Wei, Wei

Buckwheat (Fagopyrum esculentum Moench) is a fast-growing, low-input crop in the Polygonaceae family, cultivated on about 2 million hectares worldwide, mainly in Russia, China, and Ukraine by FAO (2023). In Taiwan, it is grown as a winter cover crop during fallow periods and is valued for improving soil health, suppressing weeds, and supporting sustainable farming systems. It is also utilized in various food products due to its rich fiber content and gluten-free properties. In May 2024, 147 out of 3000 plants (Taichung No. 5) corresponding to an incidence rate of approximately 5% exhibiting floral abnormalities, including phyllody, virescence, and flower sterility, were observed in Xinshe District in Taichung, central Taiwan (Fig. 1). These symptoms were indicative of possible phytoplasma infection. For further investigation, floral and leaf tissues were randomly collected from 10 symptomatic and 10 asymptomatic buckwheat plants. Plant total DNA was extracted using the DNeasy Plant Mini Kit, and PCR was performed targeting the 16S rRNA gene using universal phytoplasma primers P1A/P7A, followed by a semi-nested reaction with P1A/16S-SR (Lee et al. 2004). All PCR amplicons of approximately 1.5 kb were obtained only from symptomatic samples. PCR amplicons were sequenced by Sanger sequencing, and sequences were assembled by BioEdit. All symptomatic samples yielded identical sequences and a representative sequence (1,543 bp, nearly full-length 16S rRNA gene) was deposited in GenBank (Accession no. PV487329). Based on virtual RFLP analysis using iPhyClassifier (Zhao et al. 2009), the phytoplasma strain detected in diseased buckwheat plants was identified as a ‘Candidatus Phytoplasma aurantifolia/citri’-related strain, sharing 98.27% sequence identity with the reference strain (witches broom disease of lime, WBDL, Accession no. U15442). The strain, designated as Buckwheat Phyllody (BWP) TSIPS 3-1, 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). A BLASTn search against the NCBI database revealed that BWP-TSIPS 3-1 shared 99.94% identity with the 16S rRNA gene of potato purple top phytoplasma (Accession no. KM212951, a ‘Ca. P. aurantifolia/citri’-related strain), identified in Guangdong, China (Cheng et al. 2019). To further confirm phytoplasma identity, an additional gene marker, secY gene was amplified by semi-nested PCR using group-specific primers SecYF1(II)/SecYR1(II) and SecYF2(II) /SecYR1(II) (Lee et al. 2010). The 1,263-bp amplicons were obtained from all symptomatic samples. Sequencing results revealed identical secY sequences across samples, and a representative sequence was deposited in GenBank (Accession no. PV567544). BLASTn analysis indicated 100% (1263/1263) identity with the secY gene of the phytoplasmas, including black gram witches’-broom phytoplasma in Myanmar (Accession no. AB703249), which is classified within the 16SrII-A subgroup (Win and Jung, 2012). Phytoplasma infections associated with buckwheat have been rarely reported, with only a single case documented in India, though the short 16S rRNA gene sequence limited subgroup classification (Bandakkanavar et al. 2023). Similar symptoms, such as phyllody, have also been observed in Ukraine, but the causal agent remains unconfirmed due to the absence of molecular diagnostics (Demchenko et al. 2016). Our study confirmed the first identification of a ‘Ca. P. aurantifolia/citri’-related strain (16SrII-A subgroup) in buckwheat in Taiwan. Phytoplasmas of the 16SrII-A subgroup have been widely documented across Taiwan, including in Taichung (Tseng et al. 2012). Given the rising popularity of buckwheat for its dietary benefits and its increasing incorporation into crop rotation systems, especially as a winter cover crop, the presence of phytoplasma presents potential concerns. Infected buckwheat plants may inadvertently serve as hosts or reservoirs for phytoplasmas and their insect vectors, indicating a need for ongoing monitoring, especially in areas where buckwheat is part of crop rotations.

‘Candidatus Phytoplasma trifolii’ is a cell wall-less phytopathogenic bacterium that infects many agriculturally important plant species such as alfalfa, clover, eggplant, pepper, potato, and tomato. The phytoplasma is responsible for repeated outbreaks of potato purple top (PPT) and potato witches’ broom (PWB) that occurred along the Pacific Coast of the United States since 2002, inflicting significant economic losses. To effectively manage these phytoplasmal diseases, it is important to develop diagnostic tools for specific, sensitive, and rapid detection of the pathogens. Here we report the development of a DNA endonuclease targeted CRISPR trans reporter (DETECTR) assay that couples isothermal amplification and Cas12a transcleavage of fluorescent oligonucleotide reporter for highly sensitive and specific detection of ‘Candidatus Phytoplasma trifolii’-related strains responsible for PPT and PWB. The DETECTR assay was capable of specifically detecting the 16S-23S ribosomal DNA intergenic transcribed spacer sequences from PPT- and PWB-diseased samples at the attomolar sensitivity level. Furthermore, the DETECTR strategy allows flexibility to capture assay outputs with fluorescent microplate readers or lateral flow assays for potentially high-throughput and/or field-deployable disease diagnostics.

The genus ‘Candidatus Phytoplasma’ was proposed to accommodate cell wall-less bacteria that are molecularly and biochemically incompletely characterized, and colonize plant phloem and insect vector tissues. This provisional classification is highly relevant due to its application in epidemiological and ecological studies, mainly aimed at keeping the severe phytoplasma plant diseases under control worldwide. Given the increasing discovery of molecular diversity within the genus ‘Ca. Phytoplasma’, the proposed guidelines were revised and clarified to accommodate those ‘Ca. Phytoplasma’ species strains sharing >98.65 % sequence identity of their full or nearly full 16S rRNA gene sequences, obtained with at least twofold coverage of the sequence, compared with those of the reference strain of such species. Strains sharing <98.65 % sequence identity with the reference strain but >98.65 % with other strain(s) within the same ‘Ca. Phytoplasma’ species should be considered related strains to that ‘Ca. Phytoplasma’ species. The guidelines herein, keep the original published reference strains. However, to improve ‘Ca. Phytoplasma’ species assignment, complementary strains are suggested as an alternative to the reference strains. This will be implemented when only a partial 16S rRNA gene and/or a few other genes have been sequenced, or the strain is no longer available for further molecular characterization. Lists of ‘Ca. Phytoplasma’ species and alternative reference strains described are reported. For new ‘Ca. Phytoplasma’ species that will be assigned with identity ≥98.65 % of their 16S rRNA gene sequences, a threshold of 95 % genome-wide average nucleotide identity is suggested. When the whole genome sequences are unavailable, two among conserved housekeeping genes could be used. There are 49 officially published ‘Candidatus Phytoplasma’ species, including ‘Ca. P. cocostanzaniae’ and ‘Ca. P. palmae’ described in this manuscript.

Abstract The reference strain of 'Ca. Phytoplasma trifolii' is the causative agent of clover proliferation (CP) disease of alsike clover (Trifolium hybridum). The CP disease was first reported in Canada in the early 1960s when the aetiological agent was mistakenly presumed to be a yellows-type virus (Chiykowski, 1965). Subsequent investigations revealed that the disease was associated with infection by a mycoplasma-like organism (Chen and Hiruki, 1975>; Hiruki and Chen, 1984), now termed phytoplasma, strain CPR (Hiruki and Wang, 2004). Later, phytoplasmas of the same lineage (subgroup 16SrVI-A) were found in the USA, Mexico, and many countries in Europe and Asia, causing diseases in diverse leguminous and vegetable crops, responsible for significant yield losses and quality reductions. Phytoplasmas of the same lineage also caused disease in elm trees in the USA. Phytoplasmas of closely-related lineages (various subgroups of group 16SrVI) also have wide distributions around the world.