Utilization of 16Sr RNA and secA genes for molecular discernment of 'Candidatus Phytoplasma australasiaticum' strain associated with linseed germplasm in India.

The growing prevalence of phytoplasma associated symptoms on linseed or flax (Linum usitatissimum L.) germplasm at Indian Council of Agricultural Research- National Bureau of Plant Genetic Resources (ICAR-NBPGR) fields was noticed during the 2019-22 growing seasons. The characteristic phytoplasma symptoms of phyllody, stem fasciation, stunting, along with floral and capsule malformations were observed in 41 linseed accessions grown at experimental fields of ICAR-NBPGR, Delhi. During 3 years, the presence of phytoplasma in symptomatic linseed accessions was confirmed by nested-PCR assays utilizing 16S rRNA and secA gene-specific primers. The 16S rRNA and secA gene sequences of linseed phytoplasma strains from the representative symptomatic 41 linseed accessions exhibited 100% sequence identity among themselves and 99.93% and 99.82% sequence homology with reference strain, 'Candidatus Phytoplasma australasiaticum' (GenBank Accession: Y10097). Phylogenetic analysis of 16S rRNA and secA gene sequences clustered the linseed isolates with the peanut witches' broom group belonging to 'Ca. P. australasiaticum' strains. The virtual RFLP analysis of 16S rRNA F2nR2 fragment (~1.2 kb) of linseed phytoplasma strains further classified it into 16Sr group II, subgroup D. Our results suggested confirmation of the association of 'Ca. P. australasiaticum' strain (16SrII-D) in the linseed germplasm accessions from North India, which is the first report from India. The phytoplasma infection also reduced the growth and yield parameters of two linseed accessions (IC0498748 and EC0718851).