First Report of Candidatus Phytoplasma asteris related strain (16SrI-B Subgroup) associated with phyllody disease of moth bean in world.

Moth bean (Vigna aconitifolia), a drought and heat-resistant legume from the Fabaceae family, is commonly cultivated in arid and semi-arid regions of the Indian subcontinent In September 2022, phyllody symptoms (Figure 1) were observed on 50-days-old moth bean plants at the ICAR-NBPGR research farm in Jodhpur, Rajasthan, India. The disease incidence ranged from 10 to 25%. To investigate the cause, ten symptomatic VacoJod (1-10) and ten asymptomatic VacoJod (11-20) Vigna aconitifolia plants were collected. Insect populations were also collected from the vicinity using the sweep-net method to examine the role of insect vectors. The leafhopper was identified based on morphological characterization as Empoasca sp. at the Division of Entomology, ICAR-IARI, New Delhi. DNA was extracted from midribs of all collected plants and the Empoasca sp., using Qiagen DNeasy Plant Mini Kit and Blood and Tissue kit, respectively. Nested polymerase chain reaction (Nested-PCR) with universal primers P1/P7 and R16F2n/R16R2 (Deng and Hiruki, 1991; Gundersen and Lee, 1996), and secA gene primers (secAfor1/secArev3 and secAfor2/secArev3) (Hodgetts et al., 2008) were employed to determine phytoplasma species association. Out of the 10 symptomatic plants and 10 leafhopper samples, 6 leafhopper samples and all symptomatic plants produced expected band sizes for the 16S rRNA (approximately 1.25 kb) and secA gene (480 bp). The PCR products were cloned, sequenced, and sequences (two each from moth bean and leafhopper) were submitted to NCBI GenBank with accession numbers OP941130, OP941132, OP941133 and OP941134 for 16S rRNA and OP958868, OP958869, OP958870, and OP958871 for secA gene sequences. Nucleotide BLAST analysis of 16S rRNA sequences revealed a minimum of 99.92% similarity with 'Primula acaulis' yellows phytoplasma (KJ494340) from Czech Republic. All 100% hits corresponded to 16SrI-B group phytoplasmas, for example rapeseed phyllody phytoplasma (CP055264) from Taiwan. Similarly, nucleotide BLAST analysis of secA sequences revealed a minimum of 99.15% sequence similarity with Paulownia witches'-broom phytoplasma (secA) (OP124308) from China. All 100% hits were of 16SrI-B group phytoplasmas, for example Ageratum conyzoides yellowing phytoplasma (MW401697, secA) from India. Phylogenetic analysis using MEGA11 (Tamura et al., 2021) clustered the moth bean and Empoasca sp. phytoplasma strains with 16SrI-B phytoplasma reference strains. iPhyClassifier tool classified the 16S rRNA gene sequences into 16Sr group I, subgroup B, with a similarity coefficient of 1.0 (Figure 2a, 2b). This marks the first report of the association of 'Ca. P. asteris' 16SrI-B related phytoplasma strain with moth bean plants globally. The 16SrI-B phytoplasma strain is prevalent in various crops in India (Singh et al., 2023). This report emphasizes the epidemiological studies and highlights the need for further research and preventive measures to manage the spread of this phytoplasma strain, which could impact crop production and food security in hot and dry regions.