S. Kumar*, V. Singh and S. Lakhanpaul
Department of Botany, University of Delhi, Delhi-110007, India
Received: 14 Mar 2012; Published: 30 Jun 2012
Vigna unguiculata subsp. unguiculata, commonly known as the cowpea or black eyed pea, is one of the most important legumes across the semi-arid tropics valued for its pods and dried seeds. Cowpea plants severely affected with bud proliferation disease were observed during July to September 2009 in New Delhi, India. The infected plants were devoid of normal leaf patterning, flowers and fruits. The leaves present were thicker and dark green in colour compared to the healthy plants and showed extensive bud proliferation on the main shoot (Fig. 1). Most of the plants were stunted. Such features are usually associated with phloem-inhabiting wall-less bacteria, the phytoplasmas (class Mollicutes).
To investigate the association of phytoplasmas with the disease, ten plants with symptoms and two symptomless plants were collected from the botanical garden at the University of Delhi. Stem tissues were pulverised in liquid nitrogen and processed for genomic DNA extraction by the CTAB method (Doyle & Doyle, 1990). Purified DNA was used as template (10ng/µl) for the 16S ribosomal DNA amplification in a nested-polymerase chain reaction with phytoplasma specific primer pairs P1/P7 (Deng & Hiruki, 1991) and R16F2n/R2 (F2n/R2) (Gundersen & Lee, 1996). Amplification products resolved on a 1.2% agarose gel revealed amplicons of expected size (~1.25 kb) for the symptom-bearing plants only. Three PCR products were gel-extracted and purified using QIAquick Gel Extraction Kit (QIAGEN, USA), sequenced bi-directionally and aligned. All three sequences obtained shared 100% identity with each other and the consensus sequence of the Vigna bud proliferation phytoplasma was submitted to GenBank (Accession No. HM449952). BLAST searches revealed the 16S rDNA sequence of the cowpea phytoplasma to share 99% identity with those of phytoplasmas of 16SrI group, 'Candidatus Phytoplasma asteris' members, including Brassica napus phyllody (JN193482.1), periwinkle little leaf (AB646266.1) and maize bushy stunt (HQ530152.1) phytoplasmas.
Three F2n/R2 amplicons were subjected to restriction fragment length polymorphism analysis (RFLP) using six restriction endonucleases (REs), as per manufacturer’s instructions (Fermentas, Lithuania). The REs were randomly selected on the basis of their ability to assign a 16SrI member(AluI, MseI, Sau3AI, TaqI) and which could differentiate within 16SrI subgroups (BfaI, HhaI). The digestion products were run on a 6% polyacrylamide gel and visualised under a gel-documentation system (Bio-Rad, USA). The RFLP patterns obtained matched the profiles of phytoplasmas belonging to 16SrI-B subgroup reported in previous studies (Lee et al., 1998). A dendrogram constructed by the maximum parsimony method of MEGA v4.01 using reference phytoplasma 16S rDNA sequences reported earlier, confirmed that the Vigna bud proliferation phytoplasma is related to the 16SrI group (Fig. 2). A 16SrXII-B strain of 'Ca. Phytoplasma australiense' has been associated with witches’ broom and small leaves of V. unguiculata var. sesquipedalis in Australia (Saqib et al., 2006). A Vigna little leaf phytoplasma belonging to group 16SrV has also been reported (De La Rue et al., 2001). However, this is the first report of 'Ca. P. asteris' affecting Vigna in India and worldwide. The identification of symptoms of bud proliferation disease and the association with a 16SrI-B phytoplasma for cowpea is a significant tool to identify and locate infectious plants in the field and prevent spread of the phytoplasma to the whole crop.
De La Rue SJ, Padovan AC, Gibb KS, 2001. Stylosanthes is a host for several phytoplasmas, one of which shows unique 16S-23S intergenic spacer region heterogeneity. Journal of Phytopathology 149, 613-619. [http://dx.doi.org/10.1046/j.1439-0434.2001.00683.x]
Deng S, Hiruki C, 1991. Amplification of 16 S rRNA genes from culturable and non-culturable Mollicutes. Journal of Microbiological Methods 14, 53-61. [http://dx.doi.org/10.1016/0167-7012(91)90007-D]
Doyle JJ, Doyle JL, 1990. A rapid total DNA preparation procedure for fresh plant tissue. Focus 12, 13-15.
Gundersen DE, Lee IM, 1996. Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer sets. Phytopathologia Mediterranea 35, 144-151.
Lee IM, Gundersen-Rindal DE, Davis RE, Bartoszyk IM, 1998. Revised classification scheme of phytoplasmas based on RFLP analysis of 16S rRNA and ribosomal protein gene sequences. International Journal of Systematic and Evolutionary Microbiology 48, 1153-1169. [http://dx.doi.org/10.1099/00207713-48-4-1153]
Saqib M, Bayliss KL, Jones MGK, 2006. Identification of sweet potato little leaf phytoplasma associated with Vigna unguiculata var. sesquipedalis and Lycopersicon esculentum. Australasian Plant Pathology 35, 293-296. [http://dx.doi.org/10.1071/AP06028]
Kumar S, Singh V, Lakhanpaul S, 2012. A 'Candidatus Phytoplasma asteris' isolate associated with bud proliferation disease of cowpea in India. New Disease Reports 25, 28. [http://dx.doi.org/10.5197/j.2044-0588.2012.025.028]
©2012 The Authors