First Report of ‘Candidatus Phytoplasma asteris’ (16SrI) from Cassia fistula Showing Symptoms of Flat Stem and Witches’-Broom in India

Cassia fistula (family Fabaceae), known as golden shower tree, is a deciduous tree with greenish-gray bark, complex leaves, and clusters of yellow blossoms. It is used in traditional medicine for a laxative, treating abdominal pain, ulcer healing, and antirheumatic properties (Pawar et al. 2017). Flat stem symptoms and unopened flower buds (suspected phyllody-like) were randomly observed in C. fistula plants in the Indian Institute of Science Education and Research, Thiruvananthapuram, Kerala, India, in March 2022 and flat stem and witches՚-broom symptoms in Sher-e-Kashmir University of Agricultural Sciences and Technology, Jammu, in May 2022, indicating possible infection with phytoplasma. A surge of leafhoppers was observed in and around the C. fistula trees in Kerala, but few were observed near C. fistula trees in Jammu at the time of collection. Leaf samples were collected from three symptomatic trees and one sample from an asymptomatic tree at both sites. Leafhopper samples were collected from infected plants using a sweep net at both sites. DNA was extracted using CTAB. Nested PCR was employed to amplify the 16S rRNA gene and secA gene for the characterization and identification of phytoplasma strains. PCR primers P1/P7 followed by R16F2n/R16R2 (Deng and Hiruki 1991; Gundersen and Lee 1996) and secAfor1/secArev3 followed by secAfor2/secArev3 (Hodgetts et al. 2008) were used to amplify the 16S rRNA gene and secA gene. Amplicons of the expected size (1.2 kb from 16S rRNA gene and 480 bp from secA gene) were obtained from symptomatic plants and leafhoppers. Purified PCR products of both the genes (16S rRNA and secA) were ligated into pGEMT vector, cloned in Escherichia coli (DH5-α), and sequenced at Agri Genome Labs, Kerala, India. Comparative sequence analysis using BLASTn showed 16S rRNA sequences acquired from plant samples (GenBank OP950857, OP950858) and the leafhoppers Hishimonus phycitis (OP538583) and Orosius albicinctus (OP538584) in Kerala were identical amongst themselves and showed a minimum of 99.84% similarity with Primula acaulis yellows phytoplasma isolate (KJ494340) from Czech Republic and a maximum sequence identity (100%) with the rapeseed phyllody phytoplasma strain from Taiwan (CP055264). The sequences of 16S rRNA gene phytoplasma strains in this study from Jammu trees (OP801671, OP801672) and H. phycitis (OP801673) shared 100% similarity with each other as well as with North American grapevine yellows (KX236148) and a minimum of 99.72% with bitter gourd little leaf phytoplasma from Myanmar (AB741631). Similarly, the secA gene sequences of the same samples from both sites were also submitted to GenBank as OP806224 (Jammu), OP806225 (Jammu), OP806226 (H. phycitis Jammu), OP558017 (Kerala), OP558018 (Kerala), OP558019 (H. phycitis Kerala), and OP558020 (O. albicinctus Kerala). A 16S rRNA and secA gene sequence-based phylogenetic analysis showed that the phytoplasma strain associated with C. fistula and the leafhopper species clustered within the 16SrI phytoplasma group closest to 16SrI-B (‘Ca. P. asteris՚) subgroup strains. Virtual RFLP analysis through iPhyClassifier (Zhao et al. 2009) of results that were derived from in silico digestions of the R16F2n/R2 region of 16S rRNA gene using 17 restriction endonucleases enzymes indicated that all the samples produced virtual RFLP profiles identical to the reference strain of 16SrI-B phytoplasma subgroup (aster yellows: M30790) with a similarity coefficient of 1.0. To our knowledge, this is the first report of the phytoplasma association of ‘Ca. P. asteris’ (16SrI-B) subgroup with C. fistula in the world. This study identifies a bigger threat in the near future because of the tree’s perennial nature. To establish ways to manage and prevent the spread of the disease, more investigation is needed on the dispersion of this phytoplasma in other locations and hosts in India and the world.