First Report of Prunus domestica as the Host of a Phytoplasma Belonging to Group 16SrI, Subgroup B/L

Abstract

HomePlant DiseaseVol. 103, No. 1First Report of Prunus domestica as the Host of a Phytoplasma Belonging to Group 16SrI, Subgroup B/L PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Prunus domestica as the Host of a Phytoplasma Belonging to Group 16SrI, Subgroup B/LA. Zwolińska, N. Borodynko-Filas, D. Nowaczyk, and B. Hasiów-JaroszewskaA. Zwolińska†Corresponding author: A. Zwolińska; E-mail: E-mail Address: a.zwolinska@iorpib.poznan.plhttp://orcid.org/0000-0003-2681-2915Search for more papers by this author, N. Borodynko-FilasSearch for more papers by this author, D. NowaczykSearch for more papers by this author, and B. Hasiów-JaroszewskaSearch for more papers by this authorAffiliationsAuthors and Affiliations A. Zwolińska † N. Borodynko-Filas D. Nowaczyk B. Hasiów-Jaroszewska , Institute of Plant Protection – National Research Institute, Virology and Bacteriology Department, Poznań, Poland. Published Online:31 Oct 2018https://doi.org/10.1094/PDIS-06-18-0963-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat In July 2016 and 2017, diseased plum trees (Prunus domestica L.) were observed in the orchard localized in the Wielkopolska region of Poland. The symptoms of witches’ brooms with internode shortening, axillary bud growth, and reduced leaf size indicated a potential phytoplasma infection. Samples of shortened shoots were collected from five symptomatic and five asymptomatic plants, respectively. DNA samples were extracted from 2 g of leaf tissues using a modified cetyltrimethylammonium bromide procedure (Maixner et al. 1995) and subsequently used as templates in nested polymerase chain reaction (PCR) for the amplification of genes encoding 16S rRNA; ribosomal proteins (rp) S19, L22, and S3 and elongation factor Tu (tuf) with primers P1/P7 followed by R16F2n/R16R2; rpF1/rpR1 followed by rp(I)F1A/rp(I)R1A and fTuf1/rTuf1 followed by fTufAY/rTufAY, respectively (Duduk et al. 2013; Martini et al. 2007; Schneider et al. 1997). Amplicons of correct sizes (1.2, 1.2, and 0.9 kb) were obtained from all the diseased plants tested (samples Plum1 to Plum5), respectively. No PCR products were obtained from the asymptomatic samples. All PCR products were ligated into pGEM-T Easy Vector Systems (Promega), and plasmid DNA was sequenced by an external company (Genomed, Warsaw, Poland). The 16S rDNA, tuf, and rp genes coding sequences from Plum1 to Plum5 were identical, and therefore one representative sequence of each region was deposited in GenBank under accession numbers MH061193, MH061368, and MH061366, respectively. The R16F2n/R16R2 (MH061193) primed fragment was subjected to in silico restriction digestion using iPhyClassifier, the online tool for phytoplasma classification (https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi). The collective restriction fragment length polymorphism patterns indicated that diseased plants were infected by ‘Candidatus Phytoplasma asteris’ of subgroup 16SrI-L. Next, the 385-bp barcode fragments of the EF-Tu gene from representatives of different phytoplasma groups were retrieved from the GenBank database. The phylogenetic analysis was performed based on those sequences and the tuf sequence obtained in this study using the neighbor joining algorithm of MEGA 7 software (https://www.megasoftware.net/). The 1.2-kbp DNA segment containing rp genes was also analyzed. In the phylogenetic trees, the Polish isolate of plum witches’ broom phytoplasma clustered together with ‘Ca. P. asteris’ representatives of ribosomal subgroup 16SrI-B and 16SrI-B/L. Based on all the performed analyses, the plum witches’ broom phytoplasma (PlumWB) was classified as a member of group 16SrI, subgroup B/L, which consists of two heterogeneous 16Sr RNA genes (Jomantiene et al. 2010). To our knowledge, this is the first report of plums infected with ‘Ca. P. asteris’ representing subgroup I-B/L in Poland, suggesting that fruit trees can be perennial reservoirs of the phytoplasma, which has great significance in the pathogen’s epidemiology.