Identification of Tomato yellow leaf curl virus Naturally Infecting Common Bean in Japan

Tomato yellow leaf curl virus (TYLCV; family Geminiviridae, genus Begomovirus) is a devastating pathogen vectored by the whitefly Bemisia tabaci causing significant yield losses to tomato crops in Japan since 1998 (2). So far, there has been no report of this virus infecting common bean (Phaseolus vulgaris; family Fabaceae) in Japan. But recently, TYLCV has been reported from P. vulgaris in Spain and China (1,3). P. vulgaris is a vegetable crop commonly grown during spring to summer in Japan. In the course of a study to assess virus incidence on P. vulgaris exhibiting yellowing and thickening symptoms with 20 to 30% incidence, four symptomatic and two healthy samples were collected in August, 2012 from a single mix-cropping field (where P. vulgaris was cultivated together with tomato [Solanum lycopercisum] crop and a high B. tabaci population density was observed on plants) in Komae, Japan. Meanwhile, the vector for one crop can transmit the disease to the next crop very easily. To identify possible begomovirus present in symptomatic P. vulgaris plants, total nucleic acids were extracted from plants with and without symptoms using PhytoPure Plant DNA Extration Kit (GE Amersham Biosciences, UK) and begomovirus replication was confirmed from symptomatic leaves in 3 out of 4 plants when hybridized with a specific non-radioactive probe to the coat protein region of TYLCV (5) using a Biotin DNA Labelling Kit (Fermentas). To confirm the identity of the virus detected, leaf samples were further tested by PCR using the TYLCV specific detection primers (TYF/TYR) corresponding to the V2/CP region and a DNA fragment of an expected size was obtained from the samples positive for TYLCV in Southern hybridizations (4). The PCR products were sequenced directly and these sequences showed the highest nucleotide sequence identity >99% to the TYLCV isolate AB116630 reported from Japan. Rolling circle amplification (RCA) was done on all samples to produce concatamers and with Sac I restriction enzyme an amplicon of ~2.7 kb was obtained and cloned into PUC118 vector (Takara, Japan). Multiple clones were obtained showing the same restriction pattern with different restriction enzymes, and three of them were randomly selected and sequenced by Macrogen (Japan). Sequence analysis showed all three clones were identical and one sequence (J18.11) was submitted to GenBank. This sequence (GenBank Accession No. KJ585666) was 2,774 nt long and displayed the arrangement of four ORFs (AC1, AC2, AC3, and AC4 in complementary sense) and two ORFs (AV2 and AV1) on virion sense) typical of the genome of a begomovirus. This whole genome sequence was compared with those of other reported begomoviruses and exhibited greater than 99% nt sequence identity to a previously reported TYLC sequence (JN183876). Efforts to identify the presence of an additional begomovirus components or DNA satellites either by PCR and/or RCA were negative, suggesting that this is a monopartite begomovirus and an isolate of TYLCV. This is the first report of a natural infection of TYLCV in P. vulgaris in Japan. The presence of TYLCV on P. vulgaris could therefore represent a serious threat for this valuable crop in Japan. Thus, it is important to develop an effective way to control this virus to reduce the further losses. References: (1) Y. H. Ji et al. Plant Dis. 96:1229, 2012. (2) K. Kato et al. Ann. Phytopathol. Soc. Jpn. 64:552, 1998. (3) J. Navas-Castillo et al. Plant Dis. 83:29, 1999. (4) M. S. Shahid et al. J. Phytopahol. 161:205, 2013. (5) M. S. Shahid et al. Viruses 6:189, 2014.