First Report of 'Candidatus Phytoplasma asteris' Associated with "Witches'-Brooms" on Jointleaf Rush (Juncus articulatus) in Poland.

Juncus articulatus L. (Juncaceae) is a species of rush occurring in Eurasia, Canada, and the United States. In 2013, symptoms of "witches'-brooms," similar to those associated with phytoplasma infection in other plants, were observed on jointleaf rush plants in Lower Silesia (southwest Poland), with some pests feeding on them. Livia junci (Liviinae, Hemiptera) is a small plant-feeding sap-sucking insect that affects monocotyledonous plants. To confirm the presence of phytoplasma in 15 examined plants, total DNA was extracted from 100 mg of inflorescence and leaf samples collected in July 2013 in Bogatynia, Poland, from six symptomatic and six asymptomatic plants using a DNeasy Plant Mini Kit (Qiagen, Syngen Biotech, Wrocław, Poland) according to the manufacturer's protocol. Additionally, three leaf samples from asymptomatic rush plants, collected from a location where the disease was not observed (Wrocław, Poland), as well as water blank samples were included as negative controls. Moreover, thirty-two insects were collected from symptomatic plants and preserved in ethanol (75%). DNA from L. junci specimens (the imago and the last larva stage) was extracted using DNeasy Blood and Tissue Kit (Qiagen, Syngen Biotech). Extracted nucleic acids were used as templates for PCR employing a) phytoplasma universal rRNA primer pairs P1/P7 followed by R16F2n/R16R2 (1), b) primers rp1-rp2 followed by rp3-rp4, allowing amplification of fragments of ribosomal protein rpl22 and rps3 genes (3), and c) primers AYsecYF1/AYsecYR1 (2) for amplification of the secY gene. The phytoplasma was detected in all tested insects as well as in all six symptomatic and four out of six asymptomatic plant samples (10 out of 12 plant samples from Bogatynia were positive). No amplification products were detected in negative control samples from Wrocław or in water blanks. The fact that we detected the pathogen in some asymptomatic plants indicated that a low concentration may have been present prior to the development of disease symptoms. Amplicons representing three genetic loci were sequenced in an AbiPrism 3100 Genetic Analyzer apparatus (Applied Biosystems, USA), at the Maria Skłodowska Memorial Cancer Center and Institute of Oncology, Warsaw, Poland. To avoid sequencing errors, all DNA samples were sequenced twice in both directions. The obtained sequences were nearly identical, and representative sequences of 16S rDNA fragments (Accession Nos. KF774297, KF774298, and KF774299), secY gene (KJ394481, KJ394482, and KJ394483) and ribosomal protein gene (KJ394484, KJ394485, and KJ394486), isolated from two plants and one insect, were deposited in GenBank. BLAST analysis of the sequenced 16S rDNA fragments revealed that tested strains shared more than 99% sequence identity with the sequences of phytoplasmas from the aster yellows group (e.g., KJ556903, KJ494330, and KJ491100). The same analysis performed for ribosomal proteins and secY genes confirmed the highest identity (99%) of analyzed sequences with those of 'Candidatus Phytoplasma asteris' (HM626105 and KC354611, respectively). The impact of the detected phytoplasma in the regional ecosystem and the role of L. junci as a possible vector of this pathogen are being assessed. References: (1) I. M. Lee et al. Int. J. Syst. Evol. Microbiol. 48:1153, 1998. (2) I.-M. Lee et al. Mol. Cell. Probes 20:87, 2006. (3) H. Nakamura et al. Plant Dis. 80:302, 1996.