Detection, Characterization and In-Silico Analysis of Candidatus Phytoplasma australasiae Associated with Big Bud Disease of Tomato in India

Abstract

Tomato plants showing witches broom symptoms were collected from different states of India. The presence of phytoplasma infection was confirmed by PCR using phytoplasma-specific primer of 16S rRNA and SecY gene. The sequence analysis of 16S rRNA and SecY gene of eight tomato big bud phytoplasmas showed maximum nucleotide (nt) identity of 95 - 100% with Peanut WB group (16SrII). Further in-silico RFLP analysis of 16S rRNA gene of TBB-Pun1, TBB-Ban, TBB-mal, TBB-Guj and TBB-Vns showed similarity coefficient of 0.68–0.95. Since threshold similarity coefficient for classifying the phytoplasma into new subgroup is set at 0.97, the strain under study significantly distinct from the representative strains in the subgroups of pea nut witches broom. Further, the phylogenetic analysis of tomato big bud phytoplasmas revealed that, they are closely clustered with peanut witches’- broom strains (16Sr II), specifically within the 16Sr II-D and 16Sr II-A subgroups. A comprehensive recombination analysis showed the evidence of both intra and inter-species recombination in seven tomato big bud isolates with most part of their 16Sr RNA F2nR2 fragments descending from Ca.P.brasiliense (16Sr XV) as major parent, except isolate TBB-Vns which had an intra species recombination with Cactus witches-broom-16Sr II-L as major parent. Similarly, in case of SecY gene, all the seven isolates have intra-species recombination with major portion descending from Vinca virescence - [16Sr VI - A] and Potato purple top wilt - [16Sr XVIII - B]. The genetic similarities and the potential threat of this new phytoplasma belonging to 16Sr II group of Peanut witches’ broom’ group infecting tomato in India are discussed.