First Report of a ‘ Ca . P. australasiaticum’-related Strain (16SrII-A) Phytoplasma Associated with Bougainvillea Witches’ Broom in China

Bougainvillea is a widely cultivated ornamental in subtropical and tropical regions. In November 2025, Bougainvillea buttiana in Zhanjiang City, Guangdong Province, China (110°18′36″E, 21°9′36″N), exhibited symptoms including flower abortion, phyllody, and proliferation of axillary shoots with shortened internodes (Fig. S1) and the disease incidence ranged from 10% to 15%. These symptoms differed from those associated with phytoplasma infection in bougainvillea in Brazil, India and Cuba (Silva et al., 2014; Gopala and Rao, 2018; Wei W et al., 2020). Total DNA was extracted from fresh leaves of 9 symptomatic and 3 asymptomatic samples. Nested PCR was conducted with primers P1/P7 followed by R16F2n/R16R2 (Gundersen and Lee, 1996), and secYwbF1/secYwbR1 followed by secYwbF2/secYwbR2 (Wang et al. 2025) specific for phytoplasma 16S rRNA and secY gene fragments, respectively. The two gene target fragments of phytoplasma were obtained exclusively from symptomatic samples and asymptomatic samples tested negative. The PCR products were sequenced and all the gene sequences were identical (GenBank accession: 16S rRNA PX848724; secY PX853972). BLASTn search based on 16S rRNA genes in NCBI Genbank database indicated that our sequence had 99.92% identity with 16SrII-A strains Peanut witches'-broom phytoplasma NTU2011 (NZ_AMWZ01000008.1, 1238/1239 bp) and 'Vigna radiata' phytoplasma BAWM-TWN (NZ_JAOSIS010000033.1, 1238/1239 bp). Meanwhile, the secY gene sequence fragment showed 100% identity with that of phytoplasma NTU2011 (NZ_AMWZ01000013.1) and phytoplasma BAWM-TWN (NZ_JAOSIS010000010.1). Analysis conducted using the iPhyClassifier (Zhao et al., 2009) indicated that BWBZJ2025 belongs to the 16SrII-A subgroup, with a similarity coefficient of 1.00 to the subgroup reference strain L33765 (Fig. S2). Therefore, the strain was designated as strain BWBZJ2025. Phylogenetic analysis based on the concatenated 16S rRNA and secY sequences (Fig. S3) revealed that BWBZJ2025 clustered within the same subclade as the 16SrII-A reference strain PnWB, and ‘Ca. Phytoplasma australasiaticum’ related strain NCHU2014 (16SrII-A) and BAWM-TWN (16SrII-V). Taken together, these results consistently identified strain BWBZJ2025 as a member of the 16SrII-A subgroup, related to ‘Ca. Phytoplasma australasiaticum’. To our knowledge, this study is the first report of a phytoplasma associated with bougainvillea in China. Further research is needed to identify potential vectors for epidemiological monitoring.