The need to identify insect vectors and alternative hosts, which will provide theoretical guide to the control of Bamboo Witches’ Broom Disease is highlighted.
Abstract
Bambusa multiplex, one of the most acclimatized clumping-bamboo species, has been broadly cultivated in China and has significant economical, ecological and ornamental importance (Yuan et al. 2009). In November 2025, more than 30% of B. multiplex plants exhibiting typical witches’ broom, internode shortening, leaf proliferation and clustering of leaves were observed in a horticultural landscaping field in Zhangjiang city, Guangdong Province, China. To confirm the presence of a phytoplasma, total DNA was extracted from fresh leaves of ten symptomatic and four asymptomatic plants using the CTAB method. Conventional nested PCR was performed using primer pairs P1/P7 followed by R16F2n/R16R2 (Gundersen and Lee, 1996), and expected fragment amplified only from the symptomatic plants. The PCR products were sequenced, and deposited in GenBank under the accession number PX998769. BLAST analysis revealed that the sequence shared 99.60% identity with that of the SPLL subgroup reference strain ‘Candidatus Phytoplasma australasiaticum subsp. Ipomoeae’ o7C (GenBank accession no. GCA_024425275.1, 1,244/1,249 bp) (Rodrigues Jardim et al. 2023). Further virtual RFLP pattern analysis of the 16S rRNA sequence by iPhyClassifier (Zhao et al. 2009) verified that the phytoplasma strain, designated as BMW-ZJ, was a member of the 16SrII-A subgroup with a 1.00 similarity coefficient to the reference phytoplasma strain (GenBank accession no. L33765). Moreover, the secY gene was amplified using primer pairs secYwbF1/secYwbR1 followed by secYwbF2/secYwbR2 (Wang et al. 2025), and the sequence(GenBank accession no. PZ135411) shared 100% identity to several ‘Ca. P. australasiaticum’ strains (NTU2011, BAWM-TWN and BAWM-THA-CLP). Phylogenetic analysis based on the 16S rRNA and secY genes conducted by MEGA 12 (Kumar et al. 2024), confirmed that BMW-ZJ was related to ‘Ca. P. australasiaticum’(Bertaccini et al. 2022; Rodrigues Jardim et al. 2023). Earlier, 16SrII phytoplasma associated with bamboo species Dendrocalamus giganteus (Che et al. 2024), and 16SrI phytoplasma associated with Sasa fortunei (Zhang et al. 2009) were reported in Hainan and Shaanxi Province, China, respectively. To our knowledge, this is the first report a ‘Ca. P. australasiaticum’ -related strain associated with Bamboo Witches’ Broom Disease (BWB) in Guangdong, China. The BWB caused by Ca. Phytoplasma (16SrI, 16SrII, 16SrVIII and 16SrXIV group), has been confirmed in several bamboo species in Southeast Asian countries such as India (Yadav et al. 2016; Ravi et al. 2022), Philippines (Dolores et al. 2023), Indonesia (Prasetya et al. 2020) and China (Zhang et al. 2009; Che et al. 2024). This study highlight the need to identify insect vectors and alternative hosts, which will provide theoretical guide to the control of Bamboo Witches’ Broom Disease.
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.
Little ironweed (Cyanthillium cinereum; syn. Vernonia cinerea) is an herbaceous plant in the Asteraceae family (Trang et al. 2024). The plant is distributed in Asia, Australia and Africa, and has been used as a traditional medicine (Trang et al. 2024). In January 2026, little ironweed plants exhibiting phyllody symptoms (Fig. S1) were observed in a grass-covered area (measuring 3 m × 6 m) in Qishan District, Kaohsiung, Taiwan. Around 8% (22/285 plants) of the population showed symptoms. Six symptomatic and four asymptomatic plants were collected. Flower-like tissues from symptomatic plants were collected for DNA extraction with a Synergy 2.0 Plant DNA Extraction Kit (OPS Diagnostics). For asymptomatic plants, capitula were used instead. One DNA sample was extracted for each plant. The samples were first tested with primers specific to the plant’s 26S rDNA (28KJ/28C; Cullings 1992), and all of them produced the target amplicon (~0.69 kb), indicating sufficient quality for polymerase chain reaction (PCR). Nested PCR targeting phytoplasmas’ 16S rDNA was conducted with outer and inner primer pairs P1/P7 and fU5/rU3 (Lorenz et al. 1995). In all PCR assays, negative controls (nuclease-free water) were included. All samples from symptomatic plants amplified the outer and inner amplicons (~1.81 kb and ~0.88 kb), whereas DNA from the asymptomatic plants did not. After assembling sequences of the outer and inner fragments for each symptomatic sample, identical 16S rDNA sequences were found. A representative sequence was deposited in GenBank (1,459 bp; accession no. PZ193724), and BLASTn search against GenBank revealed that it was identical to that of a reference phytoplasma strain belonging to subgroup 16SrII-A (NCHU2014; accession no. CP040925; 100% identity, 100% query coverage; Rodrigues Jardim et al. 2023). A 1,248-bp fragment within the obtained sequence was further analyzed using iPhyClassifier (https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi), and its virtual RFLP pattern was the same as 16SrII-A’s reference pattern (similarity coefficient = 1.00; accession no. L33765). The detected phytoplasma’s identity was further confirmed by amplification and sequencing of the elongation factor Tu (tuf) and protein translocase (secY) genes. Fragments containing tuf and secY were respectively amplified by primer sets TUF-II-F1/TUF-II-F2/TUF-II-R1 (for semi-nested PCR; Al-Subhi et al. 2017) and SecYF1(II)/SecYR1(II) (for PCR; Lee et al. 2010). The tuf sequences were obtained from the inner amplicon, whereas sequences of secY were obtained by sequencing followed by primer walking (accession nos. PZ377067 and PZ377068). The detected phytoplasma and the reference strain NCHU2014 (‘Ca. Phytoplasma australasiaticum’-related strain; 16SrII-A; accession no. CP040925) shared identical sequences for both tuf (986 bp; partial) and secY (1,263 bp; complete) fragments. This is the first report of little ironweed infected by a 16SrII-A phytoplasma in Taiwan; this specific host-pathogen association has only been reported in Hainan Province of China (Yu et al. 2024). Because 16SrII-A phytoplasma can infect economic crops in Taiwan (Wu et al. 2026) and little ironweed is widely distributed in the island, it is possible that the weed may represent a potential reservoir host. Thus, monitoring and eradication of symptomatic little ironweed is advisable in crop production areas.
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