First Report of a Tospovirus in Mulberry.

Mulberry (Morus alba L.) is an economically important crop grown widely throughout Asia. Various virus-like symptoms including mosaics, vein banding, and chlorotic ringspots have been observed and reported on mulberry trees in China and Japan for decades. However, the etiology of mulberry viral diseases is generally understudied, although two mulberry-infecting viruses, Mulberry latent virus (genus Carlavirus) (2) and Mulberry ringspot virus (genus Nepovirus) (3), have been partially characterized. In a recent (2010 to 2011) field survey in Guangxi Province, China, supported by the local government, the incidence of virus-like diseases of mulberry ranged between 40 and 80%. To identify the viruses infecting mulberry, deep sequencing of small RNAs (4) was conducted using an Illumina Genome Analyzer. Small RNAs were isolated from five samples of mulberry leaves showing various virus-like symptoms and sequenced. Among the contigs assembled, a 445-bp contig (GenBank Accession No. JX268597) was found to share 76.6% nucleotide identity and 83.0% amino acid identity to Groundnut bud necrosis virus (genus Tospovirus, family Bunyaviridae; Accession Nos. U42555 and AAC55521). To obtain a longer cDNA fragment of this virus, a reverse transcription (RT)-PCR was done with primers MV-N-F (5'-AAGCCATCAATGTGCCTCCGGA-3') and MV-N-R (5'-AACACCATGTCTACCGTCCGTC-3') that align to the S-RNA sequence encompassing the nucleocapsid (N) gene and a portion of the intergenic region (IGR) of the Tospovirus. PCR products of about 1,000 bp were successfully amplified from the total RNA of the three mulberry samples (sl-1, xcsy-1, and xcsy-4) showing vein banding symptoms, but not from asymptomatic mulberry (jk-1). These PCR products were cloned and sequenced. The lengths of the amplicons were 1,027 bp (isolate sl-1, JX173786), 987 bp (isolate xcsy-1, JX173787), and 979 bp (isolate xcsy-4, JX173788) and the partial IGRs of the sl-1, xcsy-1, and xcsy-4 isolates were 187 bp, 147 bp, and 139 bp, respectively. The coding regions for the N protein were 831 bp and the deduced proteins of 277 amino acid residues were 100% identical for all three isolates. Since the N protein of this virus shared up to only 74.4% identity to other tospoviruses (74.4% to Capsicum chlorosis virus, ABB83818; and 71.5% to Watermelon bud necrosis virus, ABY79095), it may represent a new member of the Tospovirus genus, temporarily named Mulberry vein banding virus (MuVBV), according to the species demarcation criteria for the Bunyaviridae (1). To the best of our knowledge, this is the first report of a Tospovirus infecting M. alba. In an RT-PCR screening of 48 randomly selected mulberry samples suspected to be virus-infected, 32 were MuVBV-positive. Giving the high incidence and the high yield loss associated with Tospovirus and the presence of thrips, suspected vectors for the virus, MuVBV may represent a substantial threat to the silkworm industry in China. References: (1) M. Q. K. Andrew et al. Virus Taxonomy: 9th Report of the ICTV. Elsevier Academic Press, San Diego, 2012. (2) T. Tsuchizaki. Annu. Phytopath. Soc. Japan 42:304, 1976. (3) T. Tsuchizaki et al. Annu. Phytopath. Soc. Japan 37:266, 1971. (4) Q. Wu et al. PNAS. 107:1606, 2010.