BIOLOGICAL AND MOLECULAR CHARACTERIZATION OF A NEW ISOLATE OF CUCUMBER GREEN MOTTLE MOSAIC VIRUS (CGMMV) IN INDONESIA

Transcription

1 BIOLOGICAL AND MOLECULAR CHARACTERIZATION OF A NEW ISOLATE OF CUCUMBER GREEN MOTTLE MOSAIC VIRUS (CGMMV) IN INDONESIA Budi Setiadi Daryono, Tri Joko 2, Alin Liana, Utari Saraswati Genetics Laboratory, Faculty of Biology, Gadjah Mada University, Yogyakarta 5528 Indonesia (Corresponding bs_daryono@mail.ugm.ac.id) 2 Laboratory of Agricultural Biotechnology, Faculty of Agriculture, Gadjah Mada University, Yogyakarta 5528 Indonesia Faculty of Mathematics and Natural Sciences STKIP Pembangunan Indonesia Makasar, Makasar Indonesia ABSTRACT Cucurbits grow throughout the Java island of Indonesia as dry season crops. However, the cucurbits cultivation has been faced with virus infections that reduce their production. Cucumber green mottle mosaic virus (CGMMV), which a member of the Genus Tobamovirus, has been reported infecting cucurbits in Indonesia. The most common symptoms of infected plants are leaf mosaic and mottling, growth stunting and malformation of leaf and fruit. The purpose of this research were to find out the occurrence of CGMMV infecting cucurbits in Daerah Istimewa Yogyakarta (DIY) and to identify the biological and molecular characterization. Survey and samples collection were conducted in several fields in Sleman and Kulon Progo, DIY during March June 29. Serological analysis confirmed that two angled loofah (Luffa acutangula L.) were positively infected by a virus related to Cucumber green mottle mosaic virus (CGMMV), and called CGMMV-ILa isolate. The reaction of indicator plants to CGMMV-ILa were the necrotic local lesion on Chenopodium amaranticolor, Chenopodium quinoa, and Momordica charantia. The mosaic symptoms on Citrullus lanatus, Cucumis sativus, Luffa acutangula, Nicotiana tabacum var. Virginia, and the chlorotic symptoms on Cucumis melo, but the CGMMV-ILa did not infect Lycopersicon esculentum. The coat protein (CP) of purified CGMMV-ILa detected with molecular weight of about 6 kda by SDS- PAGE analysis. Viral and complementary CGMMV-specific primer sets were designed for spanning the genome using previously reported CGMMV sequence. About 5bp of Coat protein (CP) gene of CGMMV- ILa was amplified by RT-PCR. The nucleotide sequence of CP gene of CGMMV- ILa shared 8% identities with several isolate of CGMMV. Based on these result, we identified CGMMV- ILa as a CGMMV isolate of Luffa acutangula, a member of Tobamovirus. Keywords: CGMMV, cucurbits, coat protein, RT-PCR INTRODUCTION Cucurbit crops, including melon (Cucumis melo L.), watermelon (Citrullus lanatus L.), cucumber (Cucumis sativus L.), angled loofah (Luffa acutangula L.) and bitter gourd (Momordica charantia L.), produced throughout Yogyakarta, a province in Indonesia, are important vegetable fruit crops. Java is one of the main cucurbit growing areas of Indonesia and more than 8% of national production is produced by five provinces in Java i.e. East Java, Central Java, Jakarta, West Java, and Yogyakarta (BSCI, 2). The yield of the crop of cucurbit plants depends not only on agricultural and meteorological condition, but also on the diseases including those caused by plant viruses. Plant viruses have become a problem in cucurbits production in Indonesia. Daryono and Natsuaki (29) reported that melon and cucumber plants grown in Java Indonesia during 2-2 were severally affected by a new kind of mosaic disease.

2 The genus Tobamovirus is one of the biologically and molecularly best-characterized groups with many species and strains varying widely in host range and its interaction with host plants (Yoon et al., 22). Phylogenetic analysis of recognized and tentative tobamovirus species has revealed four sub-groups within the genus, which corresponded well with groupings based on host range. These four tobamovirus sub-groups are those that infect () solanaceous plants; (2) brassicas; () cucurbit or legumes, and (4) Malvaceous (Adkins et al., 2). Tobamoviruses constitute a group of plant viruses with single positive-sense RNA molecules as genomes and rod-shaped virions consisting of viral RNA surrounded by a single coat protein (CP). Members of the genus Tobamoviruses form rigid, rod-shaped particles approximately nm in length and 8 nm in width. The genomes are capped at the 5 end and have a trna-like structure at the end (Lartey et al., 996). Tobamoviral RNAs encoded four polypeptides. Two large polypeptides, and 8-kDa proteins, function in viral RNA replication, and are translated from the same 5 proximal start codon. The -kda movement protein (MP) required for cell-to-c ell movement and the 7-kDa CP are translated from co-terminated sub-genomic mrnas derived from the portion of the tobamoviral RNAs (Lewandowski, 2). Cucumber green mottle mosaic virus (CGMMV) is a member of the genus Tobamovirus, a serious disease agent of cucurbit crops and causes significant economical losses in several countries (Tan et al., 2). This virus was firstly found in Great Britain (Ainsworth,95). Surveys of viral diseases of cucurbitaceae plants conducted in Java, Indonesia during March June 29 showed that amongst cucurbit plants observed, angled loofah crops grown in the area were severely infected by a new kind of mosaic disease. The infected plants showed growth retardation, mottle and mosaic on the leaves and water soaked spots on the surface of fruits. Sometimes malformation of fruits was also observed. The objectives of this study were to characterize a new isolate of CGMMV in Indonesia biologically, morphologically and serologically and to complete the sequence of coat protein gene of the isolate. MATERIALS AND METHODS Virus source, maintenance and electron microscopy. Some cucurbits plant leaf showing mosaic mottle and suggestive of virus infection were collected in the traditional fields in Sleman and Kulon Progo-Yogyakarta, Indonesia. The leaves were homogenized in mm sodium phosphate buffer (ph 7.) to transmit the viral agent to Chenopodium amaranticolor leaves mechanically. Cholorotic local lesions appearing in inoculated C. amaranticolor were used as inoculum source and for maintenance. Enzyme-linked immunosorbent assay analysis. Serological characters among cucurbits were compared by DAS-ELISA as described by Clark and Adams (977). All samples were tested at the same time in a single assay. The antisera and enzyme conjugated antibodies against CGMMV, respectively were used at :2 dilutions according to the manufacture instruction (Agdia Inc., Madison, USA). To compare absorbance values among plates directly, a CGMMV infected and a healthy melon were included in ELISA analysis. Absorbance at 45 nm was determined with a plate reader (Zenix). Host range determination. To determine the host range of CGMMV-ILa, crude sap from leaf samples of the virus, infected angled loofah were mechanically inoculated to 8 plant species representing families (Chenopodiaceae, Cucurbitaceae, and Solanaceae) that were pre-dusted with Carborundum (6 mesh). Plants were grown in glass house under continuous illumination (8 lux) at 5 o C and observed weekly for symptom development. Coat protein analysis. CGMMV-ILa coat proteins extracted from infected angled loofah leaves were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

3 on.5% SDS-PAGE gels and stained with Coomassie brilliant blue G-25 according to protocols described by Sambrook and Russel (2). Analysis of the virus coat protein gene. Viral RNA was extracted from upper leaf of mechanically inoculated angled loofah. First strand cdna was synthesized by one-steps RT-PCR Kit (SBS Genetech Co., Ltd. China) reverse transcriptase at 45 o C for minutes with a primer reverse 5 -AACTAAGCTTTCGAGGTGGTAG- and stop the reaction by incubating at 94 o C for 5 minutes. This primer and a forward primer 5 -GATGGCTTACAATCCGATCACA - were used to amplify the cdna product following by PCR conditions: 5 cycles of minute at 94 o C, minute at 48 o C and 2 minute at 72 o C. PCR products were fractionated in 2% agarose gel, which was then stained in ethidium bromide solution. Bands of the expected size were purified using purification kit (Bioron). Pure DNA were direct sequencing analysis on st BASE manufacture, Singapore. Nucleotide sequence was analyzed and compared using Software Sequence Scanner v.. and subjected to BLAST searches to identify related sequences available from the DDBJ database. The obtained sequence and available Tobamovirus sequence data were initially aligned with CLUSTAL W (Thompson et al., 994) to assemble the complete CGMMV-ILa CP gene, and Kyuri green mottle mosaic virus (KGMMV-YM) was used as the out group. Phylogenetic trees were obtained from the data by distance method using the PAUP 4. phylogentic package. The distance matrix was analyzed by algorithms based on Saitou and Nei s neighbor-joining (Saitou and Nei, 987). The strength of the internal branches from the resulting tree was statistically tested by bootstrap analysis from, bootstrap replications. RESULTS The Incidence of viral diseases and serological relationships. The cucurbits plants showing specific symptoms of growth retardation, mottle and mosaic on the leaves and fruit malformations were collected from many areas of cucurbits growing field in Sleman and Kulon Progo, Yogyakarta, Indonesia between March and June 29 (Table and Table 2). Serological analysis among cucurbits was compared by Double Antibody Sandwich Enzyme-Linked Immunosorbent Assay (DAS-ELISA) and all samples were tested at the same time in a single assay. The result showed that two samples of angled loofah (Luffa acutangula L.) were positively infected by CGMMV. Tests were conducted using several negative controls of cucurbit plants. Therefore, the range of absorbance values of negative controls varied from.6 to.84 at 45 nm, whereas positive samples gave absorbance value of.5 to.29 (Table ). Furthermore, RT-PCR results showed a specific DNA band of coat protein gene of CGMMV-LA, around 486 base pair (Fig. 2). Table. Numbers of fields surveyed and samples collected and tested in each district/village during survey in March June 29 District, Villages No. of fields No. of samples DAS-ELISA* surveyed collected CGMMV Sleman Cangkringan Ngemplak Kadirojo Tempel Brebah Kulon Progo Karang Sewu Congot Glagahsari Total % infection * DAS-ELISA reading (absorbance at 45 nm) of sample extracts / / /5 /6 2/2 / /2 /

4 Table 2. The incidence of viruses on cucurbit plants grown in Yogyakarta Spesies No. of samples tested CGMMV* Cucumis melo Cucumis sativus Citrullus lanatus Momordica charantia Luffa acutangula Total 5 2 Percent (%) Infected - 4 * DAS-ELISA reading (absorbance at 45 nm) of sample extracts Table. Range of absorbance values of negative control, positive and negative samples Absorbance values Plant Negative controls Positive samples Negative samples Angled loofah Sample Sample Host range determination. Results of an experimental host range for CGMMV-ILa were shown in Table 4. No evidence of infection was detected in Lycopersicon esculentum, but symptoms were detected in Nicotiana benthamiana. Symptoms were produced on inoculated leaves of five species in the Cucurbitaceae (Luffa acutangula, Cucumis melo, Cucumis sativus, Citrullus lanatus, and Momordica charantia). Chenopodium amaranticolor and Chenopodium quinoa commonly used as indicator hosts for Tobamovirus developed necrotic local lesions on inoculated leaves. Table 4. Comparison of experimental host range between isolates of CGMMV Family Species Symptoms of CGMMV Chenopodiaceae Cucurbitacaeae Solanaceae Chenopodium amaranticolor Chenopodium quinoa Cucumis melo Cucumis sativus Citrullus lanatus Luffa acutangula Momordica charantia Lycopersicon esculentum Nicotiana benthamiana NLL NLL C M, D Mo, D M NLL Mo, M, D M: Mosaic; C: Chlorotic; Mo: Mottle; NLL: Necrotic local lesions; D: Leaf deformation; : No symptom.

5 Fig.. SDS-PAGE of CGMMV-ILa viral coat protein. H: Healthy; CG: CGMMV-ILa; M: Protein standard markers (BIO-RAD). Arrows indicate approx. 6 kda coat protein for CGMMV-ILa. Fig. 2. RT-PCR products of coat protein genes of CGMMV strains by 2% agarose gel electrophoresis. H: Healthy leaf; CP: CGMMV-ILa; CP2: CGMMV; M: DNA marker (Roche). Arrow indicates the 486 bp of CGMMV-ILa CP gene. Coat protein analysis and CP sequence analysis. Coat protein of CGMMV-ILa purified from infected leaf samples were analyzed by SDS-PAGE. The coat protein

6 molecular weight was ~6 kda (Fig. ). The CP gene encoded a protein consist of 6 amino acids with a predicted translation product of ~6 kda, consistent with the size of SDS-PAGE. The only sequence producing significant alignments from initial BLAST query was tobamovirus coat proteins including many strains of CGMMV. The CP of CGMMV-ILa was composed of 486 nucleotides and 6 amino acid residues, as same as CP of CGMMV-SH. Coat protein of CGMMV-ILa shared 8.9% nucleotide identities with CGMMV-SDAU-TANG, CGMMV-LN, and CGMMV-GR7 (Table 5). Result of phylogenetic tree for CP gene analyses displayed CGMMV-ILa presents in clusters of cucurbits infecting tobamovirus (Fig. ) together with KGMMV-YM and strains of CGMMV. Table 5. Homology of nucleotides sequence identities of CP gene between CGMMV-ILa and other CGMMV strains Homology GenBank Viruses* Accession no. Nucleotides (%) Amino Acid (%) Number of total amino acid CGMMV-SH DI CGMMV-SDAU- GQ TANG CGMMV-BG FJ CGMMV-LN EU CGMMV-Zucchini AB CGMMV-AL AJ CGMMV-GR7 AJ CGMMV-MC- EF KGMMV-YM AB *CGMMV; Cucumber green mottle mosaic virus, KGMMV; Kyuri green mottle mosaic virus Fig.. Comparison of coat protein sequences of CGMMV angled loofah isolate and the recognized species in the genus Tobamovirus.

7 DISCUSSION An Indonesian isolate of angled loofah infecting Tobamovirus (CGMMV-ILa) was originally detected and isolated from a field of angled loofah in Sleman and Kulon Progo, Yogyakarta, Indonesia, and it appears to be a new isolate of CGMMV based on several criteria. The virion morphology of CGMMV-ILa is consistent with those of tobamoviruses. Furthermore, the experimental host range of CGMMV-ILa was identical symptoms with previously reported of CGMMV isolates. CGMMV- ILa induced necrotic local lesions on Chenopodium amaranticolor. CGMMV- ILa not infected on Lycopersicon esculentum by symptom (Table ). The difference in the response of Chenopodium amaranticolor and Lycopersicon esculentum to CGMMV- ILa indicates that these strains are not identical in host reactions although they shared their natural host preference. Alignment of CGMMV-ILa CP with CP sequences of previously reported tobamovirus species shares 56. to 8.% amino acid identity and 5.9 to 8.9% nucleotide identity. Based upon CP sequence, CGMMV-ILa is most similar to CGMMV- SDAU-TANG and CGMMV-LN, sharing 8. and 8.9% of amino acid and nucleotide identity, respectively. The relationships between CGMMV-ILa and other recognized tobamovirus species based on the amino acid sequences of the CPs gene with phylogenetic tree analysis resulted in a closer relationship of CGMMV-ILa than to KGMMV-YM as to other tobamovirus species. CGMMV-ILa may have a common ancestor which could infect some cucurbit plants and then diversified or differentiated its coat protein gene and host range separately. Since CGMMV is very stable and is mechanically transmissible during pruning or harvesting, or through infected seeds, it is essential to survey the occurrence of cucurbitinfecting tobamoviruses in Asia and produce commercially available resistant cucurbits. For protection, it is also important to sow virus free seeds and use virus free irrigation water and soil. ACKNOWLEDGEMENT This research was funded by The Indonesia Toray Science Foundation (ITSF) Science and Technology Research Grant for 29. We would like to thank Gadjah Mada University-Indonesia for supporting research facilities. REFERENCES Ainsworth, G.C. 95. Mosaic disease of cucumber. Ann.Appl. Biol.22: Bureau of sentral statistics, Indonesia (BSCI). 2. Statistical pocket book of Indonesia, Jakarta, Indonesia. Clark M.F., and Adams, A.N Characteristics of the microplate method of enzymelinked immunosorbent assay for the detection of plant viruses. J. Gen. Virol. 4:

8 Daryono, B.S., Somowiyarjo, S., and Natsuaki, K.T. 25. Biological and molecular characterization of melon-infecting Kyuri green mottle mosaic virus in Indonesia. J. Phytopathology 5 (): Daryono, B.S., Somowiyarjo, S., and Natsuaki, K.T. 26. Biological characterization and complete nucleotide sequence of coat protein of Kyuri green mottle mosaic virus isolated from Angled Loofah in Indonesia. J. Agricultural Science, Tokyo University of Agriculture.5 (): Inoue, T., Inoue, N., Asatani, M., and Mitsuhata, K Studies on cucumber green mottle mosaic virus in Japan (in Japanese). Nogaku Kenkyu 5: Lee, S.H., Lee, Y.G., Park, J.W., Cheon, J.U., Lee, K.W., and Choi, Y.C. 2. Nucleotide sequence of coat protein gene of Kyuri green mottle mosaic virus (KGMMV) isolated from zucchini (Cucurbita pepo) in Korea. Plant Pathol. J. 6 (2): Rosner, A., Bar-Joseph, M., Moskovitz, M., and Mevarech. M, 98. Diagnosis of specific viral RNA sequences in plant extracts by hibridization with a polynucleotide kinase-mediated, 2 P-labeled, double-stranded RNA probe. Phytopathology 67: Saitou, N. and Nei, M The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol 4: Sambrook, J., and Russell, D.W. 2. Molecular cloning: A laboratory manual. rd ed. Cold Spring Harbor Laboratory. Cold Spring Harbor, NY. Tan, S.H., Nishiguchi, M., Murata, M., and Motoyoshi, F. 2. The genome structure of Kyuri green mottle mosaic tobamovirus and its comparison with that of cucumber green mottle mosaic tobamovirus. Arch. Virol. 45: Thompson, J.D., Higgins, D.G., and Gibson, T.J CLUWTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequencing weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: Yoon, J.Y., Min, B.E., Choi, S.H., and Ryu, K.H. 2. Completion of nucleotide sequence and generation of highly infectious transcripts to cucurbits from fulllength cdna clone of Kyuri green mottle mosaic virus. Arch. Virol. 46: