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

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.

Detection and molecular characterization of a phytoplasma associated with Cucumber (Cucumis sativus) and its first report from India

“…(Dolye and Doyle 1990). The DNA extracted from the phytoplasma infected bitter gourd (16SrI, KX179474) and bottle gourd (16SrI, MT386510) was included as positive control in PCR assays (Venkataravanappa et al 2017; Ashwathappa et al 2020). Using universal 16Sr RNA gene specific primers pairs (P1/P7, (R16mF2/R16mR2), the phytoplasma infection in cucumber was confirm in both direct and nested PCR assays (Deng and Hiruki 1991; Schneider 1995); (Gundersen and Lee 1996).…”

“…(Dolye and Doyle 1990). The DNA extracted from the phytoplasma infected bitter gourd (16SrI, KX179474) and bottle gourd (16SrI, MT386510) was included as positive control in PCR assays (Venkataravanappa et al 2017;Ashwathappa et al 2020). Using universal 16Sr RNA gene specific primers pairs (P1/P7, (R16mF2/R16mR2), the phytoplasma infection in cucumber was confirm in both direct and nested PCR assays (Deng and Hiruki 1991;Schneider 1995); (Gundersen and Lee 1996).The 16S rRNA gene sequence is highly conserved and insufficient for finer level differentiation of phytoplasma strains, therefore housekeeping genes such as translocase subunit secY and ribosomal protein (rp) was amplified by PCR using primer pairs (SecYF1/SecYR1) (Lee et al 2010) and (rp(II)F1A/rp(II)R1A) (Martini et al, 2013).…”

“…Using universal 16Sr RNA gene specific primers pairs (P1/P7, (R16mF2/R16mR2), the phytoplasma infection in cucumber was confirm in both direct and nested PCR assays (Deng and Hiruki 1991;Schneider 1995); (Gundersen and Lee 1996).The 16S rRNA gene sequence is highly conserved and insufficient for finer level differentiation of phytoplasma strains, therefore housekeeping genes such as translocase subunit secY and ribosomal protein (rp) was amplified by PCR using primer pairs (SecYF1/SecYR1) (Lee et al 2010) and (rp(II)F1A/rp(II)R1A) (Martini et al, 2013). PCR reactions were carried out in a thermal cycler (Master cycler, Profelex, Hamsburg, Germany) and the cycling protocol used for PCR assay was followed as earlier described (Venkataravanappa et al 2017).Then five microliters of PCR products were analysed in 0.8% (W/V) ethidium bromide stained agarose gel, and visualized with a UV transilluminator. The amplified PCR products of 16SrRNA, SecY and rp genes were purified from agarose gel using Qiaquick gel extraction kit (Qiagen, Hilder, USA) and cloned into pTZ57R/T cloning vector according to manufacturer's directions (MBI Fermentas, USA).…”

Molecular detection and characterization of phytoplasma in association with begomovirus in eggplant

“…Further, the SecY gene was amplified by PCR using SecYF1/SecYR1 (Lee et al, 2010) of eggplant phytoplasma, which has been proven to be useful for fine scale differentiation of phytoplasma strains. All the PCR reactions were carried out for DNA amplification as described by Venkataravanappa et al (2017). PCR amplified products of 16S rRNA (1.8 kb) and SecY gene (1.5 kb) of eggplant phytoplasma were excised from the gel and purified by Qiagen gel extraction kit (Qiagen GmbH, Germany) and cloned into the pTZ57R/T vector (Fermentas, Germany) following the manufactures instructions.…”

‘Candidatus Phytoplasma’ belonging to the 16SrVI phytoplasma group, is associated with witches broom disease of Azadirachta indica in India

“…The DNA isolated from a known phytoplasma (16SrVI group) (Brinjal) was used as positive control. The genomic DNA samples were tested for phytoplasmas by PCR using universal primers P1/P7 (Deng and Hiruki 1991) followed by R16mF2/R16mR1 (Gundersen and Lee 1996) nested primers as previously described by Venkataravanappa et al (2017). The resulting PCR amplicons of 1.8 kb and nested PCR amplicons of 1.4 kb corresponding to the phytoplasma 16S ribosomal RNA were obtained.…”