Archives of Biological Sciences 2017 Volume 69, Issue 4, Pages: 707-714
https://doi.org/10.2298/ABS170223017V
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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 (CHES, Chettalli, Indian Institute of Horticultural Research, Hessaraghatta Lake PO, Bangalore, India)
Narasimha Reddy Lakshminarayana Reddy C. (University of Agricultural Sciences(B), College of Sericulture, Department of Plant Pathology, Chintamani, Karnataka, India)
Polam Swarnalatha (Indian Institute of Horticultural Research, Hessaraghatta Lake PO, Bangalore, Karnataka, India)
Subbanna Shankarappa K. (University of Horticultural Sciences, College of Horticulture, Department of Plant Pathology, Bangaluru, Bagalkot, Karnataka, India)
Manem Krishna Reddy (Indian Institute of Horticultural Research, Hessaraghatta Lake PO, Bangalore, Karnataka, India)
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.
Keywords: bitter gourd, phytoplasma, group 16SrI, PCR, in silico analysis