VietnamJ.A^ Set 2016. Vot14. No. 9:1312-1322 TgpchiKH NGngnghi§pVi$tNam2016. t * 14.s69:1312-1322 www.vnua.edu.vn
BAC TRUNG PHAN TUT NAM IHyeosphaerella beiKeleyl GAY BENH BOM DEN LAC TAI NGHE AN
Ngo Thi Mai Vi'", Ha Giang\ Tran Thi Nhw HoaS Nguyin VSn Vien^
'Khoa Nong lam ngir, Tru&ng dgi hgc Vinh
'Trung tam Nghien ciru Benh cay Nhift d&i, Hgc vien Nong nghifp Vift Nam 'Khoa Nong hoc, Hge vien Nong nghiep Vift Nam
Email: maivitp-(@yahoo.com
Ngay gui bai: 05.09.2016 Ngay chfip nhan: 30.10.2016
TOM TAT
Nghign ciru nfiy dfi Ifin dfiu tien xfic djnh duvc trinh ty viing ITS (Internal Transcribed Spacer) cOa 11 mfiu nfim dfim den thu thfip trgn Igc nfim 2013, chu ydu tgi tinh Nghg An. Trinh tif gen ITS cua tfit cfi cfic mSu nfim dfiu ddng nhfit 100% vfli nhau vfi ddng nhfit tfl 99.7 - 100% vd'l lofii tAycosphaarella bert<eleyHt^n GenBanl(. K9 thufit PCR diing mdi thifit kd trgn cfic chudi Ifip (Rep-PCR, repetitive sequence primed PCR) vo'i 3 bfi mdi duKyc thifit kfi trfin cfic chudi Ifip cua vi khufin gdm chuii Ifip ddi song viing khflng mfi hfla (REP, repetitive extragenic palindromic), chudi Ifip bfio thu vung lifin gen (ERIC, enterobacterial repetitive intergenic consensus) vfi chudi BOX cQng dfi dupc si>
dgng dfi dfinh gifi mirc dfi da dgng di truyfin cua 33 mfiu nfim ddm den thu thfip trgn Igc, chu ydu tgi tinh Nghg An.
Phfin flng PCR diing bfl mdi BOX da tgo nhidu bfing sfin phfim da hlnh. Phfin tich Rep-PCR da chflng td qufin thfi nfim ddm den khflng da dgng vfi di truyfin. Khflng cfl mdi quan h# giOa cfic nhflm nfim duvc xfic djnh dya trSn phfin tich Rep-PCR vfi ngudn gdc thu thfip mfiu cung nhu dfic difim hlnh thfii.
Tfl khfla: Mycosp/iaerete bericetey;, ddm den Igc, Nghg An, Vigt Nam, Rep-PCR, ITS.
Molecular Characterization of Mycosphaerella berkeleyi Causing Late Leaf Spot of Groundnut in Nghe An
ABSTRACT
This study identified the ITS (Internal Transcritied Spacer) sequence of 11 groundnut late leaf spot fungal isolates collected from Vietnam, mainly from Nghe An province in 2013. The ITS sequences of the 11 isolates were identical among them and highly identical (99.7 and 100%) with two available isolates of Mycosphaerella berkeleyi on the GenBank. Rep-PCR technique (Repetitive sequence primed PCR) with 3 sets of primers designed on the repetitive sequences of bacterial genome including the repetitive extragenic palindromic (REP) sequence, enterobacterial repetitive intergenic consensus (ERIC) and BOX element, was used to investigate the genetic diversity of 33 M. berkeleyi isolates. PCR using BOX primers (BOX-PCR) produced polymorphic bands from tested fungal Isolates. Rep-PCR analysis proved that fungal populations in Nghe An were of low genetic diversity. There was no correlation between Rep-PCR defined fugal groups with the origin ot the isolates (location of sampling.
groundnut cultivars) and morphological characteristics of the isolates (color and size of cultures) Keyvrards: Mycospliaerella berkeleyi. late leaf spot, groundnut, Nghe An, Vietnam, Rep-PCR, ITS.
1. DAT VAX DE ' ^ ' " ^ ' ' 'rong cac benh hai la nguy hi^m nhfi't doi vdi cay lac tren tofin the' gidi. B$nh dom den hgi Benh dom den do nam Phaeoisariopsis Igc cd the lam giam tdi »Q\ nfing sufi't lgc personata (giai dogn vd tfnh) hay (Grichar et al, 1998; McDonald et al., 1985;
M.vn>spbaereUa berkeleyi (giai doan hflu tinh) Miller et al, 1990).
1312
IJli;
Ngo Thi Mai W, Hfi Giang, Trfin Thi Nhu Hoa. fgguyin VSn Vien
Tren ddng rupng, nfiin tao ca hai giai doan smh san vd tinh vfi hflu tinh nhung hfio tu phan smh hinh thfinh tfl tan du tfin tai trong dtft la nguon benh quan trpng nhfit. Nhin chung, nfim dfim den khdng truygn qua hat nhung dUdc xem lfi tac nhan truyfin qua dfit. Khi bfit (Mu vu trong, bfio tu phan sinh nfim tfl tan du trong dat sg nhigm cac la phia dudi va nhanh chong phat tan len cac la phia tren vfi ed t h i gay tan lui bd la neu dieu kien ngoai canh thufin' Idi (McDonald et al., 1985).
Su da dang vfi cac dfic die'm sinh hoc cung nhu di truyfin cua nfim dom den ndi rieng vfi tac nhan gay benh eay noi chung cfin phai dUdc nghign cflu trude khi ap dung cac bign phap li phong chong. Cac chung nfim khac nhau ve nfin
* di truygn cd t h i cd phan flng mfin cam khae j # J i h a u dfii vdi thufie hoa hpc eiing nhu khfie nhau r "f""^ ^*y ^"^ t'^n <:^e gifing cay (Adiver et
^ a i , 2009).
if Nghign eflu vfi nfim dfi'm den h a i lae tai An 1 P . t ' * " ^ " • ^ " P ' ' ^ " "'^'^ ^ ^ D vfi isozyme
* (Adiver, 2008) cho thfiy nfim dfim den ed da dgng ve di truyfin vdi hg sfi' tuong dfing tfl 77 94% vfi mfle dp da dang di truygn ed lign quan den phan bfi dia I^r cfla mfiu nfim thu thfip.
Tgi Vi^t Nam. da dgng di truyfin nfim dfim den hai Ige chUa dupc nghign cflu. Trong nghign cflu nfiy, Chung tdi da ap dung mdt kj? thu^t phfin tfch da dang tUdng doi ddn gian (chi dua tren PCR) de tim hilu mflc do da dang di truyfin
» cua qufin the nfim dom den tai Nghg An lfi kv
" thu$t Rep-PCR.
>
Kp thufit Rep-PCR dflng mfii thifit ke trgn . cfic chufii Ifip (Rep-PCR, repetitive sequence
primed PCR = repetitive element - based PCR) dfi_ dupc flng dvng nhieu do cd Uu dilm Ifi ddn gian (ehi dua trgn PCR) vfi tin cfiy. Ky thufit nguygn ban su dyng cac mfii dUde thifit ke dua tren cac chudi Ifip trgn hp gen vi khufin nhu eac ehudi Ifip ddi song vflng khdng ma hfla (REP repetitive extragenic pahndromic) ed kfch thud<J fpD.n'' ''"• "^^ ''''"^' '*" ^^° ' • ' " ^ " e 'i«° een (liKlC, enterobacterial repetitive intergenic
«msensus) efl kich Uiudc 124 127 hp, chufii BOX cd kich thudc 54 bp. Phan flng PCR su dflng cfie mdi nfiy dupe gpi cu t h i lfi REP-PCR
ERIC-PCR vfi BOX-PCR (Rademaker ef aL, 2004). Mac dfl Gillings vfi HoUey (1997) dfi chflng minh rfing cac chudi Ifip bao thu vung lien gen khfing cd d bd gen vi sinh vat nhan chufin nhung ky thufit Rep-PCR cd t h i dudc ap dung d l n ^ e n cflu da dang nhieu lofii nfim gay benh cay nhu Exerobilum turcieum (Muiru et aL, 2010), Bhizoctonia solani (Matsumoto, 2014;
Matsumoto vfi Cuong, 2014; Toda et al., 1999), Verticillium dahUae (Komatsu et al., 2001), Fusarium spp. (Ebadi et al., 2014; Gurel et al.', 2010), Cercospora canesceas (Ferrater, 2003),' Macropbomina pbaseolina (Purkayastha et al'., 2008), thfim chl cd t h i flng diing d l phan loai nfiin d mflc loai (AbdoIIahzadeh vfi Zolfaghari, 2014; Palencia et al., 2009).
Ngoai ra, dinh danh phan tii nfim dom den hai lac cung chua dUdc nghien cflu nhilu trdn thg gidi ngoai trfl trinh tu vflng hen gen ITS (mtemally transcribed spacers) eua cum gen rDNA cua 2 mfiu phfin Ifip tfl My vdi mg GenBank AY266147 (Stewart et al., 1999) vfi tii Cpng hda Trinidad vfi Tobago vdi mfi GenBank AB435066 (Kurose et al., 2009).
Hien nay, vflng gen TTS lfi mot trong cac vflng gen phfi bifih nhfit d l nghign cflu da dang vfi xac dinh nhilu loai nfim. Cac vflng ITS do efl tfib dfi dot high nUn Chung tUdng flng vdi tdb dd biet hfla lofii (tifi-n hda bning tinh) ngn cd t h i phan bi6t mfii quan he tdi mflc lofii. Hign nay vflng ITS dupc xem lfi "ma vach - hareoding" dfii vdi nfim (Schoeh et al., 2012).
Mue tieu cua nghign cflu nfiy Ifi dinh danh phan til nam dom den hai lac cua Viet Nam dua trgn^vung TTS vfi dfinh gia mflc dd da dang di truyen cua nfim dUa tren phan ttch Rep-PCR.
2. VAT U E U VA PHUONG PHAP 2.1. Mau naxn
Nfal dfi'm den hai lae dudc phfin Ifip bfing each cfiy ddn bfio tii tfl vfit benh thu thfip trgn lac. Mfiu la lac CO vgt benh diln hinh dudC nia s^ch bfing nude vdi, sau dd bfing nude cfit vd
t ^ '
?v w"^^
^""^"^ '^° •-'
'^^-Si
trUdng VV.A ^(water agar) up ngupe. Vdi hd trd cua kinh hien vi, cac ddn hfip tii dUdc chuyin
Dfic tnmg phfin t f l nfim fcf)«jsphaere«aber*etey) gay benh ddm den Igc tgi Nghg An
bfing kim thuy tinh sang dia mdi trfldng WA mdi. Sau 2 ngay, bfio tii nam dang nay mam dfldc chuyin sang mdi trfldng PGA bd sung dich chiet la lac de dfldc mfiu nfiin thufin.
2.2. Chiet DNA nain
Khoang 50 mg tan nfim thufin dUdc nghien bfing chay nhua chuyen dung (Kontes™ Pellet Pestle) vdi 0.5 mL dem CTAB Doyle & Doyle (1987) trong fing Eppendorf loai 1.5 mL. DNA dUdc chiet 2 Ifin vdi chloroform: isoamyl alcohol (24:1). Cfin DNA dfldc rfla 2 Ifin bfing ethanol 70% va hda trong 50 uL nfldc cat 2 Ian vd triing.
Mau DNA dfldc bao quan d -20=C.
2.3. PCR va giai t r i n h tfl
Hai mfii ITS4 vfi 1TS5 (White et al., 1990) da dUdc sfl dung dg nhfin tofin bfi vung lien gen ITS cua cum gen rDNA ciia mfiu nam. Phan flng PCR dflpc thuc hifn vdi DreamTaq Polymerase cua hfing Fermentas vdi nhiet dp gfin mfii d 52°C. San phfim PCR dupc tinh chiet tfl gel agarose dflng kit tinh chiet PureLinkTM Quick Gel Extraction Kit (Invitrogen) theo hfldng dfin eiia nhfi san xufi't. Hfim lupng DNA dupe Ude lUi^ng nfing dp bfing difn di agarose. San phfim PCR dupe giai trinh tu trflc tiep 1 chieu dflng mdi PCR tgi hang Macrogen (Hfin Qufic). Trinh ti^ nucleotide dupc bien tfip vfi Ifip rap dung phfin mem Seqman (DNASTAR, LaserGene).
2.4. Phan tich t r i n h tfl
Di^a tren cfie trtnh tu thu dupc, viee tim kifim trgn ed sd dfl ligu Genbank bfing dflng phfin mfim true tuyfin BLAST tai NCBl (the National Center for Biotechnology Information) (http://www.nebi.nlm.nih.gov/BLAST/). Phan tfeh trinh tfl dupe thuc hien dflng cfic phfim mem
ClustalX (Larkin et al.. 2007) vfi MEGA 6.0 (Tamura et al., 2013)
2.5. Rep-PCR
Ba loai phan iing Rep-PCR. REP-PCR, ERIC-PCR vfi BOX-PCR da dfldc thUc hign vdi cac mfii Rep-PCR dfldc trinh bay d bang 1. Mdi phan flng Rep-PCR cd tong the ttch 15 pJL chfla 4,5 ^L nfldc sieu sach (Invitrogen), 7,5 (il GoTaq Green Master Mix (Promega), 2 jiL DNA, 0,5 pL mdi loai mdi REP IR vfi REP 21 (ddi vdi REP- PCR), 0,5 jiL radi loai moi ERIC IR va ERIC -2 (ddi vdi ERIC-PCR) vfi 1 pL mfii BOX AIR (do!
vdi BOX-PCR). Cfic moi dfiu dUdc chufin hi d ndng dp 20 (iM.
Cac phan flng Rep-PCR dfldc thflc hien tren may PCR PTC-100 (MJ Research Inc.) vdi dieu kien sau: khdi dfiu bien ttnh d 94°C trong 4 phut; tigp theo Ifi 35 chu trinh phan ling gfim bign tfnh d 94°C trong 1 phut, gfin moi d 50°C (BOX-PCR vfi ERIC-PCR) va 40''C (REP-PCR) trong 1 phut, tong hdp sdi d 72°C trong 3 phiit Phan ling dflpc ket thue vdi 5 phut d 72°C.
San phfim Rep-PCR dupe didn di trdn gel agarose 1% dUde chufin hi bfing dfm TAE vfi chfla 0,5 mg/mL ethidium bromide. Gel dflijc chay trgn thifit hi didn di Mupid-exU Mini System (Helixxtec) vdi dem TAE d dign t h i 100 V trong 30 40 phflt. Ban gel dflpc kilm tra dudi anh sang tfl ngogi vfi dfldc chup bfing may anh sfi.
Tfi't ca cae bfing xufit hien trdn ban difn di ba't ke dp sang deu dUdc ghi vfi chuyin sang dang so lieu nhi phan (0 vfi 1) d l Ifim so lieu diu vfio cho phan tfch da dang dflng phfin mim NTSYS pe 2.0. Do bd gen nfim dom den lgc tt ddn bdi vfi Rep-PCR thudc nhdm marker tr§i
Mil
Bang 1. Cac moi dfldc siif dung trong Rep-PCR
Trinh t l / (5'-3') Tham Khfio BOXA1R CTACGGCAAGGCGACGCTGACG
ERIC 1R ATGTAAGCTCCTGGGGATTCAC ERIC 2 AAGTAAGTGACTGGGGTGAGCG REP IR IIIICGICGICATCIGGC
_5EP2I ICGICTTATCIGGCCTAC
Vetsalovic e t a l , 1994 VersalovK etal. 1991 Veisalovic et al, 1991 Vereaknicstal. 1991 Versalovic6(a{,1991
1314
Ngfl Thi Mai W, Ha G i a i g . Trfin Thj N h u Hoa, N g u y i n Vfin Vifin
nen hg so phu hfl, ddn gifin SMC (simple matching coefficient) dude sii dung d l tfnh he so tfldng ddng theo cong thflc sau: S (hg so tiidng dong) = (a-M)/(a+h+c-Nl); trong dfl a la so bang giong nhau cua 2 mfiu, b Ifi so bfing chi xufi^t hipn d mfiu thfl i, c i a so bfing chi xufit hien d mfiu thfl j , c la so sfi bang khdng xufit hign d ca 2 mfiu (nhung xufit hign d cac mfiu khae). Phan tfch cum dupe thuc hien theo phUdng phap ghep efip mfiu dung khoang each trung binh so hpc i-il ngang bfing (UPGMA, Unwaited Pair Greup ul Method using Arithmetic Averages)
111!
»* 3. KET QUA VA THAO LUAN
.^ 3.1. Xac dinh cac mfiu n&n doin den la lac J t h " tai Nghe An bang giai t r i n h trf vung ITS P | k Trong nghien cflu nfiy, 8 mfiu nfim dom den P T ^ h a n lap tfl bfin vflng sinh thai cua tinh 1^ N | P A n (dfing bfing ven biln, dfing bfing, trung U; du vfi miln nui) cung nhu 3 mfiu nfim dfi'm den
„,, l^e thu tai Lfio Cai, Hfi Tinh vfi Dfing Nai dfi
^ dfldc gifil trinh tu true tig'p vflng ITS tfl san ,^ phfim PCR (Bang 2). Sau khi Ifip rap vfi loai bd
^ cac doan nhiiu d 2 dfiu, cac mfiu nfi'm diu cd
^ kich thudc doan doc dupc tfl 441 - 496 bp (Bang 2). Trinh tu cae doan dpe dUde bao phu vflng
"; ITS!, 5.8S vfi ITS2 cua chudi ITS, cho phgp xac d)nh ehfnh xae nfi'm dfl'm den tdi mfle lofii 1 (Goodwin et al, 2001).
Dfiu tien, trinh tu doc dudc cua 11 mfiu dfldc sii dung di tim kie'm cac chudi gfin giii tren GenBank. Ket qua tim kiem (BLAST SERCH) cho thay ta't ca 11 mfiu deu trflng khdp vdi 2 mfiu Af. berkeleyi duy nhfi't ti-en GenBank (ma GenBank AB435066 va AY266147). Dang chu y, ket qua tim kiem cung cho thfiy hien nay mdi chi ed 2 trinh hi vung ITS cua nfim dfim den lac trdn GenBank.
Tiep theo, trinh tu virng ITS cua 11 mfiu nain dUdc so sanh vdi nhau va vdi 30 trinh tU vung ITS eua cac mfiu nfi'm Mycosphaerella dai dien sfin co tren GenBank, k l ca 2 mfiu M.
berkeikeyi (AY266147 va AB435066). Cac mfiu nfiin tren Genbank nay Ifi cac mfiu chufin da dupe cdng hd'(Goodwin et aL, 2001) (Bang 3).
Kit qua so sanh trinh tu cho thfiy trinh tii vflng ITS cua 11 mSu nfim deu dong nhfit 100%
vdi nhau chflng td ehung diu lfi thfinh vien cua cflng 1 loai.
Khi so sanh tiinh tu cua 11 mfiu nfi'm trong nghien cflu nfiy vdi trinh tu cua 30 mfiu nfi'm GenBank thfiy chung eo mflc ddng nhfi't rfi't eao, 99,7 100%, tUdng flng vdi 2 mfiu nfi'm M berkeleyi, AY266147 vfi AB435066, sfin cd tren GenBank. Tfi't ca 11 mfiu nfi'm trong nghign cflu nfiy dgu ed mfle ddng nhfit trinh tu thfi'p hdn nhifiu, tii 69,2 94,2% ddi vdi 28 mfiu nfi'm GenBank edn lai (Bang 3).
^^^^^:^^t^^::^}^^^^±^^^^!^^^-^^ ^ ^ - « » ™a"u nfim dom den lac
MS mdu NguAn gic mSu nfim
Dia ai^m thu thip NA4,3
NA12.1 NA21.1 NA25.1 NA30.1 NA32.1 NA33.1 HT1.1 NA37,1 LC2 ONI
Nghi Th«. Nghi L«c. Ngh* An Nghi Xfi. Nghi L«c. Ngh« An Diin Thfinh. Diin Chfiu, Ngh| An Nam Thifong. Nam Din. Ngh$ An Ngoc Son, Thanh Chuong, Ngh# An Lmj San. DO Luong, N g h | An Binh Som. Anh Son, Ngh| An CdDam, Nghi Xuin, H I TTnh Tam Quang, Tirerng Dirong, Ngh| An T l Ph«, ihlnh ph4 Lio Cai Hung Thinh, Tring Bom. Ddng Nai
Gidng Viing sinh thii M l giii trinh tv L14
L14 SenNA
L14 L14 L14 L14 L14 Sen Lai
L14 L14
Odng bing ven biln Ddng bing ven biln OAng bing ven biln
Ding bing Tnjng du Trung du Miln nui Odng bing ven biln
Miln nui Miln nui Tnjng du
17B4ZAB024 17B4ZAB025 17B4ZAB026 17B4ZAB027 17B4ZAB028 17B4ZAB029 17B4ZAB030 17B4ZAB031 17B4ZAB032 17B42AB034 17B4ZAB035
Kich thu*c doan dgc du'o'c (bp) 441 493 478 464 466 482 473 486 493 496 468
Dfic mmg phan ti> n ^ Uycosphaerela berkeleyi gay benh ddm den Igc tgl Nghg An
Phan tt'eh pha he dfla tren trinh tfl viing ITS cung cho thay 11 mfiu nfiin trong nghien ciiu vfi 2 mfiu nain M. berkeleyi s i n cd tren GenBank d tren hinh thanh mfit cum loai ro ret, vdi gifi tri thfing ke boostrap 100% vfi tach biet hfin cae nfiin Mycospbaerrella khac (Hinh 1).
Kit qua so sanh trinh tfl va phan tich pha hd vung ITS da chflng td ta't ca 11 mfiu nfim
dfi'm den lac thu thfip tai Nghe An, Hfi 'finh.
Lao Cai vfi Ddng Nai Ifi thanh vign cua lofii M.
berkeleyi. Nghien cflu nay ciing chflng minh trtnh tfl vflng ITS ra't bao thii trong lofii M.
berkeleyi vfi do do khdng t h i sul dung trong phan ttch da dang nhitng lai lfi ehi thi phfin tu rfit tfi't de dinh danh n ^ dfi'm den lgc U.
berkeleyi.
Bang 3. So sanh trinh trf vung ITS cua 11 mau nfijn dom den lac vdi cac najn Mycosphaerella
Tin gjal dogn v6 tinh Ten giai dogn hOu tinh M l GenBank Mi>c ddng nhit trlnh ti/(%) 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Phaeosariopsis perstmata Phaeoisariopss personata Dottiistroma septospora Cenxkspora arachkJcola Chi/a xlc djnh Chua xlc djnh
Cenx}spora sarghi var. maydis Cercospora nicotianae (Cercospora zeae-maydis Cercospora asparagi Cercosptxa t)eticola Cercospora kikuchii Cerxspora sorghil Paracercospors fijiensis Myct)veloslella tesmaniensis Astert>meSa brassicae Cercospora kalmiae Ps0udoc6rcospora musae Ramularia brunnea Remularia tmjnnea Uwebraunie elUpsoklea Chua xlc djnh Paracetcospora ttjiensis CoMogtoeopsis moUehana Seploria trttci
Lecanosdbia acKola Uwebraunie juvenis Cladosporium nlis Cladosporium ettt^cepee Ctedospofium hertiarum
Mycosphaerella berkeleyi Mycosf^aerella berkeleyi Mycosphaerella pini Mycosf^aerella arachidis Mycosf^aereHa ahicana Mycosphaerella keriiensis Chira xdc djnh
Cht/a xdc djnh Chi/a xdc djnh Chira xdc djnh Chira xdc djnh Chira xdc djnh Chira xdc djnh Mycosphaerella fijiensis Mycosphaerella tasmaniensis Mycosphaerella brassk:icola Chira xdc djnh
Mycosf^aerella musicola Mycosphaerella fragariae Mycosphaerella fragariae Mycosphaeratta ellipsoidea Mycosphaerella marksii Mycosphaerella fijiensis Mycosf^aeralla molleriana Mycosphaerella graminicola Mycosphaerella dearnessii Mycosphaerella juven^
Mycosphaerella macrospora MycosphaereBa allii-cepae Mycosphaerella tassiana
AB435066 AY266147 AF013227 AF297224 AF173314 AF173300 AF297233 AF297230 AF291709 AF297229 AF297222 AF291708 AF291707 AF181705 AF173307 AF297227 AF297226 AF181706 AF173312 AF297235 AF173302 AF173316 AF297234 AF173301 AF181694 AF260818 AF173299 AF297231 AB026160 AJ238469
100,0 99,7 94.2 93,6 93,3 93,3 83,4 83,3 83,1 83,1 82,9 82,9 82,9 82,6 82,6 82,5 82,2 82,1 81,0 79,9 78,4 77,7 77,4 76.2 74,8 74,5 74,4 70,0 69,6 69,2
1316
Ngo Thi Mai Vi, Ha Giang, Trfin Thj N h u Hoa, Nguygn V S i Vien
i
100
>NA33.1
*'Aef*Bteyi-AY266147 INA25.1
tNA30.1
• NA12.1 MA211 M'>erte(EyMB4350e6
• L C 2 NA4.3 DN1
• NA371 Z H T I . I ' • N A 3 2 1 I — Dp»»-AF013227 5 9 1 , Mafncan3^AF173314
78^— M*eo/iBnsis.AF173300
MdhamessiAAF26D818 - Mell|)so(llesAF173302
-Mmar*sr*V\F173316 94
- CkairriaeJf297226 M/|iiBnsl»AF181705
Mfnswafa-AF181706 M«asman«ns»AF173307
M.brassia)bAF29722r
M l C ftfachMF291708 Z5fCso,gh>AF291707
'— Czaa&v7Bnfs^AF291709
"551 |CasipafagfAF2g7229
• » M Csois^ m^rtis.AF297233 9 5 ] Cft«lfcofa-AF297222
' Cncafianae-AF297230 MI|en9s.AF2g7234 Mgrana]cal^^F181694 i O O j M ftBganaeAF297235
'— MftBgana&AF173312 M 'n]a9nan3.AF173301 Mjiuvon<sAF173299
C afHxpseABOZeieO
0.02
r — - . C a t K t p a e A B O a T o o L l *<'assBna.AI2384e9
97" *«macn>^poraWF297231
Hinhl. Phan tich pha hddi^a trgn trinh ti^ toan bo vung IIS ciia ll™a- - . , - . . tHut,i Vidt Nam(dfinhd.„h^gchfiin den) v f i c . c ^ ? i Z l r t r G ? r ^ r ' ^ ' =
^^•^'^'trongl.OOOUnlfptMTSOUncictai.ap^^'lTTnl'r'"" '^""'^'"'''^
S.2. Phan tich Rep-PCR
3.2.1. Nguon gai: va dac diim binb thai cac m i u niiin dom den
Tfing ser 33 mfiu nfim dfi'm den da dUde phan lap tfl lfi lac b^nh cua 4 gifing, LI4, L26, Sen Lai vfi Sen Ngh? An (Sen NA), h^ng vu xufin nfim 2013 tai 4 tinh g^m Dfing Nai (2 mfiu), Nghg An (29 mfiu), Thanh Hda (1 mfiu), U o Cai (1 mfiu) Cfic mfiu nfim (Bang 4), sau khi duijc phan Ifip
thufin, da duoc nudi cfiy tt^n mdi taifdng PGA bo sung dich chiet la lac cho dfinh gia dfie dilm hlnh thai.
Tfit ea cac mfiu nfim dfim den diu sinh trfldng rfit chfim vdi dudng kinh tan nfim trung bmh tii 3.0 - 13,5 mm. Tan nfim eua efie mfiu nfin cd mfiu tfii, tfl mfiu xam tdi den (Bang 4) Ket qua nfiy chflng td cac mfiu nfim dfim den lac kha da dang v l dfie dilm hinh thai cung nhu tdb dp smh taudng b^n mfii trudng nhan tao.
Elfic tnmg phfin ti> nam Mycosphaerella berkeleyi gfiy bgnh ddm den lgc tgi Ngng An
Bang 4. Nguon goc va dac diem hinh thai ciia 33 mau najn dom den hai lac thu thap n a m 2013 trong phan tich Rep-PCR
TT KH Miu Oja dilm Gidng Vung sinh thii
DiKmgkinh tin nim
(mm)'
Mlu sic tin nim 1 ONI Hung Thinh. Tring Bom. Bdng Nai
2 NA13.1 Nghi Thu. Nghi L§c. Nghe An 3 NA17.1 1 DUn Hoa. Diln Chfiu. Nghe An
» NA17 2 DilnHoa. Diln Chiu. Nghe An 5 NA9.5 Nghi Phong. NgN L^c. Nghe An 6 NA11. t Nghi Hoa. Nghi LOc. Nghl An 7 NA20.3 CHin Phuc. Diln Chfiu. Nghl An 8 NA21.1 Diln Thinh, Diln Chiu. Ngh$ An 9 NA3 2 Nghi Khinh. Nghi L4c. Ngh$ An 10 NA4.3 Nghi Thir, Nghl L«c, Nghl An 11 NA12 1 NghiXI. Nghi L4c. Nghl An 12 NA14.3 Nghi TNnh, Nghi L*:, Nghl An 13 NA5.3 Nghi Dire, Nghi L|c. Ngh| An 14 NA6,4 Nghi Xuin. Nghl LOc. Nghl An 15 NA3.4 Nghi Khinh, Nghi LOc, Nghl An 16 NA7 5.1 Nghi Tn/emg, Nghi LOc, Nghl An 17 NA14.1 Nghi Thinh, Nghi LOc, Ngh| An 18 NA19.1 Diln Thjnh, Diln Chiu, Ngh| An 19 NA20 3 DilnPhuc, Diln Chiu. Nghl An 20 NA21.2 Diln Thinh. Diln Chiu. Nghl An 21 NA25.1 1 Nam Thmjmg, Nam Din. Ngh| An 22 NA23 1 Nam Hi/ng, Nam OIn, Ngh| An 23 NA26 1 Nam Hi/ng. Nam OIn, Ngh| An 24 NA31 1 Thanh Lucmg, Thanh Chuong. Nghl An 25 0N2 Himg Thinh. Tring Bom. Ddng Nai 26 LC2 T l PWri. Thinh phd Lio Cai 27 NA24.1 Nam Tin. Nam D i n . Nghl An 28 NA25 12 Nam Thi/(7ng, Nam B i n , Ngh| An 29 NAI 7 1 2 Diln Hoa, Dlfci Chlu.Ngh* An 30 NA32 1 LlfU Son, 04 Lmrng. Nghl An 31 NA7.5.2 Nghi Tnrtrng. Nghi LOc. Ngh| An 32 THI Nga Son. Thanh HOa
33 NA25 1.3 Nam Thtfpng. Nan Din. Ngh|An
L14 L14 Sen Lai SenNA L14 L14 SenNA SenNA L14 L14 L14 L14 L14 L14 Sen Lal
L14 Sen Lai SenNA SenNA SenNA L14 Sen Lai
L14 L14 L14 L14 Sen Lai
L14 Sen Lai
L14 L14 L26 L14
Tmng du Bong bing ven biln odng bing ven biln odng bing ven biln Bdng bing ven biln Odng bing ven biln odng bing ven biln Odng bing ven biln Odng bing ven biln odng bing ven biln odng bing ven biln odng bing ven biln Oing bing ven biln Odng bing ven biln 06ng bing ven biln Odng bing ven biln Bdng bing ven biln Odng bing ven biln Odng bing ven biln Odng bing ven biln
odng bing Bdng bing Odng bing Miln nui Trung du Miln nilii Ddng bing Odng bing Odng bing ven biln
Trung du Odng bing ven biln Bdng bing ven biln
Ddng bing
3.8 5.5 4 5 6.5 3,5 7,5 7,5 5,3 5,0 4 5 5,8 5,5 3.5 3,0 4,3 6.5 2,6 4,8 7.5 5,0 3.0 4 8 4,0 10,5 3.8 6.5 3,5 3.0 4.5 13,5 6.5 5.0 3,0
Xim dim Xim dim Xim nhu Xim dim X l m d i m Ben Ben X l m d ^ Xim d|m X l m d i m
Ben Ben Xim d|m
Oen Xim d|m
Ben Xim dim Xim dim Oen Xim dim
Oen Oen Xim dim Xim hdng Xim d|m Xim dim Ben Den Xim nhit Xim hdng Oen Ben Ban , i Ghi cbH: 'Dudng Unh tin nim dunc do eau diy nfm 70 ngiy vi li trung binh cua 3 lin Ifp l^i.
3.2.2. Phan tich Rep-PCR cac miu nam Ket qua phfin tich Rep-PCR eiia 33 mfiu nfim dfi^m den thu th^p dugc cho thfiy so bfing .sAn phfim cua phan flng PCR vdi bfi mdi BOX Ifi 17 vdi kich thudc tfl -- 0,15 kb tdi ~ 2 kb (Bang 5,
Hinh 2). Bd moi ERIC vfi REP khfing tao cic bfing san phfi'm.
Kiem tra eac bfing san phfim trgn gel agarose cho thfiy bfi moi BOX tao tdi 7 bfing da hinh nfim gifla vj tri 0,3 kb vfi 2 kb (Hinh 2).
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Ngo Thi Mai Vi. Ha Giang, T r i n Thi Nhu Hoa, Nguyin Van Vien
B f i n g S ^ S a n p h W u a p h a n tich Rep-PCR doi vdi 33 mau nain do^m den hai lac
Rep4>CR Sd bang hinh thanh ERIC-PCR
BOX-PCR REP-PCR
0 17 0 17
So t>ang da hinh Kich thuoc bang tdi da (kb) 0
2.0 0
j j ^ ^ ^ ^ ^ ^ ^ a i g l l 12 13 14 15 16 17 .. .. ^ „ ,, „
M 1 2 5 2« 27 2S 29 I 31 32 33
Hinh 2. Box-PCR tren 33 m i u nam d6m den hai lac M. berkeleyi thu thap d 4 tinh nam 2013
bing mm tSn. cac nang Uiam khao JSO bp vi l.SOO bp (Ml). 100 bp vs SOO bp (M2) dupc cbi ro
3.2.3. Phan ticb cum san phdm Rep-PCR Cac bfing san phfim PCR eua ho moi BOX da dudc chuyin sang so hgu nhi phan dl tao dfl heu dfiu vfio cho phan tich cum dung phfin mem NTSYS nhfim tim hilu mflc dfi da dang vfi quan , h§ eua cfie mfiu nfim dfim den. Kfit qua phan tfch cum eho bfi mfii BOX dfi tao ra 3 cum mfiu nfim (ky hieu lfi I, U, III) vdi ngudng phfin chia dtfa tren he so tUdng dfing la 83% (Hlnh 3). Cum III gfim 03 mfiu lfi NA 3.4, NA9.5, NA20.3, cum II gdm 5 mfiu THl, LC2, NA12.1, NA14.1, NA24.1, cvm I gdm 25 mfiu cdn lai (Hinh 3).
Phan tich x" da dfldc thflc hien nhfim tim hilu U^u ed mfii quan h^ gifla cac cum pha hg vdi cac ngudn gdc cung nhu dfic dilm hinh thai miu n&n Phfin tfch X-' da cho thfiy khfing ed moi quan h^
gifla cac nhdm nfiin dugc phfin biet dua tren phfin tfch Box-PCR v& ngufin gfib eua chung (4 vung sinh thai, 4 gioiig 1^). dfic dilm hmh thfii Q ^ moi
trudng nudi cfiy nhan tao (4 mau tan nfim, 4 nhdm kich thudc tan nfim) (Bang 6).
Mfic dfl chia thfinh 3 cum nhflng do (i) PCR khfing tao ra cac san phfim khi dflng mfii Rep eung nhu ERIC (chflng td ho gen cua ehung giong nhau vfi khong chfla cac vi tn' dl gfin moi) vfi (u) mflc tUdng dfing di taniygn kha cao (83 - 99%) ta^ng phan tieh dung mfii BOX nen phan tich Rep-PCR da chflng td qufin thi nfi'm doin den tai Nghg An kha dfing nhfi^t. Kit qua nfiy eung phfl hdp vdi nghign cflu eua chung tfii (phfin) lfi nfiin dfim den tai Nghe An ehi smh san vo tinh ma khfing sinh san hflu ti'nh nhu da cong Ixftrgn the gidi.
Kit qua n^uen cflu ta-en ggi y dfii vdi nfim dom den d Nghe An, cae bien phap quan ly b§nh (giong khang, thudb hda hpc, c h l phfim smh hoc) CO the dugc ap dung dfing loat tren tofin tinh vfi tao hieu qua phdng chfing gifing nhau
Dfic tnmg phfin h> n i n Uycosphaeieaa berkeleyi gfiy bgnh ddm den Igc tgi Nghg An
I — r -
0.80 0.8S
T 1 1 r—
0.90 Coemdent
0.95
DNl NA13.1 NA17.1.1 N A l l . l NA20J N A l l . l NA3.2 NASJ NA7.5.1 NA14.I NA21.2 NA23.I NA26.1 NA31.1 DN2 NA 17.1J NA32.1 X.\17.2 NA6.3 NA4J NA25.1.1 NA25.1.2 THI N.\12.1 NA14.3 LC2 NA24.1 NA19.1 NA7.5.2 I VAil.S
~ N.43.4 -XA20.3 1.00
II III
Hinh 3. P h a n tich cum dfla t r e n so lieu BOX-PCR ciia cac mau dom den t h u t a i Vidt N a m
Ghi chii: H# si'tuang ding dUffc tinb tbeo cdng tbHc Unb h$ ad'pbH bpp dan gian (Simple Matching Coericient). Cic ciy dupc vi tbeo phuang phip ghip cfp miu dUng khoang cicb trung binb aSbgc ngang bkng (UPGMA).
Bang 6. Quan he cua cfic n h o m pha hf vd»i nguon goc dia ly, giong, d a c diem hinh thfii
QuanhQ giOa nhOm phi h | (I, II, III) vOri OOty
dodf Kit luin'
li. Sen NA)
Isinh thii (Ddng bing ven biln. Odng bing. Tmng du. Miln mil) Mlutln nim (Xim nhat, Xim dim, Xim hdng, Oen)
^ ^ ^ ^ J tin nim (mm) (0 - 3,4: > 3,4 - 6,8; > 6,8-10,1; > 10,1) 6 6 6 6
1,63 10,7 3,02 6,1
0,951 0,148 0.807 0,412
KhOng quan h|
KhOng quan h|
KhOng quan h|
KhOng quan h|
G*i dill; * Wt luin dupc dtfa trin ngudng tin cfy chung lia = 0.1
4. KET LU^N
Phfin tich brinh tu viing ITS eiia 11 mfiu nfi'm dfim den l?c tai Vift Nam, chu ylu tai Nghg An dfi chflng td ehung thugc lofii
•
Mycosphaerella berkeleyi. Trtnh til viing ITS cac miu nfi'm rfi't bao thii, dong nhfit 100%
nhau.
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Phfin tieh da dang di truyin 33 miu nfim dfim den hai lac thu thfip d Vift Nam, chu yk tai Nghf An, bfing ky thufit Rep-PCR da chfli*
to chi ed marker BOX-PCR tao hfing san phin da hinh. Phan ti'eh da chflng td qufin thi nfim dom den hai lac tai Nghf An khfi dfing nhfit vi di truyin
Ngo Thi Mai W. Ha Giang. Trfin Thi Nhu Hoa. Nguyin Vai Vien
Khong cd mfii hen he gifla cac cum nfim dugc xac dinh bdi BOX-PCR vdi ngufin got thu thap mau cung nhfl dac dilm hlnh tiiai mfiu
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