First report of a 'Candidatus phytoplasma asteris' related strain (16SrI) associated with phyllody and witches'-broom symptom in Pisum sativum in Hisar, India.

Pea (Pisum sativum L.) is a leguminous vegetable crop, and India holds the fourth position in the production, primarily contributed by three major states: Uttar Pradesh, Madhya Pradesh, and Punjab (Anonymous, 2022). However, a survey conducted in February 2023 at the National Seed Corporation Farm (15 hectares) in Hisar, Haryana, revealed deformities in the growth of some pea plants. Approximately 10% of these plants exhibited a distinct bushy appearance, accompanied by phyllody and witches'-broom symptoms, characterized by deformed leaves and short internodes (Fig. 1). In response to these observed anomalies, a detailed molecular analysis was conducted at the Plant Pathology Laboratory, IARI, New Delhi. The investigation involved the collection of ten samples each from symptomatic and asymptomatic plants, and DNA was extracted from 100 mg leaf midribs using the CTAB method (Ahrens and Seemüller, 1992). The extracted DNA (100 ng/µl) along with one positive (Catharanthus roseus from glass house, 16SrII-D group) and one negative (without template DNA), served as a template for PCR reactions targeting the 16Sr RNA and secA genes. Universal primers P1/P7 and secAfor1/secArev3 were employed in the first round of PCR for the respective genes. Subsequently, the product from the 1st round was diluted and used as a template for the 2nd round of PCR with primers R16F2n/R16R2 for 16Sr RNA and secAfor2/secArev3 for the secA gene (Gundersen and Lee 1996; Deng and Hiruki 1991; Hodgetts et al. 2008). This nested-PCR approach yielded distinct bands, approximately 1.2 kb (16Sr RNA) and 480 bp (secA), from the DNA of all ten symptomatic plants and positive sample, while no bands were observed in any of the asymptomatic plants. The nested PCR products were sequenced by BBS labs (Barcode biosciences, Bengaluru). The 16Sr RNA gene sequences, showing 100% similarity, were submitted to NCBI as representative sequences (Acc. No. OQ690013, OQ690014, OQ709133, OQ709134). Similarly, secA gene sequences were submitted with Acc. Nos. OR604283-86. BLAST analysis revealed a maximum 99.76% identity with Onion yellows phytoplasma and 'Ca. P. asteris' reference strain for 16Sr RNA gene, and a maximum 100% identity with 'Elaeis guineensis' stunt phytoplasma for secA gene. The phylogenetic tree constructed using the 16S rRNA and secA gene sequences indicated that the pea phytoplasma strains of this study clustered with 'Ca. P. asteris' (16SrI-B) related strains (Fig 2a and 2b). Additionally, the 16S rRNA sequences from this study, when subjected to Virtual RFLP using iPhyclassifier (Zhao et al. 2009), exhibited a pattern (Fig. 3) matching the reference pattern of the 16S group I, subgroup B (GenBank accession: M30790), with a similarity coefficient of 1.0. Previously, Rao et al. (2021) reported several crops associated with the 16SrI ribosomal group, including eight sub-groups from India. However, this report represents the first instance of a phytoplasma 16SrI-B group associated with phyllody and witches'-broom symptoms in pea, both in India and globally. Considering the economic importance of pea as a vegetable crop, the observed disease incidence and affected area are significant. Urgent attention is required to conduct additional research and implement preventive measures to avert the potential outbreak of this disease in the near future.