First Report of 'Candidatus Phytoplasma asteris' Associated with Witches' Brooms on Sharp-Flowered Rush (Juncus acutiflorus) in Poland.

Abstract

Juncus acutiflorus L. (Juncaceae) is a species of rush widespread in Europe, North Africa, and Southwest Asia. The health of sharp-flowered rush is threatened by witches’ brooms occurrence. Typical symptoms are dense masses of shoots, usually growing from a single point, with the resulting structure resembling a broom. Witches’ brooms are typically formed in response to Livia junci (Liviinae, Hemiptera) feeding, oligofag, which typically feeds on Juncus sp. These symptoms are similar to those observed in Juncus articulatus L. (Juncaceae) related to phytoplasma infection in Silesia, Poland (Jarzembowski et al. 2015). In August 2017, samples were collected from nine symptomatic and four asymptomatic plants growing in Jagniątkow, Poland. DNA from 100 mg of inflorescence and leaf samples was extracted using a DNeasy Plant Mini Kit (Qiagen, Syngen Biotech, Wroclaw, Poland) according to the manufacturer’s protocol. Additionally, 10 L. junci specimens (adult and the last larva stage) were collected from symptomatic plants and preserved in ethanol (75%). DNA from the insects (treated as one sample) was extracted using a DNeasy Blood and Tissue Kit (Qiagen, Syngen Biotech). The extracts were used as the template in polymerase chain reaction (PCR) assays with the universal phytoplasma primers P1/P7 followed by R16F2/R16R2 (Lee et al. 1998). Additionally, primers rp1/rp2 followed by rp3/rp4, allowing amplification of fragments of rpl22 and rps3 genes (Nakamura et al. 1996), and primers AYsecYF1/AYsec for amplification of secY gene were applied. Water blank samples were included as negative controls. PCR products of expected size for each primer set were amplified from the insects and six symptomatic plants that were assayed. No amplification was observed in symptomless J. acutiflorus samples or in water blanks. Amplicons representing three genetic loci were sequenced at Genomed S.A., Warsaw, Poland. DNA samples were sequenced at least twice in both directions. Sequencing results of the PCR products confirmed that six symptomatic plants and the insects were infected by a phytoplasma. The obtained sequences were nearly identical, and representative sequences of 16S rDNA fragments (accession nos. MG976242 to MG976246), secY gene (MG983765 to MG983769), and ribosomal protein gene (MG983760 to MG983764) from four plants and one insect sample were deposited in GenBank. Determination of the phytoplasma classification group was based on the nucleotide sequence of the F2n/R2 PCR fragment within the 16S gene. Using iPhyClassifier, the online tool for phytoplasma classification (https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier_legacy.cgi), the sequence of J. acutiflorus phytoplasma shared 99.9% identity with that of the ‘Candidatus Phytoplasma asteris’ reference strain (GenBank accession no. M30790). BLAST analysis performed for ribosomal proteins and secY genes confirmed the highest identity (99%) of analyzed sequences with those of ‘Ca. P. asteris’ (CP015149 and KJ394491, respectively). To our knowledge, this is the first report of a phytoplasma of group 16SrI affecting J. acutiflorus in Poland and in the world. The impact of the detected phytoplasma in the regional ecosystem is being assessed. It is highly probable that L. junci is a possible vector for ‘Ca. P. asteris’.