Research on the Genetic Variation of Plum Pox Virus Strain M Occurred in Japan

Res. Bull. Pl. Prot. Japan No. 56：1 ～ 8（2020） Short communication

Research on the Genetic Variation of Plum pox virus Strain M Occurred in Japan

Hitoshi OYA, Yuji FUJIWARA1), Ryuichiro KASUKABE2) and Yoichi MOTOKURA1)

Research Division,Yokohama Plant Protection Station, 1-16-10 Shinyamashita, Nakaku, Yokohama 231‒0801 Japan.

Abstract: Plum pox virus (PPV) is a causal agent of plum pox (sharka), the most serious viral disease for stone fruit crops. In 2016, PPV strain M (PPV-M) was detected in Japanese apricot and peach trees in Japan. For research on the genetic variation of PPV-M that occurred in Japan, the complete genome sequences of 38 PPV-M isolates collected from the cities of Yokohama and Kawasaki were determined. Sequence analyses revealed that the nucleotide sequence diversity of the 38 Jap- anese isolates was low. In the phylogenetic analysis of the PPV-M based on complete nucleotide sequences, the Japanese and European isolates formed their own distinct monophyletic groups, and the clades of Japanese isolates were not relevant to the sample geographic origin. These results suggest that the time of first occurrence was relatively recent, as Japanese PPV-M isolates has a low accumulation of mutations. They also further suggest that the routes of viral entry into Japan were independent, and that the spread of infection occurred in a relatively short time. Key Words: Plum pox virus strain M, Prunus, genome, phylogenetic analysis

Introduction mated the origin and spread patterns within the occurrence area.

Plum pox virus (PPV) is a plant virus of the genus Potyvirus that Materials and Methods infects the genus Prunus. Ten strains of PPV (D, M, EA, C, Rec, W, T, An, CR and CV) have been described based on their biological, se- 1. Test samples rological and molecular biological properties (Garcia et al., 2014, The test samples were selected from samples collected during PPV Chirkov et al., 2018). In Japan, the occurrence of PPV was confirmed detection surveys in Yokohama and Kawasaki in 2016 - 2018 to cover in Japanese apricot (Prunus mume) in Ome City, Tokyo in 2009 all the hosts (Japanese apricot, apricot (Prunus armeniaca), peach (P. (Maejima et al., 2010), and a nationwide PPV detection survey and persica), Nanking cherry (P. tomentosa) and Japanese bush cherry (P. eradication program were started. Although the strain of PPV identi- japonica)) and the entire occurrence area (Table 1). One hundred mg fied in the survey up to the year 2015 in Japan was the D strain of single lesion tissue was cut out from one leaf, and RNA was ex- (PPV-D), the M strain (PPV-M) has been confirmed in the adjacent tracted with RNeasy Plant Mini kit (QIAGEN). It was confirmed that cities of Yokohama (Oishi et al., 2018) and Kawasaki in 2016. only the PPV-M was present by using primers specific to the D, M For PPV-D, there is a report that estimated its origin by analyzing and Rec strains (Olmos et al., 1997, Subr et al., 2004) by QIAGEN the PPV full-length genome sequences collected in the Kanto region OneStep RT-PCR kit (QIAGEN). in Japan (Maejima et al., 2011). However, there is no report on the Japanese PPV-M about its history, the time of first occurrence, and 2. Sequencing of the genome of PPV the spread of the occurrence area. Sequencing of the PPV full-length genome (9786 bp) was per- Therefore, in this study, we analyzed the PPV-M full-length ge- formed using a method (Oishi et al., 2018) which was modified from nome sequences collected from various hosts and places, and clarified the previously reported method for analyzing PPV-D (Maejima et al., the characteristics of the mutations and diversity. Furthermore, by 2011). The entire PPV genome was amplified using the RNA extract comparing with the sequences of overseas PPV-M isolates, we esti- of each sample as a template with the PrimeScript One Step RT-PCR

1) Yokohama Plant Protection Station 2) Naha Plant Protection Station 2 Res. Bull. Pl. Prot. Japan No.56

Table 1. Japanese Plum pox virus M strain isolates used in this study 3. Analyses of the genome sequences of PPV The variability of the obtained sequence and one isolate (Y1) sequenced by Oishi et al., (Oishi et al., 2018), was analyzed using the analysis software Genetyx ver.12.0.1 (Genetyx) and Mega ver.7.0 (Kumar et al., 2016). The genetic polymorphism parameters, π: nucleotide diversity (Nei et al., 1981) and θ: nucleotide polymorphism (Watterson, 1975), which are indexes indicating how diverse the nucleotide composition is, were calculated using the analysis software DnaSP ver. 6 (Rozas et al., 2017). To investigate recombination sites in each nucleotide sequence, analysis was performed using RDP (Martin et al., 2000), GENECONV (Padidam et al., 1999), BOOTS- CAN (Martin et al., 2005), MAXCHI (Smith, 1992), CHIMAERA (Posada and Crandall, 2001), SISCAN (Gibbs et al., 2000) and 3SEQ (Boni et al., 2007), which is a recombination detection program integrated in the analysis software RDP4. The phylogenetic tree was constructed using the maximum likelihood method (ML) using the TN93 model of molecular evolution (Tamura and Nei, 1993) with the software Mega ver.7.0 (Kumar et al., 2016). Thirty isolates of overseas PPV-M on the DNA database were used in the analysis (Table 3). Two isolates of PPV-D (AY912056; Fantasia, AB545926; Ou1 (ac- cession number, isolates, shown in order) were used as outgroups. The robustness of the tree topology was confirmed by other algo- rithms, neighbor joining method (NJ) and maximum parsimony method (MP). In addition, the divergence times of domestic isolates was estimated in accordance with the tutorial on Yellow fever virus divergence times estimation (https://beast.community/rates_and_dates) using the sampling year with the analysis software BEAST ver.1.8.4 (Lemey et al., 2009). The regions used for the analysis were the three gene regions that had few recombination sites by analyzing the recombination sites of overseas isolates, a partial region of the helper component protease protein (HC-Pro), the cylindrical inclusion protein (CI) and the coat protein (CP) gene (position numbers 1327- 2444; HC-Pro, 4081-5555; CI, 8975-9569; CP, position numbers refer Kit (Takara Bio Inc.). The primers (PPV1F and PPV3587R for M, to the sequence of the Y1 isolate (LC228949)). PPV2828F for M and PPV6683R for M, and PPV6204F and PPV9786R) were designed to amplify the entire PPV genome into Results and Discussion three overlapping fragments (about 3500 bp) (Table 2). A cycle sequencing reaction was performed with the BigDye Terminator v3.1 The full-length PPV genome sequences of the samples (total of 37 Cycle Sequencing Kit (Applied Biosystems) using the obtained three isolates) selected to cover the entire PPV-occurrence area in Yokoha- kinds of amplification products as templates. As primers, 6 to 8 kinds ma and Kawasaki were determined. Of these, 34 isolates were all the (Table 2) designed at intervals of about 500 bp were used. After puri- same length, but three isolates (Y11, Y15, Y25) had a three nucleo- fication, the amplification products were sequenced using 3130xl tides insertion (GAA) at the same position in the CP gene (between DNA Analyzers (Applied Biosystems), and each sequence fragment position numbers 8597-8598 of the Y1 isolate). Although no transla- was assembled using analysis software ATGC ver. 7.5.1 (Genetyx) to tion frameshift occurred, only one amino acid (Glutamic acid) was obtain a full-length PPV genome sequence. For some samples, 5 ‘/ 3’ inserted, its effect on coat protein function unknown. Both ends (24 RACE Kit, 2nd Generation (Roche) and PrimeScript II 1st strand bp on the 5 ‘side, 26 bp on the 3’ side) of five isolates (Y4, Y6, Y8, cDNA Synthesis Kit (Takara) were used to sequence both ends (total K1, Y11) that determined full-length genome sequences and one iso- 50 bp) of genome by RACE (rapid amplification of cDNA ends) late (Y1) sequenced by Oishi et al., (Oishi et al., 2018) was consid- primers (Table 2). ered to be highly conserved because they were all the same by comparison with overseas isolates, and thus both ends of the other isolates December. 2020 Oya et al.: Research on the Genetic Variation of Plum pox virus Strain M Occurred in Japan 3

Table 2. Primers used for reverse transcription-polymerase chain reaction (RT-PCR) amplification, rapid amplification of cDNA ends (RACE), and sequencing of the complete Plum pox virus genome in this study

were not sequenced. A comparison of the nucleotide diversity of 38 formative site; Pis). The variability, π and θ of each functional gene domestic isolates with overseas isolates showed that the average in were higher for the 6 kilodalton first protein gene (6K1) (Table 4). Slovakia was 0.0111 (maximum 0.0273) and the average in Turkey Except for the three isolates that had a 3-nucleotide insertion, all oth- was 0.0147 (maximum 0.0207), whereas the average for domestic er amino acids were 3140 aa, with 98 sites (3.12%) variability and 24 isolates was 0.0041 (maximum 0.0072). This suggested that the nu- sites (0.76%) Pis. 6K1 (11.54%) was also higher in the variability of cleotide diversity of domestic isolates was lower than that of overseas each protein (Table 4). 6K1 is implicated in viral replication (Cui and isolates. The detection of recombination sites by seven detection pro- Wang, 2016) and is also involved in PPV pathogenicity determination grams integrated in the software RDP4 (Martin et al., 2015) did not (Sáenz et al., 2000) by binding to a third protein (P3). Analysis of detect any recombination sites in 38 domestic isolates. This may sug- mutants that inhibited the normal processing of P3 and 6K1 revealed gest that the density of PPV-M isolates distribution in Japan is not that binding of P3 and 6K1 was not essential for virus survival high enough to cause mixed infections and genetic recombination, but (Riechmann et al., 1995). Although it is possible that mutations are further investigation is needed. likely to occur, in a report by Maejima et al. (2011) analyzing the di- As a result of calculating the variability for each functional gene, versity of 37 isolates for the PPV-D in the same manner, the variabili- there are 498 sites (5.09%) in the genome of all isolates, 107 sites ty of the 6K1 protein (3.85%) was slightly higher than the average (1.09%) of which were found from 2 or more isolates (Parsimony-in- (2.96%). Comparing the amino acid sequences of the domestic and 4 Res. Bull. Pl. Prot. Japan No.56

Table 3. Foreign Plum pox virus M strain isolates used in this study

Table 4. Genome diversity in 38 Japanese isolates of Plum pox virus

foreign isolates, there were four mutations (two sites for the first pro- rus. The KITC motif (Blanc et al., 1998) and PTK motif (Peng et al., tein (P1), one site for each of CI and nuclear inclusion body b (NIb) 1998) in HC-Pro, which are considered to be important structures for protein) unique to domestic isolates, but no mutation at nine polypro- aphid transmission, and the DAG motif (Lopez et al., 1999) in CP, tein cleavage sites (Salvador et al., 2006) characteristic of the Potyvi- was conserved in all domestic isolates except one isolate (Y21). Only December. 2020 Oya et al.: Research on the Genetic Variation of Plum pox virus Strain M Occurred in Japan 5

Fig. 1. Maximum-likelihood phylogenetic tree based on complete genome sequences of the Plum pox virus (PPV)-M isolates including the 38 Japanese isolates. Two PPV-D isolates, Fantasia and Ou1 were used to root the tree. Numbers at the nodes represent the percentage of bootstrap values obtained for 1,000 replicates (only values above 75% are shown). The scale bar indicates the number of nucleotide substitutions per site. in the Y21 isolate, K (Arginine) of the KITC motif was mutated to R MP methods also showed the similar topology as the ML tree, with (Lysine), but the RITC motif is also thought to be involved in aphid high bootstrap values. It is possible that Japanese isolates originated transmission (Blanc et al., 1998). This suggested that any of the test- from a Greek isolate, but more accurate estimation requires the full- ed Japanese isolates could be transmitted by aphids. length genome sequence of PPV-M occurrence countries (France, Ita- As a result of constructing a phylogenetic tree using the ML meth- ly, Bulgaria and Syria, etc.) other than the overseas isolates used in od to examine the phylogenetic relationship between 38 Japanese iso- this study. Based on the topology formed by the Japanese isolates, lates and 30 overseas PPV-M, Japanese isolates formed one group they were divided into seven groups, and checked against the sam- different from overseas isolates and were most closely related to pling points, there was no clear relationship with the sampling points Greek isolates (Fig.1). Phylogenetic trees constructed by the NJ and (Fig.2). The short length of branches of the phylogenetic tree sug- 6 Res. Bull. Pl. Prot. Japan No.56

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和文摘要

国内で発生したウメ輪紋ウイルス M 系統の遺伝的変異の調査

大矢仁志・藤原裕治 1）・粕壁隆一郎 2）・本蔵洋一 1）

横浜植物防疫所調査研究部

Plum pox virus（PPV）は、核果類の重要なウイルス病であ離株は単独の異なるクレードを形成し、日本の分離株のクレーるウメ輪紋病の病原体である。 2016 年、日本のアンズとモモドは試料採取地点で明確なグループに分かれることはなかっから PPV-M 系統が検出された。日本で発生した PPV-M の遺た。これらの結果は、日本の PPV-M 分離株は変異があまり蓄伝的変異に関する調査のために、横浜市と川崎市から収集され積されておらず、初発生の時期は比較的近年であること、さらた 38 株の全長ゲノム配列を決定した。遺伝子解析により、日に、日本へ侵入経路は独立しており、発生地域内の感染拡大も本分離株の塩基配列の多様性が低いことが明らかになった。全比較的短期間であることが示唆された。長配列による PPV-M の系統解析では、日本とヨーロッパの分

1) 横浜植物防疫所 2) 那覇植物防疫事務所