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First Report of Plum pox virus Strain M in Japan

Oishi, M., Inoue, Y., Kagatsume, R., Shukuya, T., Kasukabe, R., Oya, H., Hoshino, S., Ushiku, S., Fujiwara, Y., Motokura, Y., Maeda, Y.
Plant disease 2018 v.102 no.4 pp. 829
Plum pox virus, Prunus mume, RNA, apricots, enzyme-linked immunosorbent assay, fruit crops, genes, home gardens, immunoaffinity chromatography, leaves, monoclonal antibodies, national surveys, nucleotide sequences, orchards, parks, peaches, phylogeny, plums, reverse transcriptase polymerase chain reaction, reverse transcription loop-mediated isothermal amplification, roadsides, trees, Europe, Japan, Mediterranean region
Plum pox virus (PPV) is a causal agent of plum pox (sharka), the most serious viral disease for the stone fruit crops (Prunus spp.) (García et al. 2014). Nine PPV strains are known, out of which strains D and M are the most widespread and economically important. In 2009, PPV-D was detected from Japanese apricots (Prunus mume) in Japan (Maejima et al. 2011). A nationwide survey was then started, followed by an eradication program. In the program, the collected samples were tested by a PPV immunochromatographic assay kit (NIPPON GENE, Japan), and only those with positive results were further tested by the reverse transcription loop-mediated isothermal amplification method (RT-LAMP) using a PPV detection kit (NIPPON GENE). In June 2016, some Japanese apricots and apricot (P. armeniaca) trees were found with typical PPV symptoms such as ringspot and mottle on leaves, along with some peach (P. persica) trees with mild mottle on leaves, in Yokohama, Japan. These trees were distributed in a small area in Yokohama city. Most of them were grown in home gardens, and some were on roadsides, parks, and orchards. The ages of these trees varied, ranging from those estimated to be about 5 years old to 20 years or more. The leaves sampled from these trees were tested by immunochromatography and RT-LAMP. Although immunochromatography gave positive results on 133 samples, only weak or no reactions were observed by RT-LAMP. Because the RT-LAMP kit was designed and improved to detect PPV-D in Japan, the negative results suggested the existence of strains other than D strain. To make clearer the presence of PPV and its strains, the samples were tested by reverse transcription PCR with polyvalent PPV primers P1/P2 (Wetzel et al. 1991) as well as some specific primers for strain D P1/PD (Olmos et al. 1997), strain M P1/PM (Olmos et al. 1997), and strain Rec mD5/mM3 (Subr et al. 2004). As a result, only the polyvalent primers P1/P2 and strain M specific primers P1/PM gave positive results. Subsequently, these samples were analyzed by double antibody sandwich indirect ELISA with PPV-M specific monoclonal antibody line AL (Agritest, Italy), and all of them (133 samples) showed positive results. Among the 133 positive samples, the proportions of P. mume, P. persica, and P. armeniaca were 114 (85.7%), 3 (2.2%), and 15 (11.2%), respectively, and one (0.75%) was P. tomentosa. As an example of infection rate, in the investigated park, 38% (11/29) of the Japanese apricot trees were infected. Total RNAs were extracted from one of the infected P. mume trees, and its whole genome sequence was determined by direct sequencing method (Maejima et al. 2011) with minor modification to the primers used. As a result, 9,786 bp of the complete nucleotide sequence was obtained (LC228949) and showed 98% identity with PPV-M isolates (AJ243957, M92280) throughout its genome. While phylogenic analysis based on CP gene sequence indicated that this isolate was relatively close to PPV-M isolates from Mediterranean countries compared with isolates from central-eastern European countries, the origin of M strain in Japan remains unresolved. This is the first detection of PPV-M in Japan, and the relevant area was added to the national eradication program in February 2017.