First report of 'Candidatus Phytoplasma asteris' associated with witches'-broom disease of Pinus yunnanensis in China

Pinus yunnanensis is an evergreen tree belonging to Pinaceae. The species is distributed in the east of Tibet, southwest of Sichuan, southwest of Yunnan, southwest of Guizhou and northwest of Guangxi. It is an indigenous and pioneer tree species for barren mountain afforestation in southwest China. P. yunnanensis has important value to both the building and medicine industries (Liu et al. 2022). In May 2022, P. yunnanensis showing witches'-broom symptom were found in Panzhihua City, Sichuan Province, China. The symptomatic plants had yellow or red needle, and exhibited plexus bud and needle wither. The lateral buds of infected pines developed into twigs. Some lateral buds grew in clusters and a few sprouted needles (Fig.1). The disease was named the P. yunnanensis witches'-broom disease (PYWB) and was found in some areas of Miyi, Renhe, and Dongqu. More than 9% of the pines showed these symptoms in the three areas surveyed, and the disease was spreading. A total of 39 samples were collected from three areas, including 25 symptomatic plants and 14 asymptomatic plants, respectively. The lateral stem tissues of 18 samples were observed under a scanning electron microscope (Hitachi S-3000N). Spherical bodies were found in the phloem sieve cells of symptomatic pines (Fig.1). Total DNA was extracted from 18 plant samples using the CTAB method (Porebski et al. 1997) and subjected to nested-PCR testing. Double-distilled water and DNA extracted from asymptomatic plants were used as negative controls, and DNA extracted from Dodonaea viscosa affected by the D. viscosa witches'-broom disease was used as positive control. Nested PCR was employed to amplify the pathogen's 16S rRNA gene (Lee et al. 1993; Schneider et al. 1993) and 1.2 kb segment were produced (GenBank accessions OP646619; OP646620; OP646621). PCR specific to the ribosomal protein (rp) gene yielded segment of approximately 1.2 kb (Lee et al. 2003)(GenBank accessions OP649589; OP649590; OP649591). The fragment size from 15 samples was consistent with the positive control, confirming the association of phytoplasma with the disease. A BLAST analysis of the 16S rRNA sequences of P. yunnanensis witches'-broom phytoplasma showed that it shared 99.12% ~99.76% identity with that of Trema laevigata witches'-broom phytoplasma (GenBank accession MG755412). The rp sequence shared 99.84%~99.92% identity with that of Cinnamomum camphora witches'-broom phytoplasma (GenBank accession OP649594). An analysis with iPhyCla