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 Plum Pox 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, genus Potyvirus, family Potyviridae) is the most important virus pathogen of stone fruit cultures of the genus Prunus 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 aphid Brachycaudus 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 aphids 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 insect 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].
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