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AGRICULTURAL BIOLOGY, ISSN 2412-0324 (Еле» ed. Online)
2015, V. 50, № 5, pp. 529-539
(SEL’SKOKHOZYAISTVENNAYA BIOLOGIYA) ISSN 0131-6397 (Russian ed
v_____________________________________' ISSN 2313-4836 (Russian ed. Online)
Reviews. Advances and challenges
UDC 634.22:632.3:578.864(470+571) doi: 10.15389/agrobiology.2015.5.529rus
doi: 10.15389/agrobiology.2015.5.529eng
GENETIC DIVERSITY AND POPULATION STRUCTURE OF Plum pox virus IN RUSSIA
(review)
S.N. CHIRKOV1, Yu.N. PRIKHOD’KO2
lM.V. Lomonosov Moscow State University, Department of Virology, Biological Faculty, 1/12, Leninskie gory, Moscow, 119234 Russia, e-mail [email protected];
2Ail-Russian Plant Quarantine Center, Federal Agency of Scientific Organizations, 32, ul. Pogranichnaya, pos. Bykovo, Ramenskii Region, Moscow Province, 140150 Russia, e-mail [email protected] Acknowledgements:
The authors thank I.V. Mitrofanova for the photos of PPV symptoms on peach and nectarine leaves.
Supported by Russian Science Foundation, grant № 14-24-00007 Received February 17, 2015
Ab s t r a c t
Plum pox virus (PPV) is the causal agent of Sharka that is considered the most detrimental viral disease of stone fruit crops. Regular monitoring of stone fruit plantings using enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction for PPV detection and identification resulted in the findings of numerous focuses of the disease in European Russia. The virus was found in collections, variety test plots, nurseries, fructiferous and abandoned orchards, decorative plantings, private gardens, wild stone fruit trees growing in urban and rural areas. PPV was detected in naturally infected plum (Prunus domesica), peach (P. persica), nectarine (P. peisica var. nec-taiina), myrobalan (P. cerasifera), blackthorn (P. spinosa), downy cherry (P. tomentosa), sour cherry (P. cerasus), sweet cherry (P. avium), apricot (P. armeniaca) and Canadian plum (P. nigra). PPV has been reported from Petersburg, Novgorod, Tver, Moscow, Tula, Voronezh, Tambov, Lipetsk, Belgorod, Rostov, Samara, Saratov, Volgograd, Astrakhan, Stavropol, Krasnodar, Karachay-Cherkessia, Dagestan, and Crimea regions. Six of the nine known PPV strains (D, M, Rec, W, C, CR) have been revealed in European Russia. Most isolates belong to the strains D (38 %), W (25 %), CR (23 %), M (7 %) and C (7 %). Two distinct PPV-Rec isolates have been found on myrobalan and plum in Crimea and Stavropol regions. Population of PPV in European Russia and, probably, all over the European part of the former USSR seems to be the most diverse in the world due to wide spread of PPV isolates belonging to the strains W, C, and CR that were never detected or only sporadically identified in other countries until now. Phylogenetic analysis of their genomes shows that these three strains constitute the supercluster divergent from other PPV strains. This evolutionary branch originated from a common ancestor and apparently developed mainly in Russia. The wide dissemination of PPV in Russia is a potential threat for newly bred stone fruit cultivars and further selection and biotechnological works.
Keywords: plant viruses, Plum pox virus, Sharka disease, genetic diversity, strains.
Plum pox virus, PPV (Potyvirus, Potyviridae) is the causal agent of Sharka disease of stone fruit crops. Because of reactive oxygen species produced in plants as a response to viral infection, the chloroplasts and photosynthetic apparatus are damaged resulting in visible symptoms appeared on leaves (Fig. 1), fruits, flowers and seeds [1]. PPV can infect, naturally and experimentally, many (or perhaps all) stone fruit crops from genus Prunus (Rosaceae family) and also from other taxa [2-5]. PPV is considered the most detrimental viral pathogen in stone fruit plants which causes significant yield losses in peach, apricot, plum, etc., as up to 100 % of fruits drop prematurely, being worse in quality and bad processed. In the susceptible varieties annual growth of the infected trees is suppresses. As a result, a lot of infected varieties are not in use despite high agronomic properties and quality indices [6, 7]. Annual losses in EUR reach hundreds of millions, and the millions of infected trees are eliminated [8]. PPV can
be transmitted mechanically, under vegetative propagation and by different aphids. Seed transmission is not found. For long distances PPV can be mainly transmitted via infected plants. The disease is recorded all around the world, except Australia, New Zeeland, South Africa and California [9-11]. In Russia PPV is a limitedly spread quarantine virus.
Fig. 1. Sharka symptoms on nectarine (a), peach (b), plum (c), apricot (d), cherry (e, f), felt cherry (g, h), cherry-plum (i). PPV strains: D (a-e, g), CR (e), C (f), W (h), Rec (i). Photo (a, b) courtesy of I.V. Mitrophanova.
Molecular biology of PPV. PPV virions are filamentous particles 750 nm long with 15 nm in diameter in which the +RNA of 9.8 thousand nucleotides is packed into 2,000 copies of virus capsid 36.5 D protein. PPV genome structure is typical for potyviruses. In the genome RNA there are two
open reading frames, 5' and 3'non-coding regions (NCR) of 146 and 217 nucleotides, respectively, the viral protein (VPg) is covalently linked at 5'end and poly(A) sequence is attached to 3 'end. In infected cells RNA is expressed into single precursor polyprotein than processed by virus-specific proteases into 10 functional proteins (P1, HcPro, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb and capsid protein CP). Another protein, the PIPO (Pretty Interesting Potiviridae ORF), is expressed due to the fusion of P3N and PIPO resulting from the shift in the P3 gene reading frame [9].
100
100
100
T
M
D
100
Rec
An
100
100
EA
- W
— C
CR
0.02
Fig. 2. Dendrogram of Plum pox virus (PPV). Grouped by the «neighbor-joining» method for full genome sequences of typical strains. The results of bootstrap analysis (%) for 1000 random samples are shown. Scale indicates the number of substitutions per nucleotide. An (AL-11pl, HF674399), T (AbTk, EU734794), M (SK68, M92280), D (Ou1, AB545926), Rec (BOR-3, AY028309), EA (AM157175), W (LV-145bt, HQ670748), C (BY181, HQ840518), CR (RU-17sc, KC020124) were analyzed (strain name and the record in GenBank are indicated). MEGA 5 was used for phylogenetic analysis [12].
To date, the analysis of full genome sequences of PPV allows distinguishing 9 strains. These are Dideron (D), Marcus (M), Recombinant (Rec), Cherry (С), Cherry Russian (CR), El Amar (EA), Winona (W), Turkish (T) and Ancestral (An) [9, 13], clustered into monophylic groups of closely related isolates. Recombination plays important role in the PPV evolution together with spot mutations. M and T probably appeared due to independent recombination between isolates of D and An strains [9]. Rec strain considered to be a recombinant of D and M isolates [14]. In the genome of the W317 isolate of W strain, there is an apparent evidence of recombination with D and М strains [15]. Most probable relationships are shown in the dendrogram based on full genome sequences of typical isolates of each strain (Fig. 2). The strains differ in antigenic and epidemiological properties, host plants range, geographic distribution and pathogenicity for different stone crop species. In Europe and Mediterranean basin where the majority of PPV isolates is revealed, D, M and Rec strains are spread widely while the rest one rarely and(or) endemically for specific territories. Thus, for the time being, the EA and T isolates were found only in Egypt and Turkey, respectively. The only known isolate of An strain, the AL-11pl, was found in Albania [16]. In other regions the D strain isolates are mainly detected. C, CR and W strains, with a few exceptions, are found in the ex-USSR territory.
PPV detection and identification. ELISA and different modification of RT-PCR are mostly used [17-19]. In ELISA the viral CP can be assayed. Most virus specific and strain specific epitopes are located at N-end of CP, which is exposed on the surface of the viral particle being the most variable
part of the molecule [9]. Numerous assay kits for PPV diagnostics based on sand-wich-ELISA with polyclonal antibodies and conjugated alkaline phosphatase are well represented on the market. Indirect sandwich-ELISA [20] with 5B monoclonal antibodies to 94DRDVDAG100, a universal epitope found till now in the CPs of all studied isolates [21], seems to be the most reliable. In RT-PCR any PPV isolate can be found with universal primers specific for CP gene or 3'-NCR [22, 23]. The amplification results in 243 and 220 bp fragments. Immune-specific RT-PCR is deemed most specific detection technique [24]. Variability of PPV isolates can be studied differently [25]. In particular, the strain can be identified in indirect sandwich ELISA-test with monoclonal antibodies to D, M, C and EA [20, 26-28], in RT-PCR with primers specific to D, M, C, CR, EA, Rec, and W [29-34], and by sequencing CP gene or its hyper variable N-terminal fragment followed by phylogenetic analysis. Sometimes the PPV strain can be identified by sequencing and phylogenetic analysis of 243 bp product along with its restriction analysis using RsaI and AluI endonucleases [22]. Note, the strain specific monoclonal antibodies can reveal the epitopes located at the CP N-end, while the strain specific primers are designed to different regions of the PPV genome 3'-end, mostly for the CP gene. However, the An, T, Rec and М distinguishes are mainly located in the genome 5'-end. Besides, even single nucleotide or amino acid replacement may result in incorrectness of strain identification [13]. Therefore, several methods should be used, of which the phylogenetic analysis of full genome sequences is deemed the most reliable. Optimized protocols for ELISA, ER-PRC and other methods for PPV diagnostics and identification are approved by European and Mediterranean Plant Protection Organization (France) [35, 36].
PPV prevalence and genetic diversity in Russia. Charka symptoms in stone fruit crop plantations in the Russian territory were observed at least since 1970th. Nevertheless, till 2000th the end no molecular evidence of viral infection was reported with no disclosed isolate characteristics represented. Due do modern techniques of PPV diagnostics and identification the systemic resulted in the findings of numerous focuses of the disease in European Russia [37-39]. The virus was found in collections, variety test plots, nurseries, fructiferous and abandoned orchards, decorative plantings, private gardens, wild stone fruit trees growing in urban and rural areas. Collections and variety test plots where the cuttings for nurseries are produces seem to be most probable source of infection [37]. The isolates are mostly found in old gardens planted when the importance of total diagnostics and identification of plant viral infections were not understood enough and the means for its monitoring were not developed.
Till now, PPV has been reported from Petersburg, Novgorod, Tver1, Moscow, Tula, Voronezh, Tambov, Lipetsk, Belgorod, Rostov, Samara, Saratov, Volgograd, Astrakhan, Stavropol, Krasnodar, Karachay-Cherkessia, Dagestan, and Crimea regions. Of note, no PPV was disclosed in about 130 samples from Kursk, Orel, Ryazan’ and Penza regions.
PPV was detected in naturally infected plum (Prunus domestica), peach (P persica), nectarine (P persica var. nectarina), myrobalan (P cerasifera), blackthorn (P spinosa), downy cherry (P tomentosa), sour cherry (P. cerasus), sweet cherry (P avium), apricot (P armeniaca) and Canadian plum (P nigra). Of 370 PPV isolates, shown in 2007-2014 by experts All-Russian Plant Quarantine Center, 90 % were detected in plum and cherry trees. Nevertheless, these were the crops mostly surveyed. To date, no PPV was reported from P pumila, P. fruti-cosa, P. mahaleb, P. triloba, and in the hybrids of P fruticosa and P. maackii.
Six of the nine known PPV strains (D, M, Rec, W, C, CR) have been revealed in European Russia. Most isolates belong to the strains D (38 %), W (25 %), CR (23 %), M (7 %) and C (7 %). Since D strain prevails in the
world, obviously, Russia is not an exception. D strain has been mainly detected in plum, and few isolates have been disclosed in apricot, myrobalan and downy cherry. In Crimea a big number of D strain isolates were found in peach and nectarine plant collections. About 7 % of Russian isolates belong to the M strain. Importantly, the M-isolates have been revealed only in commercial plantation of plum (84 %) and peach (16 %) trees. Two distinct PPV-Rec isolates have been found on myrobalan and plum in Crimea and Stavropol’ regions.
Wide prevalence of W, С and CR strains which are rare or even not found in other territories in the world seems to be most significant distinction of the PPV population in Russia. Thus, the molecular study of the Russian PPV isolates makes it possible to contribute stigmatically to our knowledge about PPV genetic diversity.
First isolates of W strains (W3174 and UKR44189) have been disclosed in Canada and USA but in the trees of the Ukrainian origin [40, 41]. For the long time, these isolates remained the only known representatives of new strain W. In our investigations the numerous genetically distinct isolates were revealed in Middle Russia, Chernozem zone, Volga region and in South Russia in all types of plantations and in a wide host plant range, including plum, apricot, myrobalan, downy cherry, blackthorn and Canadian plum [38, 42-44]. Few W-isolates have also been revealed in blackthorn and plum in Latvia [15]. For W-isolates poor manifested symptoms or symptomless infection is characteristic. Sequence analysis of genomes or their fragments approved the W strain to be most variable of all nine known PPV strains. The similarity at nucleotide or amino acid levels was 92.0-99.7 % and 96.4-99.9 %, respectively. Nevertheless, the biological properties of W-strain isolates can differ. Particularly, we have disclosed isolate RD4 in the naturally infected downy cherry [43]. However, the downy cherry can not be a systemic host plant for isolate UKR44189 [41]. Monoclonal antibodies to W strain [45] failed to distinguish the Russian W-isolates as the first asparagine acid (D) in the 2DEEDD6 epitop is changed to asparagine (N) [43]. High genetic diversity along with wide prevalence in the European Russia territories and very wide host range allow to suggest the strain W to be one of the oldest in Russia.
C-strain isolates make about 7 % of all up-to-date PPV Russian isolates. These isolates were found in different types of cherry and sweet cherry plantings in Leningrad, Moscow, Belgorod, Samara, Saratov and Volgograd regions. They differ genetically that have been shown by molecular methods. The first C-strain isolate was disclosed in Moldova in cherry [46]. Few C-strain isolates have been revealed in Belarus [47]. Despite the relatively small number of the revealed isolates, the findings indicate broad spreading C strain in Russia and the neighboring countries.
First CR-isolates have been recently disclosed in cultivated cherry plants in Saratov and Samara regions, and in Moscow in wildly growing trees [29, 48, 49]. For CR-isolates the extremely low genome variability is characteristic, making 0.6-0.9 % for the whole genome sequences and 0.0-0.8 % for CP gene. Nevertheless, the isolates from Moscow and Volga regions are reliably distinct phylogen-tically under full genome sequence analysis. Their findings are located at the hundreds kilometer distance thus indicating wide area of CR strain in European Russia. A new strain disclosed improves scientifically the knowledge about genetic diversity in PPV. Moreover, until this finding, C strain considered the only strain able to cause a systemic infection in cherry (P cerasus). Comparative study of two PPV isolates infecting cherry can possibly detect determinants involved in host range control [29]. An important peculiarity of CR-isolates consists in the 96DRDVDAG102 universal epitope modification due to replacement of the asparagine acid (D) at 96 to glutamic acid (E). Such isolates can not be identified by ELISA with 5B monoclonal antibodies specific to the universal epitope and earlier
considered the universal testers for any PPV isolates. It obviously means the necessity to develop new universal monoclonal antibodies to provide reliable ELISA diagnostics of PPV.
Thus, to date there are six PPV strains disclosed in Russia. Three of them, the D, M and Rec, prevailed in Europe and Mediterranean regions. It is quite reasonable to assume their appearance in Russia due to introduction of European stone fruit crops infected by PPV. Sharka was first detected in plum in Bulgaria in 1917 [50], then Balkan PPV appeared in Europe, Mediterranean basin, Asia, North and South America [9]. For instance, wide dissemination of Rec strain in Europe was possibly caused by infected tolerant plum varieties from the former Yugoslavia [14]. M-strain isolates were found in peach in Krasnodar Krai and in plum in Stavrolol’ region in the trees imported from the former Yugoslavia [38]. It is a probable reason of the M strain occurrence in the commercial garden only. Close similarity of the Russian and the European isolates are confirmed by phylogenetic analysis of their genomes.
The С, CR and W PPV strains are practically not found out of the ex-USSR, and to date the CR is found only in Russia. Two W-isolates from Canada and USA are of Ukrainian origin [40, 41]. Moreover, the isolate UKR44189, detected by the US quarantine guard in the in vitro plum culture hand carried from Ukraine is related closely to LV-145bt from Latvia [41]. LV-145bt and other Latvian W-isolates are obviously derived from Russia and Ukraine [15, 49]. C-isolates are described for sweet-cherry in Italy [51] and in cherry in Croatia, with the Croatian isolate being probably identic to that isolated in Moldova [52]. It also must be emphasized that sporadic findings of C and W PPV out of ex-USSR are in contrast with their frequency in Russia and neighboring countries.
Nevertheless, on the dendrograms based on partial or total genome sequences these strains form a distinct supercluster (see Fig. 2). Phylogenetic analysis indicates the common ancestor for W, C and CR strains, and this branch further evolved, mainly or exclusively, in the territory of contemporary Russia. Assumptions of East-Europe ancestry of W and C strains have been reported [13, 40, 53]. A presumable ancestor could appear in Russia or be introduced from Front and Middle Asia with infected plants. In this connection the two closely related D-isolates found in wild apricot and cultivated plum trees in Kazakhstan are of interest. These isolates posses an unique deletion of six nucleotides in the CP gene corresponding to CP N-end unknown in all PPV isolates for which this genome segment is sequenced. Wild apricot trees from Tien Shan are considered the most probable source of PPV isolates with this genetic marker [54].
It should be mentioned that for the long time in an single economic area of ex-USSR Russia the viral strains can move unlimitedly with infected planting material. Occurrence of W-isolates in Latvia and Ukraine [15, 40, 41], C-isolates in Moldova and Belarus [31, 46, 47], and D strain in Lithuania and Ukraine [55, 56] allows to assume the single PPV population formed in European Russia. Its characteristic feature is the highest genetic variability due to wide range of W, C and CR strains which are extremely rare or even not found in other regions in the world. The reasons of W, C and CR absence in other European countries are unknown. Probably, it may be due to limited practical use of stone fruit crop varieties derived from Russia. Thus, occurrence of two genetically distinct M-subpopulations in Europe of which one covers the area of France, Italy, Greece, and Cyprus, while another includes Bulgaria, Czech Republic, Serbia and Slovakia) one explains by no exchange with planting material for the long period of confrontation [57]. Also the areal of these strains is possibly limited by climatic factors at its western edge.
So, the recent results of Plum pox virus (PPV) monitoring its show prevalence all over the European Russia territory. PPV population in Russia and probably in European ex-UUSR is characterized by the highest genetic diversity in the world due to wide range of D, M, Rec, W, CR and C strains. Molecular studies of the Russian PPV isolates have already contributed significantly to the knowledge about PPV biodiversity and evolution, though the stone fruit trees in the territories to the east from Volga, in Ural and Altai regions, in Siberia and Far East still remained unstudied with special reference to PPV distribution. Our data show that such investigations of PPV isolates can result in numerous unexpected findings, since in Russia a probability of disclosing new isolates with unknown properties is extremely high. On other side, so broad spreading PPV in Russia is a potential threat for newly bred stone fruit cultivars and further selection and biotechnological works.
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