MOLECULAR CHARACTERIZATION AND DETECTION OF PHYTOPLASMA INFECTING PULSES
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Date
2015
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Acharya N.G. Ranga Agricultural University, Guntur
Abstract
The present study was carried out for molecular characterization phytoplasmas infecting pulses and studies on their detection and host range. Phytoplasma infected samples of blackgram, greengram, pigeonpea, chickpea, cowpea and fieldbean were collected from Chittoor and Kurnool districts. DNA from plant sample was isolated following CTAB method. The isolated DNA was successfully amplified by nested PCR with phytoplasma specific primers P1/P7 and R16F2n/R16R2. The resulting amplicons of six isolates were cloned, sequenced and data was assembled. The amplicon was determined as 1250bp. Sequence analysis revealed that blackgram phyllody phytoplasma shared highest identity of 98% with ‘Candidatus Phytoplasma australasiae’ (Ca. P. australasiae) 16S rDNA II-D followed by sesame phyllody 16S rDNA II-D (98%) whereas greengram phyllody and cowpea phytoplasma shared maximum identity of 99.9% with sesame phyllody 16S rDNA II-D, ‘Ca. P. australasiae’ 16S rDNA II-D and papaya mosaic phytoplasma (Y10096). Chickpea phytoplasma showed highest sequence identity (99.4%) with sesame phyllody 16S rDNA II-D, ‘Ca. P. australasiae’ 16S rDNA II-D and papaya mosaic phytoplasma (Y10096). Fieldbean bud proliferation phytoplasma had shown highest identity of 99.5% with sesame phyllody 16S rDNA II-D, ‘Ca. P. australasiae’ 16S rDNA II-D and papaya mosaic phytoplasma (Y10096) and pigeonpea little leaf phytoplasma shared maximum identity of 100% with sesame phyllody 16S rDNA II-D, ‘Ca. P. australasiae’ 16S rDNA II-D and papaya mosaic phytoplasma (Y10096).
The important finding of the work was identification of the 16S rDNA-II phytoplasma with fieldbean, cowpea and pigeonpea for the first time from India. The present results with partial 16S rDNA gene sequence alignment and construction of phylogenetic tree using 16S rDNA gene sequences had clearly established that the phytoplasma infecting blackgram, greengram, redgram, cowpea, chickpea and fieldbean belongs to ‘Candidatus phytoplasma aurantifolia’ (16S rDNA-II) in Andhra Pradesh. Totally 32 other crop plants and weed hosts were collected for host range studies of phytoplasma. The DNA was isolated from 32 weeds and other crops showing phytoplasma disease symptoms and subjected to PCR amplification with phytoplasma specific primers R16F2n/R16R2. The result shows that R16F2n/R16R2 primer amplified 1250bp product in 19 weeds and other crop species. They are Cleome gynendra, Solanum duclamara, Portulaca oleracea, Pergularia daemia, Aerva lanata, Celosia argentea, Parthenium hysterophorus, Tephrosia purpurea, Solanum melongena, Solanum lycopersicum, Sesamum indicum, Cleome viscosa, Croton bonplandianum, Saccharum officinarum, Citrullus lanatus, Capsicum annuum, Borreria hispida, Cassia auriculata and Arachis hypogaea. The notable contribution in the present study was identification of six new hosts for phytoplasma for the first time in the Andhra Pradesh. They are Cleome gynendra, Solanum duclamara, Portulaca oleracea, Aerva lanata, Celosia argentea and Pergularia daemia in Andhra Pradesh. The above results indicate that phytoplasma have wide host range. Greengram variety LGG-460 was used to detect the phytoplasma by PCR at different stages of crop growth. Phytoplasma infected three greengram plants were collected for DNA isolation at 30, 45, 60 and 75 DAS from the same plants and performed PCR with R16F2n/R16R2 primers. Samples collected at 30 and 45 DAS gave good amplification of expected product size in PCR with R16F2n/R16R2 primers, but weak bands were observed in samples collected from 60 and 75 DAS, thus indicating that the optimum age for the detection of phytoplasma in greengram is 3045 DAS. The present result shows that the PCR techniques described here allows rapid, sensitive and accurate detection of phytoplasma in plants that are showing typical phytoplasma disease symptoms.
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D5366
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