Detection and characterization of ‘Candidatus Phytoplasma asteris’ associated with littleleaf disease of bitter gourd from India by 16S rRNA phylogenetic and RFLP (in vitro and virtual) analysis

Venkataravanappa Venkataravanappa, Lakshminarayana Reddy C. Narasimha Reddy, Polam Swarnalatha, Subbanna K Shankarappa, Manem Krishna Reddy

Abstract


Bitter gourd plants showing symptoms of little leaf disease are prevalent in farmers’ fields in the Bangalore rural district, Karnataka state, India. Twenty leaf samples from different locations were collected to determine the etiology of the disease. Using PCR and phytoplasma 16S rRNA gene-specific universal primers, we observed positive amplification for the phytoplasma specific primers in five out of twenty samples. The amplified products were cloned, sequenced and nucleotide (NT) sequence comparisons were made with the available phytoplasmas’ 16S rRNA gene NT sequences in the NCBI database. The 16S rRNA gene NT sequence of bitter gourd phytoplasma shared highest identity of 81.7-96.0% with ‘Candidatus Phytoplasma asteris’ (Ca. P. asteris) 16Sr I group isolates from different parts of the world. This was supported by close clustering of phytoplasma of the current study with the Ca. P. asteris 16Sr I subgroup by phylogenetic analysis. The virtual restriction fragment length polymorphism (RFLP) pattern generated for the Phytoplasma from bitter gourd was in congruence with the in vitro RFLP pattern for the six enzymes. This was typical to Ca. P. asteris from the 16Sr I group. Further, virtual RFLP analysis with 11 more enzymes used for RFLP pattern prediction revealed differences only in the Mse I RFLP pattern, with a similarity coefficient of 0.91, which is less than the threshold similarity coefficient for a new subgroup. We propose that the phytoplasma detected in the present study that infects bitter gourd and causes littleleaf disease should be considered as a new subgroup of group 16Sr I (Ca. P. asteris). This is the first report of phytoplasma associated with littleleaf disease of bitter gourd from India.

https://doi.org/10.2298/ABS170223017V

Received: February 23, 2017; Revised: May 7, 2017; Accepted: May 10, 2017; Published online: June 12; 2017

How to cite this article: Venkataravanappa V, Narasimha Reddy LRC, Polam S, Subbanna SK, Manem Krishna R. Detection and characterization of ‘Candidatus Phytoplasma asteris’ associated with littleleaf disease of bitter gourd from India by 16S rRNA phylogenetic and RFLP (in vitro and virtual) analysis. Arch Biol Sci. 2017;69(4):707-14.


Keywords


bitter gourd, phytoplasma, group 16SrI, PCR, in silico analysis

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