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Virus Detection from Local Banana Cultivars and the First Molecular Characterization of Banana bunchy top virus in Indonesia

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Virus Detection from Local Banana Cultivars

and the First Molecular Characterization

of Banana bunchy top virus in Indonesia

By

Noriko F

URUYA

*, Susamto S

OMOWIYARJO

** and Keiko T. N

ATSUAKI

***

(Received May ,., ,**./Accepted July -*, ,**.)

Summary : For banana plants (Musa spp.), occurrence of viruses is serious threat not only for production but also in conservation of banana species/cultivars as genetic resources. In this study, we collected 02 samples of -2 banana cultivars during - years of survey and carried out the detection of viruses by serological and/or molecular methods in a banana germplasm garden of central Java, Indonesia. Banana bunchy top virus (BBTV) and Cucumber mosaic virus (CMV) were confirmed from ,+ samples and - samples, respectively, though Banana bract mosaic virus (BBrMV) was not detected in this survey. Thus it was made clear that the major constraint virus is BBTV in this germplasm garden where it should be free from any virus infection. The occurrences of BBTV were confirmed in +, cultivars and of CMV were confirmed in - cultivars among -2 cultivars along with viral symptoms though all of tested cultivars were grown under the same condition for long time. Based upon this results, it was supposed that these +, and - cultivars are more sensitive to BBTV and CMV infection, respectively. Two BBTV isolates (BBTV-IG-- and IG0.) in this germplasm garden and one isolate of BBTV (BBTV-IJs++) from a village nearby the germplasm garden were sequenced for their DNA-+ and DNA-- and shown to have high homologies among them in full-length of each component (32 to 33ῌ and 33 to +**ῌ, respectively). In major common region (CR-M) of DNA-+, the Indonesian isolates showed higher homology obviously with isolates from Japan, Taiwan, the Philippines, China and Vietnam than with isolates from Australia and Fiji (3- to 3/ῌ and 0- to 0/ῌ, respectively). Therefore, molecular characters of these Indonesian BBTV isolates, which were classified into the Asian group (KARANet al., +33.), were firstly reported and analyzed in this study.

Key words : virus detection, Indonesia, banana cultivar, Banana bunchy top virus, nucleotide sequence ῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍

Introduction

Banana (Musa spp.) is one of the most important tropical and subtropcal crops and its origin is known as Malay Peninsula and the neighborhood. In Indonesia, since geographic location is close to the origin of banana, the diversity of banana plants is observed in local regions. Consequently, various cultivares of both dessert- and cooking-type bananas were produced total -.1 million tons, approximately +*ῌ share of it in Asia, in a year ,**- mainly for domestic market (FAO). In

Indonesia, under the supervision of Gadjah Mada Uni-versity, more than -** cultivars of banana have been collected from all over the country and grown for conservation of biological resources. However several virus-like symptoms have been observed in the germplasm collections.

Two banana viruses have been previously reported prevalent in Indonesia+*῎

. Among them, Banana bunchy top virus (BBTV), genus Babuvirus, is the most destruc-tive banana virus in the world except American region and causes banana bunchy top disease. The isometric

* ** ***

Department of International Agricultural Development, Graduate School of Agriculture, Tokyo University of Agriculture Faculty of Agriculture, Gadjah Mada University

Department of International Agricultural Development, Faculty of International Agriculture and Food Studies, Tokyo University of Agriculture

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virions of +2ῌ,* nm in diameter contain at least six circular ss-DNA components of approximately + kb each and are transmitted by banana aphid, Pentalonia nigronervosa Coq. Another virus, Cucumber mosaic virus (CMV), belongs to Cucumovirus and has spherical particles ,2ῌ-* nm in diameter including tripartite genome with ss-RNA. CMVhas worldwide distribu-tion and is a causative virus of banana mosaic disease. Host range of CMVis wide and is transmitted by more than 0* species of aphids. Moreover, in Indonesia, it has been known one more disease, banana streak disease, only by symptoms+*ῌ

. However, in this study, we did not work on Banana streak virus (BSV), genus Badnavirus, because the characterization of BSVis under development due to integration to host genome and high serological and genomic variability0, ., 1ῌ

. Additionally two potyviruses, Banana bract mosaic virus (BBrMV) and Abaca mosaic virus (AbaMV), are known to occur on banana in some parts of the Asia+*ῌ

, though the occurrence of them has not been reported in Indonesia.

This work reports the occurrence of viruses in local banana cultivars detected by serological and/or molec-ular methods to show the viral epidemiology in Indone-sia and find sensitive cultivars against viruses. The sequences of DNA-+ and DNA-- of isolates of BBTVare also studied as the first report of molecular analysis of Indonesian BBTV.

Materials and methods

Collection of banana cultivars

We collected banana leaves showing symptoms of viral diseases like bunchy top, mosaic, and mottle or no symptoms in the banana germplasm garden at Yog-yakarta District, YogYog-yakarta Special Territory, Indone-sia once a year from +332 to ,***. Along with sixty-eight samples of -2 cultivars, + sample, observed typical bunchy top symptoms in a village nearby the banana germplasm garden was stored in ῍-*῎ until use. Among them, BBTVs which were detected from three samples of banana (IG--, IG0. and IJs++) were used for molecular analysis. Virus isolates IG-- and IG0. were obtained from banana cultivars of Mas and Ambon Warangan, respectively, and IJs++ was from a banana plant of unidentified cultivar. These banana samples from Indonesia and one abaca plant from the Philip-pines were imported under the permission of the Minis-try of Agriculture, ForesMinis-try and Fisheries, Japan. Detection of BBTV, CMV and BBrMV by ELISA

Three ELISA kits (Agdia, USA) for three banana viruses (Banana bunchy top virus ; BBTV, Cucumber

mosaic virus ; CMVand Banana bract mosaic virus ; BBrMV) were used according to the manufacturer’s instructions. The sample which showed - times higher absorbance value at A.*/ nm than healthy banana sample was evaluated as positive to each of the viruses. Detection of BBTV by PCR

Total DNA was isolated from small pieces of banana midribs (*.+ g) using a PhytoPure DNA extraction kit (Nucleon, UK). Primers FPCR. (/῎ TTC CCA GGC GCA CAC CTT GAG AAA CGA AAG -῎, nts ,2.ῌ-+-) and F-(/῎ GGA AGA AGC CTC TCA TCT GCT TCA GAG ARC -῎, nts ,21ῌ,/2) designed by KARANet al.++ῌand TaKaRa Ex TaqTM(TaKaRa, Japan) were used in PCR to ampli-fy complete BBTVDNA-+. PCR conditions were . min at 3.῎ followed by -* cycles of + min at 3.῎, + min at 0+῎, , min at 1,῎ and finally an extension time of +* min at 1,῎ in a thermal cycler (Gene Amp PCR System 30**, Perkin Elmer). To avoid possible interference by latex or other banana leaf components during PCR reaction, *.+ῌ skimmed milk was added to the PCR cocktail-ῌ

.

Detection of potyviruses by RT-PCR

RT-PCR detection by potyviruses universal primer sets was conducted on banana samples which showed positive reaction to BBrMVELISA kit (Agdia, USA). After small pieces of banana leaves (*.+ g) were ground with sterilized PBS, the crude sap including putative virus particles depend on sample was incubated in PCR tube to be trapped to the wall. Total nucleic acids were derived by washing with sterilized PBST and heat shock. First-strand cDNA was synthesized using ReverTra Ace῍ (TOYOBO, Japan) according to the manufacture’s instructions with Oligo d(T) primer, as the initial primer. PCR was carried out using TaKaRa Ex TaqTM (TaKaRa, Japan). Primers Poty /῎ (/῎ GGA TCC GGB AAY AAY AGY GGD CAR CC -῎) and Poty d (T) -῎ (/῎ CAC GGA TCC CTT TTT TTT TTT TTT TTT V -῎), from Gibbs and Mackenzie/ῌ

with minor modific-ation, were used to amplify approximately +.1 kbp nu-cleotides including coat protein gene of Potyviridae. One abaca (Musa textilis), infecting BBrMV-aP21 from the Philippines, was used as a positive control. Cloning, sequencing and homology analysis of BBTV DNA-+ and

DNA--Complete DNA-+ and DNA-- of three BBTVisolates (IG--, IG0. and IJs++) were cloned and sequenced. Am-plification of full-length DNA-- was carried out under the same PCR condition for DNA-+ except for an anneal-ing temperature of .3῎ and the specific primers+0ῌ

:

FURUYA, SOMOWIYARJOand NATSUAKI

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CBT-F.PRI (/῍ GGT ATT TCG GAT TGA GCC TAC -῍, nts -/0ῌ-10) and CBT-R.PRI (/῍ TTG ACG GTG TTT TCA GGA ACC -῍, nts -//ῌ-..). PCR reactions were subjected to electrophoresis in agarose gel and stained with ethidium bromide. Each approximately +.+ kbp fragment was recovered using a QIAquickTMGel Ex-traction kit (QIAGEN, Germany). Three isolates (IG--, IG0., IJs++) of BBTV DNA-+ and DNA-- were ligated into the pGEM-T Vector (Promega, USA) and trans-formed into E. coli DH/a (Life Technologies Inc., USA). The positive clones were selected by X-gal and IPTG screening. At least three clones of each PCR fragment were sequenced in both directions using an automated ABI PRISM -11 DNA Sequencer with an ABI Prism BigDyeῌ Terminator Cycle Sequencing Kit (Applied Biosystems, USA). Sequence alignment and homology analysis were carried out using AssemblyLIGNTM +.*.3 c (Accelrys, USA) and CLUSTAL W package+-῍

with Mac Vector 0./.- (Accelrys, USA).

Sequences obtained in this study were submitted to DDBJ/EMBL/GenBank database with accession num-bers fromAB+203,. to AB+203,3. The reported se-quences of BBTV DNA-+ and DNA-- in various parts of the world were obtained fromDDBJ/EMBL/GenBank database and used for comparisons : Japan (JN. : AB +*2./,/AB+*2..3, JK- : AB+*2./-/AB+*2./*, JY+ : AB +*2./0/AB+*2./+), Taiwan (Tw : AF.+0.02/AF+.23.,), Philippines (Ph : AF.+0.03/AF+.2*02), China (C-NS : AF ,-221. / AF ,-2210, C-NSP : AF ,-221/ / AF ,-2211), Vietnam(V0 : AF++-0/3/AF++-00+, V+. : AF++-00*/AF ++-00,), Australia (Au : S/0,10/L.+/1.) and Fiji (Fj : AF .+0.00ῌAF+.23..)ῌ

Results

Detection of viruses by ELISA and PCR and/or RT-PCR

BBTV was detected from ,+ samples of +1 cultivars, by ELISA and PCR, and CMV was detected from -samples of - cultivars, by ELISA (Table +). Mix infec-tion of BBTV and CMV was not confirmed in these samples. Eight samples which were slightly reacted to BBrMV in ELISA were not amplified by RT-PCR for Potyvirus universal primers even though an approxi-mately +.1 kbp band of BBrMV-aP21 was shown in BBrMV positive sample.

Among +1 cultivars fromwhich BBTV were detected, there were / cultivars (Kepok Gabu, Raja Entos, Raja Trunpong, Rejang and Tanduk Hijau) without viral symptoms such as bunchy top observed in other +, cultivars. As for CMV, mosaic symptom, but not with necrosis, was observed in all - cultivars (Klutuk Susu, Raja Bandung, Turi). While both BBTV and CMV were not detected, rosette and chlorosis symptoms which had probability of virus infection were observed on ‘Gading’ and ‘Jiwel’, respectively. Among total -2 cultivars used in this study, BBTV and CMV were not detected from +0 cultivars. Especially from / cultivars (Barly, Klutuk Warangan, Koja Srimentak, Pinang and Raja Polo), the viruses were not detected during con-secutive survey for - years.

Sequence analysis of BBTV

BBTV DNA-+ and DNA-- of two isolates (IG-- and IG 0.) fromthe germplasmgarden and one isolates (IJs++) fromneighbor village were amplified by PCR and

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sequenced after cloning. All DNA-+ and DNA-- frag-ments were consisted of ++*- to ++*. nts and +*/2 nts, respectively. In both components, the stem-loop se-quences were observed in all isolates. Two open read-ing frames (ORFs) were found on the DNA-+, O RF + of 2/2nts and ORF , of +,0 nts, and one ORF of /+* nts was found on the DNA-- of the Indonesian isolates as previously reported for other BBTVs+, +/ῌ

.

Sequences of all isolates were compared with previ-ously reported sequences. Among Indonesian isolates, though . nucleotides of IG-- in DNA-+ were di#er from IG0. and IJs++ in approximately 3* nucleotides in major common region (CR-M) of DNA-+, nucleotide sequence in CR-M of DNA-- were same completely, and they were apparently close to the Asian group than the South Pacific group (Fig. +).

Table , Sequence homologies (ῌ) of Indonesian BBTV isolates and their homologies with the other geographical isolates in DNA-+ and

DNA--Fig. + Comparison of nucleotide sequences in CR-Ms of BBTV : A) DNA-+, B) DNA--. Dashes mean gaps introduced to maximize alignment

FURUYA, SOMOWIYARJOand NATSUAKI

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The three Indonesian isolates were shown to have high homologies within them in full-lengths of DNA-+ and DNA-- (32 to 33ῌ and 33 to +**ῌ, respectively, Table ,). In CR-M of DNA-+, the nucleotide sequence of Indonesian isolates had a high homology with isolates of the Asian group (3- to 3/ῌ) but a low homology with the South Pacific group (0- to 0/ῌ, Table ,). Amino acid sequence in CP gene of three Indonesian isolates agreed completely in each other and it was also same even though in a nucleotide level (Table ,). When compared with foreign BBTV in amino acid sequences, Indonesian isolates showed a high degree of homology with foreign isolates except C-NSP from China (32 to +**ῌ and 3.ῌ, respectively).

Discussion

In Indonesia, banana bunchy top disease has oc-curred since +312 and been considered to reach epide-mic level in most banana growing provinces+,, +.ῌ

. On CMV, though the occurrence on tobacco was known since +31,, it is said that infection in banana plantings is localized and seldom causes serious outbreaks+,, +.ῌ

. However the epidemiology of the causal viruses in local banana cultivars based on serological or molecu-lar measures and the molecumolecu-lar characterization of them have not been surveyed. This study revealed that viruses from bananas with or without viral symp-toms were identified from -2 local cultivars in Indone-sia, and the partial genetic status of BBTV was deter-mined and evaluated.

In the germplasm garden surveyed, viruses detected were only two species, BBTV and CMV, and the popu-lation of BBTV was 1 times higher than that of CMV in this garden. Therefore it was shown that the major constraint virus is BBTV in this germplasm garden. Though BBrMV was not detected in this study, it is necessary to continue taking precautions against it because BBrMV is reported to occur on banana and abaca in Southeast Asia+*ῌ

. On BBTV, Damarjati,ῌ

has cited that Purnomo (+330) confirmed the disease incidence on - cultivars (Mas, Ambon Kuning and Ambon Hijau). And Nurhadi and Setyobudi+,ῌ

have referred that Muharam (+32.) showed ‘Ambon Jepang’ and ‘Ambon Putih’ are very susceptible to the virus, meanwhile SULYO et al. (+33,)

reported . banana cultivars (Klutuk, Jimbluk, Kapas and Seribu) showed resistance to BBTV out of -* cultivars tested. However, the methods of virus detec-tion in these studies were not precisely reported. Our study using ELISA and PCR showed that the occur-rences of BBTVs in +1 cultivars and CMVs in - cultivars among -2 cultivars and this is particular interesting

because they were grown under the same condition for long time and occurrence might reflect the resistance of cultivars against virus infection. Among the cul-tivars shown to be infected of BBTV or CMV by sero-logical and/or molecular detection, viral symptoms were observed on +, cultivars out of +1 cultivars for BBTV and - cultivars out of - cultivars for CMV. Thus it was supposed that these +, and - cultivars are more sensitive to BBTV and CMV infection, respectively, than other latently infected cultivars. Further study by artificial inoculation test is essential for accurate screening of virus resistant cultivars.

We selected DNA-+, which encodes the master-Rep protein gene3ῌ

, and DNA--, which encodes the coat protein gene+/ῌ

, and determined and analyzed their se-quences of Indonesian BBTV isolates. In our study, the Indonesian BBTVs had a common structure with previ-ously reported BBTVs on their nucleotides in DNA-+ and DNA--. And the translated CP genes, which were coded in DNA--, had a high homology (over 32ῌ) with the all isolates from Japan, Taiwan, the Philippines, Vietnam, Australia and Fiji, and the NS isolate from China, but not with NSP isolate from China which was reported to have a unique biological character2ῌ

. Karan et al.++ῌ

identified that BBTV forms two major groups genetically. They are the Asian and South Pacific groups by comparisons of nucleotide sequences and the Asian group included isolates from three countries (the Philippines, Taiwan and Vietnam) as opposed to the South Pacific group included isolates from seven countries (Australia, Burundi, Egypt, Fiji, India, Tonga and Western Samoa). Because of geographical and historical complex backgrounds in Indonesia, it was expected to detect BBTV isolates with molecular diver-sity including both the Asian and South Pacific groups. Additionally, the banana plants in the germplasm garden have been collected from many di#erent regions in Indonesia, the detection of di#erent virus strains might be possible. In this study, however, all three Indonesian BBTVs tested belonged to the Asian group with high homology and the genetic di#erence among isolates from the germplasm garden and a vil-lage nearby the garden were not detected. Therefore, it was suggested that a single strain of BBTV, classified to the Asian group, has been introduced and spread in this germplasm garden and the neighborhood.

In this study, as we firstly analyzed Indonesian BBTV isolates for their molecular characterization, fur-ther study became possible based upon these data in-cluding development of specific primers for detection of BBTV in Indonesia.

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Acknowledgement

This study was partially funded by Nakajima Foun-dation, Japan. We are grateful to the sta#s of Labora-tory of Plant Pathology of Gadjah Mada University. We thank Dr. Narceo Bajet, University of the Philip-pines, Los Banos, the PhilipPhilip-pines, for the information of banana and abaca viruses in the Philippines.

References

+ῌ BEETHAM, P.R., HAFNER, G. J., HARDING, R.M. and DALE, J.

L., (+331) Two mRNAs are transcribed from banana bunchy top virus DNA-+. J. Gen. Virol., 12, ,,3ῌ,-0. ,ῌ DAMARJATI, D.S., (+333) Research and development of

banana in Indonesia. In : MOLINA, A.B., ROA. V.N. (ed)

Advancing banana and plantain R & D in Asia and the Pacific. Proceedings of the 3th INIBAP-ASPNET Region-al Advisory Committee meeting held at South China Agricultural University, Guangzhou, China, ,ῌ/ Novem-ber +333, IPGRI, Italy, ++,ῌ++0.

-ῌ DEBOER, S.H., WARD, L. J., LI, X. and CHITTARANJAN, S.,

(+33/) Attenuation of PCR inhibition in the presence of plant compounds by addition of BLOTTO. Nucleic Acids Research, ,-, ,/01ῌ,/02.

.ῌ GEERING, A.D.W, MCMICHAEL, L.A., DIETZGEN, R.G. and

THOMAS, J.E., (,***) Genetic diversity among Banana streak virus isolates from Australia. Phytopathology, 3*, 3,+ῌ3,1.

/ῌ GIBBS, A. and MACKENZIE, A., (+331) A primer pair for

amplifying part of the genome of all potyvirids by RT-PCR. Journal of Virological Methods, 0-, 3ῌ+0. 0ῌ HARPER, G., OSUJI, J.O., HESLOP-HARRISON, J.S. and HULL,

R., (+333) Integration of banana streak badnavirus into the Musa genome : molecular and cytogenetic evidence. Virology, ,//, ,*1ῌ,+-.

1ῌ HARPER, G., HART, D., MOULT, S. and HULL, R., (,**.)

Banana streak virus is very diverse in Uganda. Virus-Research, +**, /+ῌ/0.

2ῌ HE, Z.F., LI, H.P., XIAO, H.G. and FAN, H.Z., (,***) Cloning and sequencing of DNA component + of two BBTV strains. Acta Phytopathological Sinica, -*, -0.ῌ-03. 3ῌ HORSER, C., HARDING, R.M. and DALE, J.L., (,**+) Banana

bunchy top nanovirus DNA-+ encodes the ‘master’ repli-cation initiation protein. J. Gen. Virol., 2,, ./3ῌ.0.. +*ῌ JONES, D.R., (,***) 0 : Diseases Caused by Viruses. In :

JONES, D.R. (ed) Diseases of Banana, Abaca and Enset, CABI, UK, pp. ,.+ῌ,3-.

++ῌ KARAN, M., HARDING, R.M. and DALE, J.L., (+33.) Evidence for two groups of banana bunchy top virus isolates. J. Gen. Virol., 1/, -/.+ῌ-/.0.

+,ῌ NURHADI, A. and SETYOBUDI, L., (+332) Status of banana and citrus viral diseases in Indonesia. In : MOLINA, A.B., ROA. V.N., BAY-PETERSEN, J., CARPIO, A.T., JOVEN, J.E.A. (ed) Managing banana and citrus diseases. Proceedings of a regional workshop on disease management of banana and citrus through the use of disease-free planting ma-terials held in Davao city, Philippines, +.ῌ+0 October +332, IPGRI, Italy, +-ῌ+.2.

+-ῌ THOMPSON, J.D., HIGGINS D.G. and GIBSON T. J., (+33.) CLUSTAL W : improving the sensitivity of progressive multiple sequence alignment through sequences weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res., ,,, .012ῌ.02*. +.ῌ TRIHARSO (ed), (+332) Indonesia. In : MURAYAMA D.,

AGRAWAL, H.O., INOUE, T., KIMURA, I., SHIKATA, E., TOMARU, K., TSUCHIZAKIT., TRIHARSO(ed) Plant Viruses in Asia, Gadjah Mada University Press, Indonesia, ,21ῌ-.0. +/ῌ WANITCHAKORN, R., HARDING, R.M. and DALE, J.L., (+331)

Banana bunchy top virus DNA-- encodes the viral coat protein. Arch. Virol., +.,, +01-ῌ+02*.

+0ῌ WANITCHAKORN, R., KARAN, M. and DALE, J.L., (,***) Se-quence variability in the coat protein gene of two groups of banana bunchy top isolates. Arch. Virol., +./, /3-ῌ0*,.

FURUYA, SOMOWIYARJOand NATSUAKI

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インドネシアにおけるバナナ在来種からの

ウイルス検出およびバナナバンチ

῎トップウイルスの

分子生物学的初解析

古屋典子*

ῌSusamto S

OMOWIYARJO

**

ῌ夏秋啓子***

ῐ平成 +0 年 / 月 ,. 日受付ῌ平成 +0 年 1 月 -* 日受理ῑ 要約 : バナナῐMusa spp.ῑ にとって῍ ウイルスの存在は῍ 生産の場だけではなく῍ 遺伝資源としての種や品 種の保存においても重大な脅威であるῌ 本研究では῍ インドネシアのジョグジャカルタ特別州に位置するバ ナナ品種保存園において῍ -2 品種 02 株のバナナを - 年間に渡って採集し῍ 血清学的ῌ分子生物学的手法の 両方または一方を用いて῍ ウイルスの検出を行ったῌ その結果῍ キュウリモザイクウイルス ῐCMVῑ は - 株 のみで感染が確認されたのに対し῍ バナナバンチ῎トップウイルス ῐBBTVῑ は ,+ 株で認められたことか ら῍ 本来はウイルスフリ῎であるべき本品種保存園においても BBTV は蔓延していることが示されたῌ ま た῍ 調査を行った -2 品種は長期間同じ環境条件下で栽培されていたが῍ BBTV は +, 品種で῍ CMV は - 品 種でそれぞれ病徴を伴って感染が確認されたῌ このことから῍ これらの +, 品種と - 品種はそれぞれ BBTV と CMV に対して῍ より感受性であると思われたῌ バナナ品種保存園に発生した BBTV, 分離株 ῐBBTV-IG

--῍ -IG0.ῑ とその近郊の農村に発生した BBTV+ 分離株 ῐBBTV-IJs++ῑ について DNA-+ と DNA-- の塩基

配列を決定した結果῍ それぞれのコンポ῎ネントの全長で - 分離株は高い相同性を有していた ῐ32῏33῍῍

33῏+**῍ῑῌ また῍ DNA-+ の CR-M 領域 ῐmajor common regionῑ では῍ 既報のオ῎ストラリアおよびフィ

ジ῎の分離株よりも῍ 日本ῌ台湾ῌフィリピンῌ中国ῌベトナムの分離株と῍ より高い値を示した ῐ0-῏ 0/῍῍ 3-῏3/῍ῑῌ したがって῍ インドネシアにおける BBTV アジアグル῎プ ῐKARANら῍ +33.ῑ の発生と それらの分子生物学的性状が本研究によって初めて明確に示されたῌ キῌワῌド : ウイルス検出ῌインドネシアῌバナナ品種ῌバナナバンチ῎トップウイルスῌ塩基配列 * ** *** 東京農業大学大学院農学研究科国際農業開発学専攻 インドネシア国立ガジャマダ大学農学部 東京農業大学国際食料情報学部国際農業開発学科

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