ISSN 0096-3925, Moscow University Biological Sciences Bulletin, 2016, Vol. 71, No. 2, pp. 71–75. © Allerton Press, Inc., 2016. Original Russian Text © A.V. Zakubanskiy, A.A. Sheveleva, S.N. Chirkov, 2016, published in Vestnik Moskovskogo Universiteta. Biologiya, 2016, No. 2, pp. 8–12.

VIROLOGY

Molecular Biological Properties of New Isolates of Virus Strain Winona A. V. Zakubanskiy, A. A. Sheveleva, and S. N. Chirkov* Department of Biology, Moscow State University, Moscow, 119234 Russia *e-mail: [email protected] Received December 30, 2015

Abstract—Plum pox virus (PPV, Potyvirus, family Potyviridae) is the most important virus pathogen of stone fruit cultures of the genus from an economical standpoint. Winona strain (PPV-W) is the most variable of nine known virus strains and one of the most widespread in the European part of Russia. Six new PPV-W isolates were first discovered in green plantations of Moscow (Kp2U, Avang, Pulk, Pulk-1), in Tal- domsky district of Moscow region (Karm), and in Kovrovsky district of Vladimir region (Vlad-4) on wild trees of plum Prunus domestica. 3'-Terminal segment of genome of new isolates was notable for high variability level. The study on the relationship with other isolates of this strain by means of phylogenetic analysis of gene sequence of the coat protein showed the lack of clusterization of Russian PPV-W isolates according to geo- graphical principle. Inoculation of Nicotiana benthamiana plants by hop plant louse Phorodon humili from plum trees infected with Avang and Pulk isolates and by thistle cardui from the tree infected with Kp2U isolate led to the systemic viral infection in indicator plants, suggesting the possibility of PPV-W spread by both aphid species in nature. Transmission of PPV-W through seeds was not observed.

Keywords: plum pox virus, strain Winona, phylogenetic analysis, hop aphid Phorodon humuli, thistle aphid Brachycaudus cardui. DOI: 10.3103/S0096392516020103

INTRODUCTION this strain occur extensively [6]. Analysis of their genomes showed that PPV-W is the most variable from Plum pox virus (PPV, genus Potyvirus, family Poty- all known strains of the virus, in particular, due to viridae) is the most important virus pathogen of stone abundance of intrastrain recombination [7, 8]. Thus, fruit crops of the genus Prunus from an economical the molecular analysis of every new isolate is of inter- standpoint. The sharka disease caused by the virus est in evaluation of degree of genetic variability of PPV leads to the yield drop and to quality decrease of fruits and understanding of its evolution. Moreover, biolog- [1]. The viral genome consists of one single-stranded ical properties of PPV-W are virtually not studied. RNA of positive polarity near 9800 nucleotides long with organization typical for potyviruses. In infected In the study, six new isolates of PPV-W were dis- cells, genomic RNA is translated with production of covered on wild plum trees in Moscow, as well as in polyprotein, which is processed by virus specific pro- Moscow and Vladimir regions, were studied. Employ- teinases to 10–11 functional proteins. From plant to ing phylogenetic analysis of genomes, their relation- plant, the virus is transmitted during vegetative repro- ship with other isolates of the same strain were stud- duction, as well as by different plant louse aphid. Nine ied. The possibility of transmission of W-strain iso- strains of the virus are known: D, M, C, CR, W, Rec, lates by and through seeds was also studied. EA, T, and An. The strains differ by nucleotide sequence of genomic RNA, antigenic and epidemio- METHODS AND MATERIALS logical properties, geographical prevalence, host Avang, Kp2U, Pulk, and Pulk-1 isolates were dis- range, and pathogenicity for different species of stone covered on wild plum trees (P. domestica) in green fruit crops [2]. plantations on the north of Moscow. Karm isolate was Strain W (Winona) (PPV-W) occurs mostly in the detected on root offshoots of plum in Taldomsky dis- territory of the former Soviet Union. Two isolates trict of Moscow region, and Vlad-4 isolate was found in Canada [3, 4] and the United States [5] have detected on a plum tree in Kovrovsky district of Vlad- Ukrainian origin. Other PPV-W isolates have not been imir region. All isolates induced typical symptoms of discovered outside the former USSR so far. On the sharka on leaves of infected plants. Laboratory diag- contrary, in the European part of Russia, isolates of nostics was conducted employing enzyme-linked

71 72 ZAKUBANSKIY et al. immunosorbent assay (ELISA) using Reagent set RESULTS AND DISCUSSION SRA 31505 (Agdia, United States) and immune spe- cific polymerase chain reaction with reverse transcrip- Using ELISA and RT-PCR methods, the virus tion (RT-PCR) with universal primers, detecting any was detected in all analyzed samples of plum trees with sharka symptoms. When primers, were used in isolates of the virus [9], as was described earlier [10]. RT-PCR specific to W strain, a product of expected Strain of isolates was determined by RT-PCR with size (327 bp) was generated [13] (data is not shown). primers, developed for identification of different Amplification with primers, specific to other virus strains of the virus [11–15]. 3'-Terminal segment of the strains, was not observed. Thus, the analyzed isolates genome, including gene of the coat protein (CP) and do obviously belong to W strain. PPV-W was previ- flanking sequences of NIb gene and 3'-untranslated ously detected in various regions of European Russia, region was amplified by RT-PCR method with as well as in Latvia on plum (P. domestica), blackthorn P3dW/4CPR1 primers [16]. PCR products of 1211 bp (P. spinosa), downy cherry (P. tomentosa), and cherry were extracted from agarose gel using Cleanup Stan- plum (P. cerasifera) [6, 10, 16, 20, 21]. In Moscow, iso- dard Kit (Evrogen, Russia) and sequenced in both lates of this strain were discovered in collection plan- directions according to the Sanger’s method by Evro- tations of the Main Botanical Garden of the Russian gen Company. Sequences of CP gene of new isolates Academy of Sciences on Canadian plum (P. nigra) and were deposited in the GenBank database blackthorn [10, 22]. In green plantations of Moscow, (http://ncbi.nlm. nih.gov/) under the following num- in the north of Moscow region, and in Vladimir region bers: KU359729 (Avang), KU359730 (Karm), PPV-W was discovered for a first time. These findings KU359731 (Kp2U), KU359732 (Pulk), KU359733 are yet another confirmation of the wide distribution (Pulk-1), and KU359734 (Vlad-4). For phylogenetic of isolates of W strain in European Russia. On average, analysis, all sequences of CP gene of the following PPV- considering new isolates, sequences of CP of PPV-W W isolates available in the GenBank were used: W3174 had 5.1% divergence that corresponds to the data for (AY912055), RD4 (HG916856), STNB1 (HG916857), variability of full-genome sequences of PPV-W [8] and STNB2 (HG916858), PD2 (HG916859), P2-1 indicates a high level of genetic variability within W (HG916860), P3 (HG916861), 1410-7 (HG916862), strain. 1410-1 (HG916863), 1410 (HQ326086), LV-141pl Results of phylogenetic analysis of the CP gene of (HQ670746), LV-145bt (HQ670748), UKR44189 new PPV-W isolates are shown in Fig. 1. Avang, Pulk, (JN596110), BY (JQ970438), P1 (JQ970439), Pk and Pulk-1 isolates form one clade. Plum trees on (KC347608), P2-2 (LN852400). Analysis of genetic which these isolates were found are located hundreds diversity of the sequences and phylogenetic analysis meters from one another and are colonized by the was performed using MEGA 6.06 software [17]. same species of supposed virus vector, the hop aphid Phorodon humuli (Schr.). It is of interest that these In order to determine species of the aphids coloniz- three isolates are linked by common origin with 1410 ing sprouts of infected trees, an identification and PD2 isolates from stone fruit collection of the guide was used [18]. The possibility of transmission of Main Botanical Garden of the Russian Academy of the virus by aphids was studied as described earlier Sciences [10, 22]. It is possible that relative proximity [19]. The aphids from trees infected with Kp2U, Pulk, of habitats of host plants facilitated natural spread of and Avang isolates were transferred on Nicotiana ben- an ancestor of these isolates by aphids. One more thamiana plants in 25–40 specimens on 1–2 leaves Moscow isolate, the Kp2U, is closely related to RD4 of middle layer. Each isolate was inoculated into 5– isolate, found on downy cherry (Prunus tomentosa) in 6 plants. Virus in indicator plants was detected 2– Ramensky district of Moscow region [10]. It should be 3 weeks after inoculation by means of RT-PCR. Virus noted that all isolates representing this group were in the aphids was detected by RT-PCR using total detected in Moscow region, with the exception of Pk RNA, extracted from the aphids using RNeasy Plant isolate which was detected on a wild plum tree in Tver Mini Kit (Qiagen, Germany), as a template for reverse region [16]. transcription. 40 mg of aphids, collected from one or Although, in some cases, grouping of isolates by another tree, was homogenized in lysis buffer RTL, topographic principle can take place, in general, supplied in the kit, and then the total RNA was clasterization of Russian PPV-W isolates by geograph- extracted according to the protocol. To study the pos- ical principle is absent. Thus, Karm isolate from Tal- sibility of transmission of PPV-W by seeds, fruits of domsky district of Moscow region clasterises with BY infected trees were collected; stones were separated isolate from Belgorod region and with LV-141pl isolate from pulp, treated with potassium permanganate solu- from Latvia, and Latvian isolate is considered the tion and stratified in moist substrate during 5– closest to putative ancestor of this strain [21]. Like- 6 months at 2–4°C until sprouting. The presence of wise, Vlad-4 isolate from Vladimir region clasterises the virus in plum seedlings was detected by RT-PCR with P1 and P3 isolates from Stavropol region [8, 10] method 5–7 weeks after their planting into sterile soil and with Lv-145bt isolate from Latvia and UKR44189 substrate. isolate from Ukraine [5, 21].

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64 Pulk 99 Pulk-1 98 Avang 1410 100 99 PD2 97 P1 P3 100 Vlad-4 68 63 LV-145bt 100 UKR44189 BY 100 Karm 75 LV-141pl W3147 99 1410-1 100 1410-7 100 P2-1 P2-2 81 Kp2u 92 RD4 100 Pk 72 STNB1 100 STNB2 0.005

Fig. 1. Phylogenetic tree reconstructed by neighbor joining method based on sequences of the coat protein gene of isolates of plum pox virus of Winona strain. Isolates’ names are given at the end of branches. Isolates studied in this work are given in bold. Results of bootstrapping from 1000 of random samples (in percent) are given next to the nodes. A scale line shows number of sustitutions per one nucleotide. Accession numbers of sequences used in phylogenetic analysis in the GenBank database are given in the Methods and Materials section. Phylogenetic analysis was performed using the MEGA 6 (17) software.

The CP gene of PPV-W can consist of 990 or tree represented in this article to the tree reconstructed 993 nucleotides. This difference is due to the deletion on the basis of full genomic sequences of isolates of of T, A, G nucleotides from neighbor triplets in 5'- this strain [8]. proximal site of the CP gene in some isolates [8, 10, PPV spreads naturally from plant to plant by differ- 21]. Phylogenetic analysis showed that sequences of ent aphid species in a nonpersistent manner [2, 19]. CP gene 990 bp long (Pulk, Pulk-1, Avang, 1410, Plum trees infected with Avang and Pulk isolates were PD2, P1, P3, Vlad-4, LV-145bt, UKR44189 isolates) colonized by a hop aphid Ph. humuli, and a tree form a separate cluster (Fig. 1). However, it does not infected with Kp2U isolate was colonized by thistle include BY isolate, whose CP gene also consist of aphid Brachycaudus cardui (L.). These louse species 990 bp. It is possible that isolates from this cluster are are known as PPV transmitters [19]; however, the pos- the separate branch of evolution of PPV-W. sibility of transmission of isolates of W strain by them The W strain is the most variable of all known PPV was not studied. strains, and sequence of CP gene encoding N-termi- By means of RT-PCR with primers specific to W nal CP domain is the most variable genomic region strain [13], the virus was easily detected in leaves colo- [2]. For this reason, the phylogenetic analysis of CP nized by plant lice (Fig. 2a). Despite this, inoculation gene obviously reflects the relationships between iso- of N. benthamiana plants by Ph. humuli from plum lates reliably. This is also implied by high bootstrap trees infected with Avang and Pulk isolates and by values and similarity of topology of the phylogenetic B. cardui from a tree infected with Kp2U isolate

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Fig. 2. RT-PCR detection of plum pox virus in plum leaves colonized by (a) aphids, (b) in the aphids, and (c) in systemic leaves of Nicotiana benthamiana 3 weeks after inoculation by aphids. Isolates: (1) Avang, (2) Kp2U, (3) Pulk. Electrophoresis in 2% aga- rose gel. Staining with ethidium bromide. The arrow shows the specific RT-PCR product of 327 bp. M—GeneRuler 100 bp DNA Ladder markers (Fermentas). caused infection of indicator plants. Employing RT- ACKNOWLEDMENTS PCR, the virus was detected both in inoculated and We are grateful to Dr. Askar Akhatov for identifica- noninoculated leaves, indicating the possibility of tion of aphid species. The study was conducted with transmission of PPV-W isolates by both aphid species financial support of the Russian Science Foundation (Fig. 2c). The proportion of infected indicator plants (project 14-24-00007). depended on PPV-W isolate and was three plants of five infected for Avang and five plants of five inocu- REFERENCES lated for Kp2U. It is possible that high efficiency of transmission of Kp2U isolate is a due to significantly 1. Cambra, M., Capote, N., Myrta, A., and Llacer, G., larger amount of the virus in B. cardui aphids (Fig. 2b). Plum pox virus and the estimated costs associated with sharka disease, EPPO Bull., 2006, vol. 36, no. 2, High concentration of PPV in a vector is not typical pp. 202–204. for the nonpersistent way of virus transmission and 2. Garcia, J.A., Glasa, M., Cambra, M., and Candresse, should be studied later on. It should be noted that, T., Plum pox virus and sharka: a model potyvirus and a when transmitted PPV-W in N. benthamiana by vec- major disease, Mol. Plant Pathol., 2014, vol. 15, no. 3, tor, the infection was completely asymptomatic, as in pp. 226–241. a case of mechanical inoculation by the isolates 3. James, D., Varga, A., Thompson, D., and Hayes, S., belonging to this strain [22]. Detection of a new and unusual isolate of Plum pox virus in plum (Prunus domestica), Plant Dis., 2003, Some potyviruses can be spread by seeds [23]. PPV vol. 87, no. 9, pp. 1119–1124. isolates of D and M strains are not transmitted by 4. James, D. and Varga, A., Nucleotide sequence analysis seeds [24], but W strain was not studied in this respect. of Plum pox virus isolate W3174: evidence of a new Employing RT-PCR method, 47 plum plants grown strain, Virus Res., 2005, vol. 110, nos. 1–2, pp. 143– 150. from seeds from infected trees were analyzed. The 5. Mavrodieva, V., James, D., Williams, K., Negi, S., virus was not detected in any of the sprouts. Typical Varga, A., Mock, R., and Levy, L., Molecular analysis symptoms of virus infection did not develop. Appar- of a Plum pox virus W isolate in plum germplasm hand ently, PPV isolates of W strain are not transmitted by carried into the USA from the Ukraine shows a close seeds. relationship to a Latvian isolate, Plant Dis., 2013, vol. 97, no. 1, pp. 44–52. Discovery of new PPV-W isolates in geographically 6. Chirkov, S.N. and Prikhod’ko, Yu.N., Genetic diver- distant regions indicates wide distribution of W strain sity and population structure of Plum pox virus in Rus- in European Russia as a result of interchange of sia, S-kh. Biol., 2015, vol. 50, no. 5, pp. 529–539. infected material of stone fruit cultures and presence 7. Sheveleva, A., Ivanov, P., Chirkov, S., Prihodko, Y., of efficient virus vectors. Wide distribution of this Varga, A., and James, D., Plum pox virus W appears to strain, its absence outside the former Soviet Union, be the most variable strain of the seven recognized strains of the virus, Petria, 2012, vol. 22, no. 3, pp. 226– and high variability of genome can point to the origin 232. of W strain in Russia or neighbor countries, which is 8. James, D., Sanderson, D., Varga, A., Sheveleva, A., consistent with suppositions about the East European and Chirkov, S., Analysis of the complete genome origin of PPV-W strain [4, 6]. sequences of new isolates of the genetically diverse

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