Only bootstrap values 70% are shown

Only bootstrap values 70% are shown. isolates were performed. PPV-Rec isolates formed a homogeneous group, while PPV-M isolates split into PPV-Ma and PPV-Mb subgroups. Five separated clades were formed by the analyzed PPV-D isolates. Nucleotide sequences of the partial CP coding region of the analyzed isolates revealed a slightly higher intra-strain genetic variability in PPV-Rec and PPV-M isolates, while that of PPV-D strain isolates was higher from the reported for these strains. (PPV) is one of the most harmful diseases affecting stone (genes make the implementation of control steps very difficult. Now, Sharka disease is present in Europe, North and Central America, Asia, and North Africa. To date ten PPV strains as Dideron (D), Marcus (M), Recombinant (Rec), Cherry (C), Cherry Russian (CR), EL Amar (EA), Winona (W), Turkish (T), Ancestral (An) and Cherry Volga (CV) are distinguished (Sheveleva et al., 2018). The above PPV strains differ in their host herb range and pathogenicity on the particular host, antigenic and epidemiological properties and geographical distribution. From all pointed out strains PPV-Rec, PPV-M and PPV-D are widely spread in Europe and Mediterranean basin having an epidemiological importance (Candresse and Cambra, 2006). Sharka disease described for the first time in Bulgaria Ciclopirox (Atanasoff, 1932) has an endemic incidence in the country (Kamenova et al., 2015). From all trees were included. Our particular interest was directed also to determine the strain status of old-aged PPV infected trees of the local cultivar Kyustendilska sinya in the region of the first detection of Sharka disease. Materials and Methods Field surveys Surveys were carried out in a number of commercial orchards in thirteen different districts of Northern and Southern regions of the country in the period of 2009C2016. Leaf samples were collected from a total of 3020 different stone Ciclopirox fruit trees including plum, peach, apricot, nice cherry, sour cherry and trees growing along the roads and around the orchards. Five to 10 samples per orchard/tree with or without virus-like symptoms were taken from different parts of the tree crown, placed in an ice bag and kept at 4oC prior to testing. The positive samples were also dried under CaCl2. Serological assays All leaf samples were tested for the presence of PPV by double-antibody sandwich indirect enzyme-linked immunosorbent assay (DASI-ELISA), using the universal monoclonal antibody (MAb) 5B (Agritest, Italy) according to (Cambra et al., 1994). The samples that reacted positive were further analysed with serotype-specific MAbs against PPV-M (Agritest, Italy) and PPV-D strains (Durviz, Spain). Samples with absorbance values (at 405 nm) greater than three times the mean of the absorbance of the healthy controls were considered positive. Immunocapture reverse transcription polymerase chain reaction (IC-RT-PCR) strain typing The same extracts tested positive by DASI-ELISA were used for IC-RT-PCR assays. In total 551 isolates (321 from plum, 140 from peach, 78 from apricot and 12 from sp.Serbia and MontengroM”type”:”entrez-nucleotide”,”attrs”:”text”:”AJ243957″,”term_id”:”5650729″,”term_text”:”AJ243957″AJ243957Saenz et al. (2000)SK68-HungaryM”type”:”entrez-nucleotide”,”attrs”:”text”:”M92280″,”term_id”:”294313″,”term_text”:”M92280″M92280Palkovics et al. (1993)GR0019-GreeceM”type”:”entrez-nucleotide”,”attrs”:”text”:”FM955843″,”term_id”:”254938439″,”term_text”:”FM955843″FM955843Palmisano et al. (2009)NectagrandPeachCzech RepublicM”type”:”entrez-nucleotide”,”attrs”:”text”:”AY324839″,”term_id”:”34398225″,”term_text”:”AY324839″AY324839Glasa et al. (2004)VAR-2PeachSlovakiaM”type”:”entrez-nucleotide”,”attrs”:”text”:”AY324837″,”term_id”:”347365475″,”term_text”:”AY324837″AY324837Glasa et al. (2004)Serbia MIPlumSerbia and MontengroRec”type”:”entrez-nucleotide”,”attrs”:”text”:”AY690605″,”term_id”:”56786185″,”term_text”:”AY690605″AY690605Glasa et al. (2005)BOR-3ApricotSlovakiaRec”type”:”entrez-nucleotide”,”attrs”:”text”:”AY028309″,”term_id”:”49659680″,”term_text”:”AY028309″AY028309Glasa et al. (2004)?6PeachSerbiaRec”type”:”entrez-nucleotide”,”attrs”:”text”:”S57404″,”term_id”:”298337″,”term_text”:”S57404″S57404Cervera et al. (1993)BULGPlumBulgariaRec”type”:”entrez-nucleotide”,”attrs”:”text”:”AY324846″,”term_id”:”34398239″,”term_text”:”AY324846″AY324846Glasa et al. (2004)J4cPlumPolandRec”type”:”entrez-nucleotide”,”attrs”:”text”:”EU117116″,”term_id”:”157419767″,”term_text”:”EU117116″EU117116Malinowski et al. (2007)ALB-9PlumAlbaniaRec”type”:”entrez-nucleotide”,”attrs”:”text”:”AY553369″,”term_id”:”49188630″,”term_text”:”AY553369″AY553369Glasa et al. (2004)PENN1PeachUSAD”type”:”entrez-nucleotide”,”attrs”:”text”:”AF401295″,”term_id”:”28273147″,”term_text”:”AF401295″AF401295Mavrodieva Ciclopirox et al. (2003)PENN2PlumUSAD”type”:”entrez-nucleotide”,”attrs”:”text”:”AF401296″,”term_id”:”28273149″,”term_text”:”AF401296″AF401296Mavrodieva et al. (2003)FantasiaPeachCanadaD”type”:”entrez-nucleotide”,”attrs”:”text”:”AY912056″,”term_id”:”62766784″,”term_text”:”AY912056″AY912056James and Varga (2005)VulcanPeachCanadaD”type”:”entrez-nucleotide”,”attrs”:”text”:”AY912057″,”term_id”:”62766786″,”term_text”:”AY912057″AY912057James and Varga (2005)Dideron–D”type”:”entrez-nucleotide”,”attrs”:”text”:”X16415″,”term_id”:”1743375″,”term_text”:”X16415″X16415Teycheney et al. (1989)BIII/2PlumSlovakiaD”type”:”entrez-nucleotide”,”attrs”:”text”:”AY553370″,”term_id”:”49188632″,”term_text”:”AY553370″AY553370?ubr et al. (2010)Cdn1PlumCanadaD”type”:”entrez-nucleotide”,”attrs”:”text”:”AY953261″,”term_id”:”63029786″,”term_text”:”AY953261″AY953261Rochon et al. (2005)RD1P. tomentosaRussiaD”type”:”entrez-nucleotide”,”attrs”:”text”:”KP198599″,”term_id”:”796926241″,”term_text”:”KP198599″KP198599Chirkov et al. (2016)RD2P. tomentosaRussiaD”type”:”entrez-nucleotide”,”attrs”:”text”:”KP198600″,”term_id”:”796926244″,”term_text”:”KP198600″KP198600Chirkov et al. (2016)Kobr8PlumRussiaD”type”:”entrez-nucleotide”,”attrs”:”text”:”KP198590″,”term_id”:”796926210″,”term_text”:”KP198590″KP198590Chirkov et al. (2016)K11PeachRussiaD”type”:”entrez-nucleotide”,”attrs”:”text”:”KP198586″,”term_id”:”796926198″,”term_text”:”KP198586″KP198586Chirkov et al. (2016)Pav6aP. cerasiferaRussiaD”type”:”entrez-nucleotide”,”attrs”:”text”:”KP198596″,”term_id”:”796926232″,”term_text”:”KP198596″KP198596Chirkov et al. (2016)STNGPlumRussiaD”type”:”entrez-nucleotide”,”attrs”:”text”:”KP198602″,”term_id”:”796926250″,”term_text”:”KP198602″KP198602Chirkov et al. (2016)SoCSour cherry-C”type”:”entrez-nucleotide”,”attrs”:”text”:”AY184478″,”term_id”:”37731826″,”term_text”:”AY184478″AY184478Fanigliulo et al. (2003)SwCSweet cherry-C”type”:”entrez-nucleotide”,”attrs”:”text”:”Y09851″,”term_id”:”40252370″,”term_text”:”Y09851″Y09851Fanigliulo et al. (2003) Open up in another windowpane Multiple alignments of nucleotide and amino acidity sequences of PPV CP gene had been completed using ClustalW (Thompson et al., 1994). The alignments had been used to create phylogenetic trees from the 4933436N17Rik neighbor-joining technique, Kimura 2-model.