Abstract
Symptoms of little leaf, leaf chlorosis and leaf malformations with mosaic mottling symptoms were observed in two brinjal varieties (Pusa Shyamla and Pusa Purple Cluster) in fields of IARI, New Delhi, India during 2014–2015. Electron microscopy, PCR and sequence analysis first time provided evidence of association of Candidatus Phytoplasma trifolii with potato virus X and potato virus Y in brinjal in India.
Keywords: Brinjal, PVX, PVY, Phytoplasma
Eggplant (Solanum melongena L.) is an important vegetable grown throughout the world. Four different phytoplasma groups are reported to infect brinjal worldwide, viz. 16Sr I from Japan, Bangladesh and India [4], 16Sr II-D from Egypt, 16Sr III-J and 16Sr III-U from Brazil and 16Sr VI-C, -D and -E from China, India and USA [1]. Several potyviruses and a begomovirus are known to infect brinjal [2]. Survey of brinjal fields at Indian Agricultural Research Institute, New Delhi, during July, 2014 to June, 2015 revealed the occurrence of symptoms suspected to be caused by phytoplasma and viruses on two brinjal varieties viz. Pusa Shyamla and Pusa Purple Cluster. Symptoms like little leaf, mosaic mottling, leaf puckering and necrosis were observed in 15–20 % of plants.
Symptomatic leaves of two varieties of brinjal were examined under transmission electron microscope (TEM) using 2 % uranyl acetate. TEM results revealed mixed flexuous filamentous virus particles of size 515 × 13 nm to 720–800 × 13 nm in both the symptomatic brinjal plants. The total RNA was isolated from healthy and symptomatic brinjal leaf samples and used as a template in cDNA synthesis by using a cDNA synthesis Kit (Stratagene, LaJolla, CA, USA) following the manufacturer’s instructions. The cDNA was further used as template in PCR amplification with coat protein specific primers of potyvirus (PVYF/PVYR) and potexvirus (BM294F/BM89R) [5].
For, phytoplasma identification, the total genomic DNA was extracted by CTAB method from symptomatic and healthy brinjal leaves and subjected to PCR amplification with phytoplasma universal primer pairs P1/P6 followed by nested primers R16F2n/R16R2 [3]. Amplification of about 800, 1000 and 1250 bp were obtained with primers specific to potyvirus, potexvirus and phytoplasma, respectively. The amplified fragments were purified using the WizardR SV Gel and PCR Clean-up System (Promega, Madison, USA). Purified amplified PCR products were sequenced in both directions at Xceleris Genomics, India. The sequences of PCR products were assembled using DNA Base V.4 (http://www.dnabaser.com). The sequences were aligned with representative sequences available in GenBank using ClustalW and the consensus sequences were submitted to GenBank. The phylogenetic trees were constructed using the neighbour-joining method with MEGA 5.0 using 1000 bootstrap replications.
Pairwise sequence comparison analysis of 16Sr DNA sequences of brinjal isolates showed 99 % sequence identity with phytoplasma member strains of 16SrVI group. Phylogenetic tree constructed based on the analysis of the 16S rDNA sequences of the two brinjal isolates showed that both the isolates clustered with phytoplasma strains classified under 16SrVI group (Fig. 1). The pair wise sequence comparison of partial CP genes of the both the identified potato virus Y (PVY, GenBank KU509053, KU509054) and potato virus X (PVX, GenBank KU509055, KU509056) isolates of brinjal revealed 99 % similarity with the reference PVY (Acc. No. EF027883, JQ954350, GQ853637, JN034046) and PVX isolates (Acc. No. D87962, AY512654, KF575174), respectively (Fig. 2). Our results suggested association of ‘Ca. P. trifolii’, PVX and PVY with two brinjal varieties. This is the first report of association of phytoplasma and two viruses (PVX and PVY) in brinjal. Earlier there was no report of occurrence of PVX in brinjal. Hence, this study constitutes first report of association of PVX with brinjal. More studies are required to establish the Koch’s postulates for understanding the epidemiology of the disease.
Fig. 1.
Phylogenetic tree based on 16SrDNA constructed by neighbour-joining method showing the relationships of the brinjal phytoplasma isolates (highlighted with solid triangle) with the other isolates of phytoplasma. Acholeplasma laidlawii was used as an out group. PLL = Periwinkle little leaf, BLL = Brinjal little leaf, CaPT = Candidatus Phytoplasma trifolii, FMP = Fragaria multicipata phytoplasma, AY = Aster yellows, CaPA = Candidatus Phytoplasma aurantifolia, PFWB = Passion fruit witches’ broom and AL = Acholeplasma laidlawii
Fig. 2.
Phylogenetic tree based on coat protein sequences constructed by neighbour-joining method showing the relationships of the brinjal isolates of potato virus Y and potato virus X with the other isolates of the respective virus. Potato leaf roll virus (PLRV) was used as an out group. PVY = potato virus Y, PVA = potato virus A, PVM = potato virus M, PVX = potato virus X
Acknowledgments
The authors wish to express sincere thanks to Dr. Bikas Mandal for providing primer and laboratory facilities to carry out virus work. The authors are also thankful to the Head, Division of Plant Pathology and the Director, Indian Agricultural Research Institute for providing laboratory facilities.
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