New Disease Reports
New Disease Reports (2015) 31, 31. [http://dx.doi.org/10.5197/j.2044-0588.2015.031.031]
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Association of a 16SrII 'Candidatus Phytoplasma aurantifolia' isolate with bud proliferation disease of Lablab purpureus (lablab bean) in India

D. Vijaya Kumar Naik 1, B.V. Bhaskara Reddy 1*, J. Sailaja Rani 2, L. Prasanthi 1, R. Sarada Jayalakshmi 1, S.M. Shareef 1 and T. Giridhara Krishna 1

*bvbreddy68@gmail.com

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Received: 15 Mar 2015; Published: 21 Jun 2015

Keywords: phytoplasma, PCR

Lablab bean or lubia (Lablab purpureus) is a widely cultivated, highly drought tolerant legume vegetable crop grown in diverse environmental conditions worldwide. In India and elsewhere, the young pods, consumed as fresh vegetables and mature dry seeds are important in the diet of vegetarian people. Lablab plants severely affected with bud proliferation and little leaf disease symptoms were observed during November 2014 at the Regional Agricultural Research Station farm in Tirupati, Andhra Pradesh, India. The affected plants were showing symptoms of bud proliferation, little leaf and stunting (Fig. 1). 

To investigate the association of symptoms with a phytoplasma, total DNA was isolated from 100 mg leaf midribs from infected and symptomless plant samples using the CTAB method (Doyle & Doyle, 1990). Total DNA was used as a template for a nested-PCR assay with universal primers that target the phytoplasma 16S rRNA gene: P1/P7 (Deng & Hiruki, 1991) and R16F2n/R16R2 (Gundersen & Lee, 1996). Expected size amplicons of 1.8 kb and 1.2 kb, respectively, were amplified from symptom-bearing plants (Fig. 2), but not from the symptomless plant samples. The 1.2 kb PCR fragment was cloned into a pTZ57R/T vector (Fermentas, USA) and sequenced (GenBank Accession No. KP899065).  BLAST analysis of the partial 16S rDNA sequence showed the highest sequence identity (99%) with phytoplasma of the group 16SrII 'Candidatus Phytoplasma aurantifolia' (former peanut witches' broom phytoplasma) that included isolates like the sesame phyllody phytoplasma of subgroup 16SrII-D (KP297862), tomato big bud (JQ868448), papaya yellow crinkle (Y10097) and papaya mosaic (Y10096).

Phylogenetic analysis (Fig. 3) using MEGA version 4.0 (Tamura et al., 2007) confirmed the sequence results, and evidenced that the phytoplasma associated with bud proliferation disease of lablab bean in Tirupati, Andhra Pradesh, India, belongs to group 16SrII. Virtual RFLP patterns of the 16S rDNA sequence of the field bean phytoplasma (AcaClone, http://www.acaclone.com) using BstUI, HaeIII, HpaII and MseI endonucleases were similar to those of the papaya mosaic phytoplasma and sesame phyllody of 16SrII-D subgroup. A 16SrII-D phytoplasma has been previously identified from mung bean (Vigna radiata) affected by phyllody in Andhra Pradesh, India (Ragimekula et al., 2014), and more recently from Andrographis paniculata (Saaed et al., 2015). To our knowledge, this is the first report of the association of a 16SrII phytoplasmawith bud proliferation disease of lablab bean in India. The results have a significant phytosanitary impact for the epidemiology of the 16SrII phytoplasma disease complex in India.

Figure1+
Figure 1: Lablab bean showing symptoms of little leaf (A) and bud proliferation (B) in Andhra Pradesh, India.
Figure 1: Lablab bean showing symptoms of little leaf (A) and bud proliferation (B) in Andhra Pradesh, India.
Figure2+
Figure 2: Agarose (1%) gel electrophoresis of PCR products: lane M - 1 kb DNA ladder; lanes 1,2 - lablab bean plants affected by the 16SrII phytoplasma; lanes 3,4 - symptomless lablab bean plants.
Figure 2: Agarose (1%) gel electrophoresis of PCR products: lane M - 1 kb DNA ladder; lanes 1,2 - lablab bean plants affected by the 16SrII phytoplasma; lanes 3,4 - symptomless lablab bean plants.
Figure3+
Figure 3: Phylogenetic tree based on the partial 16S rDNA sequences of the lablab bean phytoplasma and worldwide reference phytoplasma isolates.
Figure 3: Phylogenetic tree based on the partial 16S rDNA sequences of the lablab bean phytoplasma and worldwide reference phytoplasma isolates.

References

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  2. Doyle JJ, Doyle JL, 1990.A rapid total DNA preparation procedure for fresh plant tissue. Focus 12,13-15.
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  4. Ragimekula N, Chittem K, Nagabudi VN, del Río Mendoza LE, 2014.  First report of 16SrII-D phytoplasma 'Candidatus Phytoplasma aurantifolia' associated with mung bean phyllody in Andhra Pradesh, India. Plant Disease 98, 1424. [http://dx.doi.org/10.1094/PDIS-04-14-0428-PDN]
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To cite this report: Vijaya Kumar Naik D, Bhaskara Reddy BV, Sailaja Rani J, Prasanthi L, Sarada Jayalakshmi R, Shareef SM, Giridhara Krishna T, 2015. Association of a 16SrII 'Candidatus Phytoplasma aurantifolia' isolate with bud proliferation disease of Lablab purpureus (lablab bean) in India. New Disease Reports 31, 31. [http://dx.doi.org/10.5197/j.2044-0588.2015.031.031]

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