Molecular identification of 'Candidatus Phytoplasma cynodontis' associated with Bermuda grass white leaf disease in India

Bermuda grass white leaf (BGWL) is a destructive disease of Bermuda grass (Cynodon dactylon), which is known to occur in several countries. BGWL was noticed on Bermuda grass during spring 2007 at several locations in Gorakhpur, U.P., India. The diseased plants showed typical white leaf symptoms, proliferation of axillary shoots, bushy growing habit, small leaves and shortened stolons (Fig. 1).

Samples from symptomatic plants were collected and total DNA was extracted from approximately 100 mg of leaf tissue employing a phytoplasma enrichment procedure (Ahrens & Seemüller, 1992). The amplicons of ~1500 bp and 1250 bp were amplified by PCR using P1/P6 universal primers (Deng & Hiruki, 1991) followed by nested PCR with R16F2n/R16R2 primers (Gundersen & Lee, 1996), respectively. The ~1250 bp amplicon was cloned, sequenced and data submitted to GenBank (Acc. No. EU032485) which shared highest identity 99% with isolates of 'Candidatus Phytoplasma cynodontis': Juyom Bermuda grass white leaf phytoplasma (EF444486) and Firoozabad Bermuda grass white leaf phytoplasma (EF444485) from Iran; Bermuda grass white leaf phytoplasma (AF248961), 'Ca. Phytoplasma cynodontis' (AB052871) and Brachiaria grass white leaf phytoplasma (AB052872) from Thailand; and Bermuda grass white leaf phytoplasma (AJ550984) from Italy.

A white leaf disease on C. dactylon had previously been reported to occur in India on the basis of symptomatology, remission of symptoms after tetracycline treatment of affected plants, and electron microscopic observations (Singh et al., 1978). However, molecular identification of the causal agent and its phylogenetic relatedness to other phytoplasmas has not previously been reported. This is the first molecular identification of 'Ca. Phytoplasma cynodontis'(16SrXIV group) associated with Bermuda grass white leaf disease in India.