SURVEY ON THE OCCURRENCE OF VIRUSES INFECTING CUCURBITS INYOGYAKARTAANDCENTRALJAVA

ABSTRACT Cucurbits are grown throughout the Java Island as dry season crops. Plants having mosaic, mottling, chlorosis and leaf distortion symptoms were frequently found in most of the cucurbit fields during the survey which conducted in Central Java including Sleman,Kulon Progo, and Klaten during July-September 2000 and 2001. Usingdouble antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA)Cucumber mosaic virus (CMV), Cucumber green mottle mosaic virus(CGMMV)and Kyuri green mottle mosaic virus (KGMMV)were found infecting cucurbits.CMV was widespread, infecting 48.9% of the samples tested followed by CGMMV (12.8%) and KGMMV (6.4%), while others samples (31.9%) were not tested, double infections were common with 8.5 % of the samples being infected with two viruses (CGMMV and KGMMV) and 34% with three viruses (CMV, CGMMV, and KGMMV). Severe mosaic and mottle symptomswere associatedmost often with single infectionofCGMMV andKGMMVrespectively. In addition, these are the first detections of CGMMVand KGMMV infecting cucurbit plants in Indonesia. Key words: CGMMV,CMV,Cucurbits, DAS-ELISA, KGMMV INT/SARI Tanaman labu-labuan umumnya tumbuh sepanjang musim kemarau di Pulau Jawa. Tanamanlabu-labuan dengan gejala mosaik, klorosis, mottling dan bentuk daun serta buah yang berubah banyak dijumpai selama survei yang dilakukan di Kulon Progo, Sleman dan Klaten pada bulan Juli sampai September tahun 2000 dan 2001. Deteksi menggunakan metode double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELlSA) telah berhasil mengetahui keberadaan dan infeksi Cucumber mosaic virus (CMV), Cucumber green mottle mosaic virus (CGMMV) dan Kyuri green mottle mosaic virus (KGMMV) pada tanaman labu-labuan di tiga kabupaten tersebut. CMV menginfeksi tanaman labu-labuan tinggi yaitu 48,9% dari jumlah sampel tanaman yang dikoleksi, kemudian CGMMV (J 2,8%) dan KGMMV (6,4%), sedangkan sebanyak 14 sampel tanaman (31,9%) tidak dideteksi. 1nfeksi ganda banyak ditemukan dan 8,5 %sampel tanaman terinfeksi oleh dua jenis virus (CGMMV dan KGMMV) sedangkan 34%sampel tanaman terinfeksi oleh tigajenis virus (CMV, CGMMV, dan KGMMV). Gejala mosaik dan mottling sering terjadi pada tanaman labu-labuan yang terinfeksi ganda oleh CGMMV dan KGMMV Hasil penelitian merupakan deteksi pertama CGMMV dan KGGMV pada tanaman labu-labuan di Indonesia. Kata kunei: CGMMV, CMV, Cucurbits, DAS-ELlSA, KGMM

Survey on the Occurrence of Viruses Infecting Cucurbits in YOGYAKARTA and Central Java

Cucurbits are grown throughout the Java Island as dry season crops. Plants having mosaic, mottling, chlorosis and leaf distortion symptoms were frequently found in most of the cucurbit fields during the survey which conducted in Central Java including Sleman, Kulon Progo, and Klaten during July–September 2000 and 2001. Using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA); Cucumber mosaic virus (CMV), Cucumber green mottle mosaic virus (CGMMV) and Kyuri green mottle mosaic virus (KGMMV) were found infecting cucurbits. CMV was widespread, infecting 48.9% of the samples tested followed by CGMMV (12.8%) and KGMMV (6.4%), while others samples (31.9%) were not tested, double infections were common with 8.5 % of the samples being infected with two viruses (CGMMV and KGMMV) and 34% with three viruses (CMV, CGMMV, and KGMMV). Severe mosaic and mottle symptoms were associated most often with single infection of CGMMV and KGMMV respectively. In addition, these are the first detections of CGMMV and KGMMV infecting cucurbit plants in Indonesia.Tanaman labu-labuan umumnya tumbuh sepanjang musim kemarau diPulau Jawa. Tanaman labu-labuan dengan gejala mosaik, klorosis, mottling dan bentuk daun serta buah yang berubah banyak dijumpai selama survei yang dilakukan di Kulon Progo, Sleman dan Klaten pada bulan Juli sampai September tahun 2000 dan 2001. Deteksi menggunakan metode double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) telah berhasil mengetahui keberadaan dan infeksiCucumber mosaic virus(CMV),Cucumber green mottle mosaic virus (CGMMV) dan Kyuri green mottle mosaic virus (KGMMV) pada tanaman labu-labuan di tiga kabupaten tersebut. CMV menginfeksi tanaman labu-labuan tinggi yaitu 48,9% dari jumlah sampel tanaman yang dikoleksi, kemudian CGMMV (12,8%) dan KGMMV(6,4%), sedangkan sebanyak 14 sampel tanaman (31,9%)tidak dideteksi.Infeksi ganda banyak ditemukan dan 8,5 % sampel tanaman terinfeksi oleh dua jenis virus (CGMMV dan KGMMV) sedangkan 34% sampel tanaman terinfeksi oleh tiga jenis virus (CMV, CGMMV, dan KGMMV). Gejala mosaik dan mottling sering terjadi pada tanaman labu-labuan yang terinfeksi ganda oleh CGMMV dan KGMMV. Hasil penelitian merupakan deteksi pertama CGMMV dan KGGMV pada tanaman labu-labuan di Indonesia

Characterization of potato and tobacco isolates of Cucumber mosaic virus from Syria and the first report on CMV satellite RNA from potato

Cucumber mosaic virus (CMV) has been reported from potato production areas in Europe, USA, Japan and more frequently in regions with warm climates such as Egypt, India, Saudi Arabia and Syria. As it is considered as an uncommon virus in potato, the characterization of potato isolates of CMV is far behind those from other hosts. In addition to potato, CMV is a common virus infecting many crops in Syria, but nothing is known about its molecular characteristics. The present study aimed to characterize Syrian CMV isolates collected from potato and neighboring tobacco fields. All potato isolates of CMV (total of four) co-infected potato plants with Potato virus Y (PVY) which is the most frequent potato virus in Syria. According to the sequence analyses of the coat protein (CP) coding region, three potato and three tobacco CMV isolates were found to be closely related regardless of the host species or geographic origin, and all belonged to the IA strain subgroup of CMV. A potato CMV isolate, PoCMV7-5, readily infected solanaceous plants in which it induced systemic infection, but was less infectious to other hosts including those of Leguminosae and Cucurbitaceae. When inoculated on potato plants, PoCMV7-5 alone or with various PVY strains was able to cause local but not systemic infection in all potato cultivars inoculated. PoCMV7-5 contained heterogeneous variants of satellite RNA which varied in length due to A or/and T deletion/insertion at approximate nucleotide position 225‒240. This is the first report on CMV satellite RNA from potato

フィリピンサン パパイヤ ユテン ウイルス ノ セイシツ ト ケンシュツホウ

フィリピンのルソン島バタンガスで採集した輪紋症状を示すパパイヤ(Carica papaya)からウイルスの1分離株(PRSV-P-B)を得た。このウイルスを各種植物へ汁液接種したところ,全身感染したパパイヤ,ズッキーニ(Cucurbita pepo),メロン(Cucumis melo),シロウリ(Cucumis melo var. conomon),ニホンカボチャ(Cucurbita moschata),およびキュウリ(Cucumis sativus)と局所感染したC. amaranticolorの7種に感染した。電子顕微鏡観察ではパパイヤ病株や汁液接種したズッキーニに幅12nm,長さ700～800nmのひも状ウイルス粒子と細胞質封入体の破片とが認められた。PRSV-P-BはELISAでパパイヤ輪点ウイルス抗血清(ATCC)と陽性反応を,ズッキーニ黄斑モザイクウイルス抗血清(ATCC)および真岡哲夫博士より分譲を受けたパパイヤ奇形葉モザイクウイルス(PLDMV)の抗血清とは陰性反応を示した。ウイルス外被タンパク質の分子量は約36Kであった。以上の性質から,フィリピンのパパイヤから分離されたPRSV-P-Bはパパイヤ輪点ウイルスと同定,確認された。PRSV-P-Bに対する抗血清を作製し,健全ズッキーニ葉汁で吸収してからELISAに用いた。PRSV-P-Bは新鮮なあるいは乾燥したパパイヤあるいはズッキーニ病葉からELISA,DIBA,そしてTIBAによって検出された。さらに,PRSV-P-Bは渡辺ら(1998)によるPRSV特異的合成プライマーを利用したRT-PCRによって増幅された。PLDMV(真岡哲夫博士から分譲)は同プライマーで増幅されなかったので,RT-PCRはPRSVとPLDMVの迅速な類別に有効と考えられた。An isolate of virus (PRSV-P-B) from papaya (Carica papaya) showing ringspots on leaves collected at Batangus, Luzon, the Philippines was characterized. The virus was manually inoculated and showed to be infectious to 7 species of plants in 3 families including papaya, zucchini (Cucurbita pepo), two types of melons (Cucumis melo, Cucumis melo var. conomon), pumpkin (Cucurbita moschata), and cucumber (Cucumis sativus), as systemic hosts, and Chenopodium amaranticolor as a local lesion host. Filamentous virus particles of 700～800nm length and 12 nm width and debris of inclusion bodies were observed in diseased papaya or manually inoculated zucchini under electron microscope. PRSV-P-B was positively reacted with antiserum to Papaya ringspot virus (ATCC) but showed negative reactions with antisera to Zucchini yellow mosaic virus (ATCC) and Papaya leaf distortion mosaic virus (Gift from Dr. T. Maoka) in ELISA. Molecular weight of coat protein was approximately 36K. Based upon these results, the virus (PRSV-P-B) obtained from papaya plant collected in the Philippines was identified as Papaya ringspot virus. Serum against PRSV-P-B was produced and used for ELISA after absorption with healthy sap of zucchini. PRSV-P-B was detected by ELISA, DIBA and TIBA with fresh or dried leaves of infected papaya or zucchini. PRSV-P-B was also detected by RT-PCR using the synthetic primers designed by Watanabe et al. (1998) which was specific for PRSV. As RNA of PLDMV (Gift from Dr. T. Maoka) was not amplified by RT-PCR with specific primers for PRSV, it was shown that RT-PCR was useful to distinguish PRSV from PLDMV rapidly

インドネシア ニ オケル バナナ ガイライシュ カラ ノ ウイルス ケンシュツ オヨビ バナナ バンチートップ ウイルス ノ ブンシ セイブツガク テキ ショ カイセキ

バナナ(Musa spp)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。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,38品種68株のバナナを3年間に渡って採集し,血清学的・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。その結果,キュウリモザイクウイルス(CMV)は3株のみで感染が確認されたのに対し,バナナバンチートップウイルス(BBTV)は21株で認められたことから,本来はウイルスフリー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。また,調査を行った38品種は長期間同じ環境条件下で栽培されていたが,BBTVは12品種で,CMVは3品種でそれぞれ病徴を伴って感染が確認された。このことから,これらの12品種と3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。バナナ品種保存園に発生したBBTV2分離株(BBTV-IG33, -IG64)とその近郊の農村に発生したBBTV1分離株(BBTV-IJs11)についてDNA-1とDNA-3の塩基配列を決定した結果,それぞれのコンポーネントの全長で3分離株は高い相同性を有していた(98～99%,99～100%)。また,DNA-1のCR-M領域(major common region)では,既報のオーストラリアおよびフィジーの分離株よりも,日本・台湾・フィリピン・中国・ベトナムの分離株と,より高い値を示した(63～65%,93～95%)。したがって,インドネシアにおけるBBTVアジアグループ(KARANら,1994)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。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 68samples of 38banana cultivars during 3years 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 21samples and 3samples, 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 12cultivars and of CMV were confirmed in 3cultivars among 38cultivars 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 12 and 3cultivars are more sensitive to BBTV and CMV infection, respectively. Two BBTV isolates (BBTV-IG33 and IG64) in this germplasm garden and one isolate of BBTV (BBTV-IJs11) from a village nearby the germplasm garden were sequenced for their DNA-1 and DNA-3 and shown to have high homologies among them in full-length of each component (98 to 99% and 99 to 100%, respectively). In major common region (CR-M) of DNA-1, the Indonesian isolates showed higher homology obviously with isolates from Japan, Taiwan, the Philippines, China and Vietnam than with isolates from Australia and Fiji (93 to 95% and 63 to 65%, respectively). Therefore, molecular characters of these Indonesian BBTV isolates, which were classified into the Asian group (KARAN et al., 1994), were firstly reported and analyzed in this study

カンコク ノ マクワウリ ニ オケル キュウリリョクハンモザイクウイルス ノ ハッセイ ト ソノ トクセイ

韓国慶尚北道星州郡で採集したモザイク症状を示すマクワウリ(Cucumis melo L. var. makuwa Makino)に,スイカ緑斑モザイクウイルス(Cucumber green mottle mosaic virus, CGMMV),カボチャモザイクウイルス(Watermelon mosaic virus 2, WMV2)を含む複数のPotyvirus属ウイルスの単独または重複感染がELISA法で認められ,さらにモザイク症状を呈していた試料一つからはキュウリ緑斑モザイクウイルス(Kyuri green mottle mosaic virus, KGMMV)が検出された。本分離株(KGMMV-KOM)の宿主範囲は日本で分離されたKGMMV-C1と類似し,接種試験でマクワウリに潜在感染した。外被タンパク質(CP)のコード領域は486塩基で既報のKGMMVと同様だったが,移行タンパク質(MP)のコード領域は786塩基で他のKGMMV(789塩基)より短かった。KGMMV-KOMのMPとCPは,既報のKGMMVのMPとCPのコード領域の塩基配列と高い相同性を示した。韓国のマクワウリにおけるKGMMVの発生報告は本報が最初のものである。Mosaic diseases were detected in oriental melons cultivated in Seongju County, Kyungpook Province, Korea. The oriental melons were infected by Cucumber green mottle mosaic virus (CGMMV), Watermelon mosaic virus 2 (WMV2) and some viruses in the genus Potyvirus, according to ELISA detection. Additionally, from one melon, a virus was isolated and identified as Kyuri green mottle mosaic virus (KGMMV) by electron microscopy, biological analysis, RT-PCR and sequencing of the movement protein (MP) and coat protein (CP) genes. Results of host reactions of the KGMMV isolate (KGMMV-KOM) were similar to that described for KGMMV-C1 isolated from Japan, while KGMMV-KOM showed latent infection on oriental melon. Although the CP gene of KGMMV-KOM comprised 486nts, the same as previously reported KGMMV, the MP gene comprised 786nts, which is shorter than that of other KGMMV (789nts). Phylogenetic analysis based on the nucleotide sequences of MP and CP genes was also conducted and KGMMV-KOM was found to belong to the major cluster of KGMMV, showing molecularly conservative nature of this species. This is the first report of KGMMV infecting on oriental melon in Korea

COLLETOTRICHUM DEMATIUMニ ヨル アガパンサスタンソビョウ:シンショウ

2004年5月茨城県取手市で激しい葉枯症状を呈するアガパンサスを採集した。病葉上には剛毛を有する分生子層が肉眼では多数の小黒粒点として観察でき,培養菌叢はPDA培地上で暗灰色～黒色,分生子は鎌形で大きさ15.0～21.5×2.5～3.7μmであった。これらの形態的特徴から,本菌をColletotrichum dematium (Per. : Fr.) Groveと同定した。接種試験による病原性の確認を行った結果,接種約1週間後に病徴が再現され,病斑部から本菌が再分離された。これらの結果から,本病はC. dematiumによって引き起こされたことが明らかになり,病名としてアガパンサス炭疽病を提言した。In 2004, a new disease was found on African lily (Agapanthus africanus (L.) Hoffmanns.) leaves, in Ibaraki prefecture, Japan. Brown to grayish brown leaf blight lesions were observed on over-wintered leaves. A fungus belonging to the genus Colletotrichum was observed on these lesions. Its acervuli had setae. The colonies were black or dark gray on PDA medium. Conidia were aseptate, hyaline, falcate, and 15.0～21.5×2.5～3.7μm (17.3×3.0μm in average). Appressoria were oval or slightly irregular, dark brown colored and 9.5～18.7×5.2～10.0μm in size. Based upon these morphological characteristics, this fungus was identified as Colletotrichum dematium (Per. : Fr.) Grove. Isolated fungus was pathogenic to the original host plant in artificial inoculation test. This is the first report describing the African lily disease caused by C. dematium in Japan. Therefore, anthracnose of agapanthus (agapanthus tanso-byo) is proposed as a new disease in Japan

ソラマメウイルトウイルス ニ ヨル エキザカム ノ イシュクビョウ シンショウ

1989年,神奈川県でエキザカム(Exacum affine)に激しい萎縮と葉巻き症状が発生した。C. amaranticolorあるいはC. quinoaに接種して得たウイルスにより宿主を調べたところ,9科21種に感染し,エキザカムでは萎縮症状が再現され,C. amaranticolorやC. quinoaでは接種葉に灰白色斑点を生じた後に全身感染し,ソラマメでは頂葉の枯死が観察された他, 各種のタバコ類にも感染した。電子顕微鏡観察では直径約27nmの小球型ウイルス粒子が観察された。また,モモアカアブラムシ(Myzus persicae)で非永続的に伝搬された。粗汁液中の安定性は,耐熱性が60-65℃(10分),耐保存性が4日であった。ウイルス粒子はソラマメウイルトウイルス(BBWV)に対する抗血清を用いた免疫電子顕微鏡法で捕捉・修飾され,パチョリ微斑モザイクウイルス(PaMMV)に対する抗血清を用いたELISA法でも検出された。ウェスタンブロット法では2種のウイルス外被タンパク質が検出され,その分子量は約42,000と約26,000であった。本ウイルスを部分純化したところその収量は感染C. quinoa葉50gあたり3.6mgであった。部分純化標品をウサギに免疫し,抗血清の作製を行ったところ,ELISA法やウェスタンブロット法に利用可能であった。以上より,本ウイルスをBBWVの1系統と同定し,BBWVによるエキザカムの病害は未報告のため,病名を萎縮病,英名をdwarfとすることを提案した。Exacum (Exacum affine) is an ornamental plant, which is popular as a pot flower in summer in Japan. In several glasshouses of growers in Kanagawa prefecture in 1989, high incidence of symptoms such as severe dwarf, upward rolling of leaves, and reduced number and size of flowers was observed. Host range of the pathogen was tested with the inocula obtained by manual inoculation from diseased exacum to Chenopodium amaranticolor and C. quinoa. Plants of 21 species in 9 families including exacum were infected. Inoculated exacum showed systemic dwarf symptom, sometimes with ring spots, about 3 weeks after inoculation. C. amaranticolor showed chlorotic local lesions followed by systemic downward leaf curling and chlorosis. C. quinoa showed chlorotic local lesions followed by severe systemic chlorosis. Broad bean showed apical necrosis. N. tabacum cv. Samsun and N. occidentalis were also infected. Electron microscopic observation of leaf dips of infected plants stained with 2% phosphotungstic acid (PTA, pH 6.0) revealed isometric particles with a diameter of approximately 27nm. Myzus persicae transmitted the pathogen from systemicaly infected C. amaranticolor to healthy C. amaranticolor after 1hr starving, 10min of acquision period and 3hr of inoculation period. Thermal inactivation point was between 60 and 65℃ and longevity in vitro was up to 4 days. Virus particles were trapped and decorated by Immuno-sorbent electron microscopy with antiserum to Broad bean wilt virus (BBWV)-serotype I (gift from Dr. V. Lisa). Cucumber mosaic virus was not detected by ELISA. Molecular weights of the coat proteins were estimated by Western blotting with antiserum to patchouli mild mosaic virus which is closely related to broad bean wilt virus. Two coat protein bands with molecular weights of approximately 42,000 and 26,000 were observed. The virus was partially purified by the modified method described by Xu et al. (1988) and the yield was 3.6mg/50g of fresh infected C. quinoa. With partially purified virus, antiserum (As-BBWV-E) to the virus was produced by a series of immunization to rabbits. As-BBWV-E could detect the virus by ELISA and Western blotting. Based upon the results, the virus, which caused severe dwarf in exacum was identified as a strain of Broad bean wilt virus (BBWV). Though patchouli mild mosaic virus (PaMMV) shares similar characters with BBWV and is serologically related to BBWV, PaMMV has narrower host range with mild symptoms on some plants. Since BBWV has two serotypes, analysis of the serotype of the virus isolated from exacum is necessary. The disease of exacum caused by BBWV has not been reported in Japan and it was named as dwarf (Ishuku byo)

Plant viruses and viroids in Japan

An increasing number of plant viruses and viroids have been reported from all over the world due largely to metavirogenomics approaches with technological innovation. Herein, the official changes of virus taxonomy, including the establishment of megataxonomy and amendments of the codes of virus classification and nomenclature, recently made by the International Committee on Taxonomy of Viruses were summarized. The continued efforts of the plant virology community of Japan to index all plant viruses and viroids occurring in Japan, which represent 407 viruses, including 303 virus species and 104 unclassified viruses, and 25 viroids, including 20 species and 5 unclassified viroids, as of October 2021, were also introduced. These viruses and viroids are collectively classified into 81 genera within 26 families of 3 kingdoms (Shotokuvirae, Orthornavirae, Pararnavirae) across 2 realms (Monodnaviria and Riboviria). This review also overviewed how Japan’s plant virus/viroid studies have contributed to advance virus/viroid taxonomy

LASIODIPLODIA THEOBROMAE ニ ヨル カカオ ノ カジツ フハイビョウ シンショウ

2005年5月,東京都小笠原村父島で施設栽培されていたカカオの果実に,初め茶褐色～暗褐色の不整病斑を生じ,のちに拡大して腐敗症状を呈する病害が観察された。その罹病部からはLasiodiplodia属菌が高率に分離され,分離菌を用いた接種試験により原病徴が再現され接種菌が再分離された。分離菌は,暗褐色から黒色の分生子殻内に,隔壁の無い側糸と,後に完熟すると2胞,暗褐色で縦縞模様をもつ分生子を形成することから,Lasiodiplodia theobromae (Pat.) Griffon & Maubl.と同定した。さらにrDNA内のITS領域を用いた分子系統解析の結果からも同定を裏付ける結果を得た。小笠原で商業的に栽培されているパッションフルーツ,パパイア,バナナ,マンゴーを用いた宿主範囲の検討では,パパイア,バナナ,マンゴーにおいて病原性が確認された。これまでカカオにおける本病害は日本において報告がないため,Lasiodiplodia果実腐敗病(英名 : Lasiodiplodia pod rot)と命名したい。Severe pod rot of cacao, Theobroma cacao, was observed at Chichijima, Bonin Islands, in May, 2005. The causal fungus was identified as Lasiodiplodia theobromae (Pat.) Griffon & Maubl. based on the morphological characteristics, inoculation experiments, and molecular phylogenetic analysis. Among the four tropical fruits cultivated in the Ogasawara Islands, cacao isolate showed clear pathogenicity on banana, mango and papaya fruits but not on passion fruit. This is the first report of Lasiodiplodia pod rot (kajitsufuhai-byo in Japanese) on cacao pods caused by L. theobromae in Japan