia nguydn lifu Dong Nam bf vd 5 kit qua dA cong bd trdn GenBank, phdn mim MEGA 4.0 dA dupe irng dung tiong vifc vd sa dd phd hf dl phan tich nidi quan hf di truyin (hinh 3). Kit qud cho thay vi kliuan thu tiiAp tgi Tdy Ninh
G^0C16_TN) va Ddng Nai (My55_DN) ndm cung nhanh tiong so dd vdi cdc mdu thu thAp tgi Binh Duong va GenBank, khdng cd sir phdn nhdnh dpc ldp cua cac mau tiong ciing khu vue, dilu ndy cho thdy sir sAp xIp cd till do ngdu nhidn. Trong khi dd, mdu vi khuan VN84_BD tiiu tiiap tgi Binh Duong hinh thdnh gdn gui vdi 4 mau thudc 3 khu vi^c khde nhau va GenBank. Bao gdm mau Comus_BD vd VN85_BD tiiu tiidp tgi Binh Duang, mdu R0C16_TN d Tdy Ninh vd mau My55_DN tgi Ddng Nai. Dilu nay cho thay khdng cd su gidi hgn rd rft vl khoang cdch dia ly giira cdc mau thu thap. Ngodi ra, sir dpc lap vd khoang each xa ciia mau K84_TN_SC thu tir Tdy Ninh qua phdn lap trdn moi trudng thgch SC vd mdu VN84_BD_S8 dupe nhan sinh khdi trdn moi trudng ldng S8 cd khoang cdch xa hon so vdi cdc nhdm mau dupe chilt true tilp tir dich mia, mAc dii mdu VN84_BD_S8 cd ciing ngudn gdc tir gidng VN84- 4137 tgi Bin Cdt, Binh Duong.
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D Q 2 3 2 S 1 0 A E O 1 0 O 2 2 R O C I S I N V N B S DD H y S 5 _ U N
D Q 2 3 2 6 1 f t A E 0 1 6 a 2 2 R O C I S I N V N 0 5 B D H y S S D N
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Hinh 4. So sdnh trinh tir ciia mdu vi khudn gdy bfnh cdn mia gdc tgi vung Ddng Nam bf vdi trinh ta;rbd gien (ma sd AE016822) vd vf til vimg 16-23S (mS sd
DQ232616) ciia Leifsonia xyh'suhsp. xyli., sir dung phdn mim CLUSTALW version 2.1. Ddu sao (*) chi
vi tri tuong ddng ciia tdt cd cdc mdu so sdnh
Tiln hdnh, khulch dgi sdn phdm PCR ciia 3 mdu vi khudn tiiu tiidp <5gi difn tir Tdy Ninh (R0C16_TN), Binh Duong (VN85_BD) vd Ddng Nai (My55_DN) bdng c$p mdi Lxl/Lx2. Dem so sdnh trinh far ciia 3 mdu ndy vdi bd gien (mA sd AE016822) vd vi tri viing 16-23S rRNA (mA sd DQ232616) cua Leifsonia xyli subsp. xyh dSi cdng bd trin GenBank (hinh 4).
Kit qud b hinh 4 cho thdy trinh tu nucleotit cua mdu vi khudn tiiu tiidp tgi Tdy Ninh (R0C16_TN) vd Binh Duong (VN85_BD) vdi chilu ddi 315 bp gidng hodn todn vdi trinh ti; chudn tir Genbank dimg tiong so sdnh. Trong khi dd, trinh tu ciia mdu thu thdp tai Ddng Nai (My55_DN) cd 6 diim khde bift b doan ddu vd cudi trinh ty. Trong dd, mdu My55_DN thim 1 nucleotit C tgi vi tri 27 vd 1 nucleotit G or vi tri 311.
Ddng tiidi, d vi tx\ nucleotit 2-3 bi hodn ddi CT tiidnh TC, ti-ong khi vi tin nucleotit 306-307 bi tiiay till AA thdnh CC. Tuy nhiln, dogn trinh tu tir vi tri nucleotit 28 din vi til 305 (ddi 277 bp) cua mdu My55-14_DN nhu hinh 4 cho thdy cd sir tuomg ddng tuyft ddi voi tdt ca cac trinh tu so sanh. Cimg vdi cac kit qua nghiin ciiu dA thao ludn or trin, cd thi khdng dinh Leifsonia A/TK subsp. jici^cung chinh la tdc nhdn giy bfnh cdn mia gdc d viing Dong Nam bp.
IV. KET LUAN
Vi khudn gdy bfnh cdn mia gdc or viing Dong Nam bp cd hinh que vdi kich thudc chilu rdng khodng 0,37 - 0,48 pm, chilu dai khoang 1,1 1,8 iim, phii hpp vdi cdc kit qud da nghiin cOru trin thi gioi vl Leifsonia xyh'suhsp. xyli.
Sir dung ky tiiudt PCR vdi c^p mdi Lxl (5'- CATrGACATTGCTGCAGAGC-SO va Lx2 (5'- CATCCACCGTrrGCTCTTAG-3') da xdc dinh dupe Leifsonia AyZr'subsp. A'y//trong trong cay mia. Mau vi khudn gdy bfnh cAn mia gdc thu thgp tgi Ddng Nam bp cd ty If tirong ddng cao (89 din 100%) vdi tiinh tir nucleotit cua Leifsonia xyh snhsp. jr^/fd Brazil, Uc va My. Ty If nay tiidp han khi so sdnh vdi kit qua nghiin ciiru a Trung Qudc, chi dgt tir 69 89%. Tuy nhiln, khdng thi hifn r6 str khde bift vl gidi hgn dia ly giiia cdc mdu vi khudn nghiin ciiu cung nhu so sdnh vdi cdc cdng bd trin GenBank.
TAlUfUTHAMKHAO
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2. Davis M. J. and Bailey R A. (2000). Ratoon stunting. In: A guide to sugarcane diseases (Rott P., Bailey R A., Comstock J. C , Croft B. J. and Saumtally A. S.). CIRAD/ISSCT.
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nov., subsp. nov. and Clavibacter xyU subsp.
cynodontis subsp. nov, pathogens that cause ratoon stunting disease of sugarcane and bermudagrass stunting disease. International Journal of Systematic Bacteriology, 34:107-117.
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A, Cole J. R and Tiedje J. M. (2000). Leifsonia poae gen. nov., sp. nov., isolated from nematode galls on Poa armua, and reclassification of ' Corynebacterium aquaticuni Leifeon 1962 as leifsonia aquatica (ex Leifson 1962) gen. nov., nom. rev., comb. nov. and Clavibacter xyli Davis et al., 1984 with two subspecies
as Leifsonia xyh (Davis et al., 1984) gen. nov., comb.
Nov. International Journal of Systematic and Evolutionary Microbiology, 50:371-380.
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7. GaoS.J., Pan Y. B., Chen R K, Chen P. H., Zhang H., Xu L. P (2008). Quick detection of Leifsonia xyli subsp. xyh by PCR and nucleotide sequence analysis of PCR amplicons from Chinese Leifsonia xyU subsp. xyh isolates. Sugar Tech. 10:
334-34.
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Clavibacter xily snhsp. xily. The causal bactieium of sugarcane ratoon. Plant Disease. 82: 285-290.
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USING PCR FOR THE DETECTION OF Leifsonia xylisuhsp. xyh, THE CAUSAL BACTERIUM OF SUG/UiC/VNE RATOON STUNTING DISEASE (RSD) IN THE SOUTH-EAST
Ha Dinh Tuan, Le Dinh Don, Bui Cach Tuyen Summary
A polymerase chain reaction (PCR) assay was developed for detecting fjeifsonia xyli subsp. y.yli from fibrovascular fluid of infected sugarcane plants and from artificial culture during 2009-2010 season. Ihe primers were Lxl (S'-CATTCACATTGGTGCAGAGC-S') and Lx2 (S'-CATCCACCGTrTGCTCT TAG-30.
The thermocycler parameters were denaturization at 95°C for 3 min, 40 cycles at 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, and final extension at 72°C for 10 min. Generic PCR products from the intergenic transcribed spacer (ITS) region of 16S-23S ribosomal RNA of Leifsonia Ay/r subsp. xyli'x's, approximately 363 bp. Based on a multiple sequence alignment, it showed highly homologous sequences from the database on GenBank, thus confirming occurrence of Leifsonia AFZT'subsp. xyli, the causal agent of sugarcane ratoon stunting disease (RSD) in the South-East of Vietnam.
Keywords: ArtiBcial culture. Sbrovascular fluid. PCR ratoon stunting disease (RSD).
Ngudi phan bifn: PGS.TS. Nguyin VSn Tudt
