First Report of a New ‘ Candidatus Phytoplasma pini’-Related Strain Associated with Witches’-Broom of Pinus spp. in Maryland

In April 2015, a pine tree located in a wooded area in Laurel, MD, was observed to have abnormal shoot branching resulting in witches'-brooms. The tree was 20 m tall with scaly dark red-brown bark and had two, occasionally three twisted needles per fascicle of 6 to 9 cm in length. The tree was tentatively identified based on morphological traits and plastid DNA markers as a mountain pine (Pinus pungens Lamb.). From this tree, a sample of needles was collected from a symptomatic branch. In the same area, needle samples were also collected from three pine trees of unknown species without witches'-brooms and used as controls. Total nucleic acids were extracted from 200 mg of each sample using, with minor modifications, a phytoplasma enrichment procedure and a DNA purification method (Ahrens and Seemuller 1992). Semi-nested and nested PCR assays for amplification of the 16S ribosomal (r) RNA gene (rDNA) sequences (primers, P1/16S-SR followed by P1A/16S-SR) and secA genomic region (primers, secAFdg/secARdg followed by PiniSecAF2/PiniSecARA) (Lee et al. 2004; Valiunas et al. 2015) were used to assess possible association of a phytoplasma with the disease. Amplicons of rDNA and secA were of expected sizes, indicating possible phytoplasmal infection of the individual symptomatic pine while no PCR products were obtained from the three control samples. Nucleotide sequencing confirmed that the amplicons were derived from a phytoplasma, termed strain MDPP. The nucleotide sequences obtained from the amplified 1.5-kbp rDNA and 2.0-kbp secA genomic regions were deposited in GenBank under accessions KU242428 (rDNA) and KU242429 (secA region). The phytoplasma was classified as a member of group 16SrXXI using i PhyClassifier, an interactive online tool for phytoplasma classification and taxonomic assignment (Zhao et al. 2013). The 16S rDNA sequence of strain MDPP shares 98.4% similarity with that of the 'Candidatus Phytoplasma pini' reference strain (Schneider et al. 2005) reported from Spain (isolate P127, GenBank accession AJ632155), which is a member of subgroup 16SrXXI-A. The phytoplasma under study was determined to be a 'Ca. P. pini'-related strain. The collective MDPP 16SrDNA F2nR2 virtual RFLP pattern is distinct from the reference patterns of all previously delineated subgroups, and is most similar to that of subgroup 16SrXXI-A, with a pattern similarity coefficient of 0.79 (GenBank accession AJ632155). Since the similarity coefficient value is lower than the thres