engleski

A case study of BN phytoplasma pathosystem: multilocus sequence typing approach

Introduction Bois noir (BN) is one of the most important grapevine yellows in Europe. Stolbur phytoplasma (16SrXII-A), the etiological agent of Bois Noir (BN), is widespread and detected in most of the Croatian grape growing regions (Šeruga Musić et al., 2009). Most important known vector cixiid Hyalesthes obsoletus Signoret (Maixner et al., 2007) has a key role in the spread of BN phytoplasma. It can transmit phytoplasma to a wide range of plants alongside grapevine. In elucidation of complex phytoplasma-vector-plant epidemiology of the BN disease, identification and characterization of phytoplasma isolates plays an important role. In addition to the widely used non-ribosomal house- keeping genes such as tufB and secY (Langer and Maixner, 2004 ; Fialová et al., 2009) BN-specific genes vmp1 and stamp encoding membrane proteins are currently proposed as genetic markers for finer differentiation of the stolbur phytoplasmas (Cimerman et al., 2009, Fabre et al., 2011).The aim of this study was to investigate the genetic variability among the detected phytoplasmas and further clarify the epidemiology of the BN pathosystems by multilocus sequence typing (MLST) approach at two chosen locations, Vukanovec and Brckovština, representing BN phytoplasma foci. Materials and methods Grapevine and weed samples together with samples of BN insect vector H.obsoletus (Vukanovec) were collected from 2 locations ; Vukanovec and Brckovština (Fig. 1). Total nucleic acids were extracted from all samples and triplex real-time PCR assay amplifying non-ribosomal house-keeping gene map was performed in order to simultaneously detect the presence of BN and FD phytoplasmas - another important GY agent (Pelletier et al., 2009). Conventional PCR/RFLP analysis of phytoplasma 16S was also performed. In order to study the variability of detected phytoplasmas MLST (mulitlocus sequence typing) analysis approach was used. Following genes were amplified from all samples: tufB, secY, vmp1 and stamp. TufB and vmp1 gene amplicons were digested with HpaII (Langer and Maixner, 2004) and RsaI (Fialova et al., 2009) respectively, whilst amplicons of secY and stamp genes were sent for sequencing and subsequent phylogenetic analyses were performed. Results and discussion In Vukanovec, the presence of BN phytoplasma was confirmed in grapevine samples as well as in insect vectors, while no phytoplasmas were detected in weed samples. In Brckovština BN phytoplasma was confirmed in grapevine samples and in one weed sample Convolvulus arvensis L. while no insect vectors were found. Restriction analysis with HpaII revealed that all tufB amplicons from grapevine, insects and C.arvensis shared the same tuf-B restriction profile. Only one type of vmp1 profile - V18 was obtained after RFLP analyses with RsaI of amplicons from grapevine and insects, while the one obtained from weed C.arvensis had a different profile – V4 (Fig.2). Phylogenetic analysis of secY gene sequences has showed that sequences from grapevine and insects belong to the same - S6 group while the one from C.arvensis belongs to the S4 group. Congruence was observed with phylogenetic analysis of stamp gene where sequence from C. arvensis belonged to ST9 phylogenetic group while sequences from grapevine and insects grouped together in different ST6 group. In comparison of results, restriction profiles of vmp1 showed correlation with phylogenetic groups of secY and stamp genes. Also, tufB proved to be less variable than the secY, vmp1 and stamp genes. MLST including RFLP and phylogenetic analyses have revealed a strong relatedness between phytoplasma detected in grapevine and insect samples showing their affiliation to the same pathosystem. BN phytoplasma found in C.arvensis from Brckovština has shown a considerable genetic variability in all of the analyzed genes. These results suggest a co-existence of different phytoplasma pathosystems at the same location.

Candidatus Phytoplasma solani; epidemiology; MLSA

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