First Report of a 16SrII-A Group Phytoplasma Associated With Witches’-Broom Disease of Soybean ( Glycine max ) in Guangdong, China

Soybean (Glycine max (Linn.) Merr.), the most widely cultivated oilseed and legume, accounts for over 60% of global oilseed production and serves as a critical component of both food systems and industrial applications (Wang et al., 2020; Fang, et al., 2022). In September 2025, typical symptoms of phytoplasma infection on Soybean variety HX10 were observed in a farmer’s field at Dongshi District (24°40′22.728″N, 115°57′13.104″E) and Pingyuan county, Meizhou city, Guangdong Province, China. Symptoms consisted of floral abnormalities, phyllody, proliferation of axillary shoots with shortened internodes, and stunting. And an field assessment showed a disease incidence ranging form 15% to 20%, withan estimated 20% yield reduction, indicating a significant threat to the Guangdong’s soybean industry. For further investigation, total DNA were extracted from fresh leaves of ten each of symptomatic and asymptomatic by the Plant Genomic DNA Purification Kit (DP305; Tiangen; Beijing; China). Conventional nested PCR was performed targeting the 16S rRNA gene using primer pairs P1/P7 followed by R16mF2/mR1 (Lee et al. 1993). All PCR amplicons of approximately 1.4 kb were obtained only from symptomatic samples. The phytoplasma strain was designated as GDPYSB2501. PCR amplicons were sequenced, and a representative sequence (1,416 bp) was deposited in GenBank (Accession no. PX583579). BLASTn analysis revealed that the sequence shared 99.86% with the SPLL subgroup reference strain ‘Candidatus Phytoplasma australasiaticum subsp. Ipomoeae’ o7C (GenBank accession no. GCA_024425275.1, 1,386/1,388 bp), and 98.70% with the PLL subgroup reference strain ‘Ca. P. fabacearum’ BAWM-027 (GenBank accession no. GCA_030586805.1, 1,370/1,388 bp), respectively (Rodrigues Jardim et al. 2023). Moreover, virtual RFLP analysis of the 16S rDNA F2nR2 fragments using iPhyClassifier (Zhao et al. 2009) revealed that the phytoplasma strain was classified within the 16SrII-A subgroup, with a similarity coefficient of 1.00 when compared to the reference strain of subgroup 16SrII-A (Accession no. L33765). To validate the results, a phylogenetic tree of the 16S rRNA sequences of GDPLSB2501 by MEGA 7.0 (Kumar et al. 2016) confirmed that the phytoplasma strain consistently clustered within the same subclade as the 16SrII group ‘Ca. P. australasiaticum’ related strain NCHU2014 (16SrII-A), BAWM-TWN (16SrII-V) and o7C (SPLL). Taken together, the phytoplasma GDPLSB2501 was related to ‘Ca. P. australasiaticum’ (16SrII-A), following the latest ‘Ca. Phytoplasma’ species description guidelines (Bertaccini et al. 2022; Rodrigues Jardim et al. 2023; Wei and Zhao 2022). Earlier, peanut witches’-broom group phytoplasma (Ca. P. australasiaticum, 16SrII-V) was recorded in green manure soybean in Taiwan Province, China (Wang et al. 2021). To our knowledge, this is the first report a ‘Ca. P. australasiaticum’ -related strain(16SrII-A) associated with witches’-broom disease of soybean in Guangdong, China. Phytoplasmas within the 16SrII group have been detected in a large range of hosts over an extensive geographic range, resulted in significant economic and ecological losses worldwide (Rodrigues Jardim et al. 2023). Nonetheless, these findings highlight the need to identify the potential insect vectors and search for alternative hosts (including economically important crops or weeds) for this phytoplasma, which will be beneficial for the epidemiological monitoring and early warning of Soybean witches'-broom phytoplasma disease.