Vigna angularis var. nipponensis was an annual wild species belonging to the subgenus Ceratotropis in the genus Vigna. It is widely distributed in East Asian and Southeast Asian countries including Korea, Japan, China, Nepal, and Taiwan. V. angularis var. nipponensis was formerly treated as a progenitor of Phaseolus or Azukia (Tateishi, 1984; Lumpkin and McClary, 1994; Yamaguchi, 1992). V. angularis var. nipponensis is expected to be a valuable genetic source for breeding of azuki bean (Siriwardhane et al., 1991; Kaga et al., 2008). It is also very important species for the study of origin of Azuki bean.

Several virus are known to infect Azuki bean including Alfalfa mosaic virus (AMV), Azuki bean mosaic virus (AzMV), Blackeye cowpea mosaic virus virus (BICMV), Bean common mosaic virus (BCMV), Bean yellow mosaic virus (BYMV), Cucumber mosaic virus (CMV), and Cowpea mosaic virus (CPMV) (Iizuka et al., 1990; Tsuchizaki et al., 1987). Among them, AMV (Heo et al., 1976; Jung et al., 2000), BCMV (Choi et al., 1989), and CMV (Choi et al., 1998; Heo et al., 1976) have been reported in Korea.

In this article, we report the occurrence of a CMV on Vigna angularis var. nipponensis in Korea, characterized on the basis of biological and complete nucleotide sequence-based properties, and named it as CMV-wVa. To our knowledge, this is the first report of CMV on V. angularis var. nipponensis in the world.

Vigna angularis var. nipponensis, showing severe mosaic, chlorotic spots, and yellow symptoms were collected from fields around Suwon, Korea (Fig. 1). The collected isolate was identified by conducting Double Antibody Sandwich-Enzyme Linked Immune Sorbent Assay (DAS-ELISA) and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) using CMV specific forward (5′-TGGTCGTCCAACT ATTAACCAC-3′), Reverse (5′-TACTGATAAACCAGTACCGGTGA -3′) primers of the coding region of coat protein gene. Each cycle included a denaturing step at 94°C for 30 sec, an annealing step at 55°C for 30 sec, and extension step at 72°C for 90 sec, and finally kept at 72°C for 10 min. DAS-ELISA was performed using CMV polyclone antibody described by Clark and Adams (Agdia, USA). Shortly, CMV antibody and its conjugate were both diluted 1:200 and all incubations were carried out at 37°C for 1 h except for the substrate that was incubated for 30 min. Quantitative measurements were examined by absorbance at 405 nm (A₄₀₅). To identify the virus isolate from V. angularis var. nipponensis, infected-leaf was macerated in 0.01 M sodium phosphate buffer, pH 7.0 and the crude sap was rubbed to the leaves of indicator plants including Chenopodium amaranticolor. Single lesions were isolated and re-inoculated on healthy Ch. quinoa three consecutive times. The isolate obtained from the third re-inoculated Ch. quinoa was designated as CMV-wVa, propagated in Nicotiana tabacum cv. ‘Xanthi-nc’, and was subjected to biological and molecular analyses.To test the infectivity of virus isolate and the symptoms on indicator plants, seedlings of test plants at the 3-5 leaf stage were inoculated by sap extracted from N. tabacum cv. ‘Xanthi-nc’. The plants were put in an insect-free greenhouse maintained at 20-25°C for 12-16 h light period. Disease symptoms were recorded four times a week for 30 days. CMV-wVa isolate caused mosaic or vein clearing symptom on the upper leaves of N. tabacum cv. ‘Xanthi-nc’, N. glutinosa, Capsicum annuum, Petunia hybrid, Cucurbita moschata, Cucumis sativus, and Cu. melo. In addition, the CMV-wVa isolate induced severe mosaic, malformation or fern leaves on upper leaves of Solanum lycopersicum (Table 1) and C. pepo (Table 1 and Fig. 2D). CMV-wVa induced necrotic local lesion on inoculated leaves and chlorotic ring local, mosaic symptoms on upper leaves of Vigna radiate (Table 1 and Fig. 2G). Local lesions were observed on Datura stramonium, Ch. amaranticolor, and Ch. quinoa. However, no symptoms were observed on Glycine max (cvs. ‘Sorok’, ‘Sodam’, and ‘Somyeong’), Brassica peckinensis L., Raphanus sativus L., and Spinacia oleracea L. (Table 1).

To compare the infectivity of CMV-wVa isolate with CMV isolates of various host, CMV-Z(zucchini), -RB(cone flower), -ZM(maize), -Pa(azuki bean), and CM-19(azuki bean) (Choi et al., 1998; Kim et al., 2010a, 2010b, 2011 and lizuka et al., 1990) along with CMV-wVa were inoculated onto 6 different plant species belonging to Fabaceae. Four to 30 plant seedlings of each test plant listed in Table 2 were inoculated with sap extracted from N. tabacum cv. ‘Xanthi-nc’. The CMV-wVa, -Z, -RB and -Pa induced similar chlorotic and necrotic symptoms as well as mottle and mosaic symptoms on inoculated or upper leaves of Vigna angularis. However, symptoms of necrotic local or pin point were observed on inoculated leaves of V. unguiculata, Vicia fava, Pisum sativum, and Phaseolus vulgaris with no systemic symptom. CMV-ZM isolate failed to infect in V. angularis and Phaseolus vulgaris (Table 2; Fig. 2). The experimental infectivity of CMV-wVa was similar to that reported for CMV isolates of zucchini, cone flower, maize and azuki bean in the host reaction of Solanaceae and Cucubitaceae (Choi et al., 1998 and Kim et al., 2010a, 2010b, 2011). However, Japan CMV isolate (CM-19) from azuki been did not systemically infect V. radiate (Iizuka et al., 1990), only CMV-wVa isolate infected systemically causing local chlorotic ring or necrosis and systemic mosaic on V. radiata leaves. These differences are considered as unique characteristics of CMV-wVa.

CMV has one of the broadest host ranges among plant viruses, and its numerous stains have been differentially characterized (Palukaitis et al., 2003). Most CMV typically infects Solanaceae and Cucurbitaceae, but either produce local lesions or do not infect legume species (Whipple et al., 1941). CMV-wVa isolate infected not only V. angularis and V. radiate but also Solanaceae and Cucurbitaceae. Despite no ordinary occurrence of CMV in wild V. angularis var. nippensis, CMV-wVa has a broad host ranges including red bean, mung bean, tomato, pepper and cucumber. So it is very important role in CMV sources for aphid transmission into commercial fields.

Total RNAs were extracted from the N. tabacum leaves that were inoculated with CMV-wVa by easy-spin^™ total RNA kit (iNtRON, Korea). Sequence specific full-length primers were designed based on previously reported genomic sequences of CMV isolates available in GenBank of National Center for Biotechnology (NCBI) (Kim et al., 2010b). RT reaction was carried out at 42°C for 30 min, and was denatured by heating at 95°C for 5 min in 5× RT buffer containing 5 μg of total RNA, 10 pmoles of the downstream primer, 2.5 mM each of four dNTPs, and 2.5 units AMV reverse transcriptase (Promega, USA). The RT reaction mixture was mixed with 1.25 units GoTaq^™ DNA polymerase (Promega, USA), 5× Green PCR buffer and 2.5 mM MgCl₂, and 10 pmoles of the upstream primer followed by amplification in a Bio-Rad Thermal Cycler (USA). The amplified PCR products were purified using PCR gel/direct extraction kit (iNtRON, Korea) and sequenced. End sequences of the genomes were obtained with the 5′/3′ rapid amplification of cDNA ends (RACE) protocol (Frohmant et al., 1988). cDNA clones containing the 5′ end of the genomes were obtained using Xec primers (5′ AAAGAATTCCCCCCCCCCCC-3′) and CMV 5′-RNA1 (5′-TCCTTTATCGCCGTGGGAGGCTAC-3′); for CMV 5′-RNA2 (5′-TCCTCGGGAGTGTCGACACC-3′); and for CMV 5′-RNA3 (5′-CCTACTGGTACCTTGGAAAGCCAT-3′) primers complementary to nucleotides 151-128, 159-140, and 144-121, respectively, of CMV strain. In addition, cDNA clones containing the 3′ end of the genomes were obtained using CMV 3′-RNA1 (5′-TTGCTCGTGCTT AGCGG3′); for CMV 3′-RNA2 (5′-ACAAAGTCCCAGCGAGAG-3′); for CMV 3′-RNA3 (5′-TTGATTCTACCGTGTGGGTGACAGT-3′), and anchor primers (5′-GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTTTV-3′).

The lengths of CMV-wVa RNAs 1, 2, and 3 were consisted of about 3.3 kb, 3 kb and 2.2 kb, respectively. CMV-wVa RNAs 1, 2, and 3 nucleotide sequences were deposited at the GenBank under accession codes JX014246, JX014247, and JX014248, respectively. So far full length three RNAs segment sequence of wild azuki bean of CMV have been not reported. This is the first record on Vigna angularis var. nipponensis. The sequences were then compared with corresponding regions of the other CMV isolates along with previously reported Korean CMV isolates (-Z, -RB, -ZM and -Pa) retrieved from the GenBank database (Choi et al., 1998; Kim et al., 2010a, 2010b, 2011 and Roossinck, 2002). The sequences of Japan CM-19 isolate from azuki bean have been not reported.

Multiple nucleotide/amino acid sequence alignment was performed by using Clustal W (MEGA 5.0). Comparative sequence analysis disclosed that the 1a, 2a, 2b, 3a, and CP ORFs of CMV-wVa had 89.1-98.8%, 89.7-98.6%, 85.1-100%, 91.3-97.6%, and 91.8-98.0% nucleotide sequence identity, respectively, with CMV subgroup I isolates at the nucleotide level (Table 3). In contrast, identities of 54.4 to 79.0% were observed with subgroup II isolates at the nucleotide level. Database comparisons of the deduced amino acid sequences showed 94.2 to 99.2% sequence identity with 1a ORF gene of CMV subgroup I. In CMV-wVa, 1a ORF showed highest sequence identity at nucleotide level with the CMV-ZM isolate (98.8%) and 2a and 2b ORFs of CMV-wVa RNA2 showed 98.6 and 100% sequence identity with the CMV-Mf and -RB isolates, respectively. Similarly, the 3a and CP ORFs of CMV-wVa showed 97.6 and 98.0% sequences identity with the CMVY and -Z isolates, respectively. To compare the sequence identity of CMV-wVa isolate with CMV-Pa from azuki bean in Korea showed lowest sequence identity (91.3%) of 3a ORF (Table 3). In general, nucleotide and the deduced amino acid sequences of five ORFs of CMV-wVa showed highest sequence identity with CMV subgroup I isolates. Phylogenetic analysis were performed employing the Maximum likehood method packaged in the MEGA 5.1 software and with the complete nucleotide sequences of three segments of CMV isolates from the GenBank database. The bootstrap maximum-likehood trees for these genomic regions are shown (Fig. 3A, B, and C). Phylogenetic analysis of RNAs 1, 2, and 3 showed that 17 CMV isolates were divided into two subgroups such as subgroup I and II. CMV-wVa isolate was belonged to subgroup I and was closely related to the CMV-ZM and -Rb in analysis with the RNA1 sequence. Analysis of nucleotide sequences with RNAs 2 and 3 showed that CMV-wVa was most closely related to the members of subgroup IA. Especially, CMV-wVa RNA1 was divided in subgroup I. Along with CMV-wVa RNA1, RNA2 and RNA3 of CMV-wVa were also assigned to subgroup I based on phylogenetic analysis (Fig. 3). Therefore, results of phylogenetic analyses of the RNA segments 1, 2, and 3 suggested that CMV-wVa belongs to subgroup I of CMV.

Vigna angularis var. nippensis is considered to be the wild counterpart of azuki bean (Lumpkin and McClary, 1994; Tateishi, 1984; Yamaguchi, 1992) and is distributed widely in Asian countries including Korea. Several azuki bean crops naturally infected by CMV have been reported (Choi et al., 1998; Heo et al., 1976; Iizuka et al., 1990).

However, there is no report of natural infection of V. angularis var. nippensis (wild azuki bean) by CMV in the world. CMV-wVa isolate causing severe mosaic, necrotic, and yellow symptoms on Vigna angularis var. nipponensis identified by serological test (DAS-ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). Results of this study indicated that CMV-wVa isolate infected systemically causing local chlorotic ring or necrosis and systemic mosaic on V. radiata and economically plants including pepper, tomato. CMV-wVa was more closely related to CMV isolates belonging to CMV subgroup I. This is the first reported show the occurrence and identification of CMV subgroup I on V. angularis var. nippensis on the basis of biological and complete sequence analyses. Further studies of comparison analysis between CMV-wVa (wild azuki bean, Korea) and CM-19 (azuki bean, Japan) will be required for identification of gene involved in pathogen and geographical relation.