First report of 'Candidatus phytoplasma asteris' (16SrI) from Cassia fistula showing symptoms of flat stem and witches'-broom in India.

Abstract

Cassia fistula commonly known as 'golden shower tree' is a deciduous tree with a greenish-gray bark and complex leaves with lovely clusters of yellow blossoms that is also utilized for several purposes in traditional medicine offer therapeutic characteristics (Pawar et al., 2017). Random spotting of flat stem symptoms along with unopened flower beds was observed in C. fistula plant during March 2022 in IISER (Indian Institute of Science Education and Research), Thiruvananthapuram, Kerala, India and during May 2022 in SKUAST (Sher-e-Kashmir University of Agricultural Sciences and Technology), Jammu, which were suggestive of phytoplasma infection (Fig. 1 a-e). Surge of leaf hoppers was also observed in and around the tree. The leaf samples were collected from 3 individual C. fistula trees showing suspected symptoms of phytoplasma and one sample from asymptomatic plant of both the states. Leafhopper (LH) species were collected using sweep net method from both the locations. DNA was extracted using CTAB (Cetyl trimethyl ammonium bromide) method and nested universal PCR primers P1/P7 and R16F2n/R16R2 for the 16S rRNA gene (Deng and Hiruki 1991; Gundersen and Lee 1996) and secAfor1/secArev3 and SecAfor2/ SecArev3 for SecA gene (Hodgetts et al. 2008) were employed for the analysis of the phytoplasma strain association. The symptomatic plants and leaf hopper species showed positive bands of 1.2kb and 480bp for 16S rRNA and SecA gene respectively along with. Purified PCR products of both the genes (16Sr RNA and sec A) were ligated into pGEM ®T vector and cloned in Escherichia coli (DH5-α) were sequenced at Agri Genome labs, Kerala, India. The comparative sequence analysis using the BLASTn tool results showed 16S rRNA sequences acquired from plant samples (GenBank Acc. No. OP950857, OP950858) and the leafhoppers Hishimonus phycitis (OP538583) and Orosius albicinctus (OP538584) of Kerala had the minimum of 99.84% of similarity with Bitter gourd little leaf phytoplasma from Myanmar and maximum sequence identity (100%) with the Rapeseed phyllody phytoplasma strain from Taiwan. The sequences of phytoplasma strains from Jammu trees (Genbank Acc. No. OP801671 & OP801672) and H. phycitis (OP801673) shared 100% similarity with each other as well as with North American grapevine yellows and a minimum of 97.65% with Beta vulgaris phytoplasma from Poland. The pairwise comparison results were completely supported by the corresponding phylogenetic sequence analysis of 16S rRNA and SecA gene sequences of all the isolates in the study which clustered with 16SrI-B subgroup related strains. Virtual RFLP analysis through iPhyClassifer results that were derived from in silico digestions of R16F2n/R2 region of 16S rRNA gene using 17 restriction endonucleases enzymes indicated that all the samples produced similar virtual RFLP profiles identical to the reference strain of 16SrI-B phytoplasma subgroup (aster yellows: Acc. No. M30790) with a similarity coefficient value of 1.0. To the best of our knowledge, this is the first report of the phytoplasma association of 'Ca. P. asteris' (16SrI-B) subgroup with Cassia fistula in the world.