‘Candidatus Phytoplasma cynodontis’ (16SrXIV group) affecting Oplismenus burmannii (Retz.) P. Beauv. and Digitaria sanguinalis (L.) Scop. in India

In India, several weed species have been reported to act as reservoir hosts of many phytoplasmas (Mall 2009). These species include Cynodon dactylon, Parthenium hysterophorus, Cannabis sativa, Achyranthes aspera, Amaranthus spp., Datura inoxia and Dichanthium annulatum (Rao et al. 2007, 2009; Raj et al. 2008). The phytoplasmas infecting the mentioned weeds, belonging mainly to 16SrI, 16SrII and 16SrVI groups, are also known to induce yellow diseases of considerable economic importance in vegetable, ornamental, medicinal, forage and fruit crop plants in India (Mall 2009). Among the most affected cultivated plants are Daucus carota (carrot), Phaseolus vulgaris (French bean), Capsicum annuum (pepper), Solanum melongena (eggplant), Cajanus cajan (pigeon pea), Sesamum indicum (sesame), Gladiolus spp. and Citrus aurantifolia (acid lime) (Ghosh et al. 1999; Khan and Raj 2006; Raj et al. 2006, 2009; Khan et al. 2007; Arocha et al. 2009). During surveys of phytoplasmal diseases of weeds in 2009, symptoms resembling those induced by phytoplasmas were observed on two grasses of the family Poaceae, namely, Oplismenus burmannii (Retz.) P. Beauv. and Digitaria sanguinalis (L.) Scop. The O. burmannii plants showed chlorotic streaks on the leaves when compared with healthy ones (Fig. 1a) at the Sugarcane Research Station campus, Gorakhpur. However, the D. sanguinalis plants exhibited chlorosis of leaves (Fig. 1b) at the Indian Agricultural Research Institute campus, New Delhi. Disease incidence ranged from 2% to 8% at both locations. Symptomatic O. burmannii and D. sanguinalis plants were examined for phytoplasma infections employing highly sensitive polymerase chain reaction (PCR) technology. The detected phytoplasma was identified and characterised using sequence and phylogenetic analyses of PCR-amplified ribosomal DNA (rDNA).

DNA was extracted from leaves of 15 diseased and 10 symptomless plants of each species examined, employing the phytoplasma enrichment procedure described by Ahrens and Seemüller (1992). For PCR amplification, the universal phytoplasma primer pair P1/P6 (Deng and Hiruki 1991) followed by nested primer pair R16F2n/R16R2 (Gundersen and Lee 1996), were used. These primer pairs amplified a 16S rDNA fragment of ~1500 and 1245 bp in length, respectively. PCR conditions and agarose gel electrophoresis analysis of PCR products were used as previously described (Gundersen and Lee 1996). All symptomatic plants of both the grass species tested positive for phytoplasma infections, whereas no PCR products were amplified from DNA extracted from symptomless plants. The R16F2n/R16R2 PCR products were separated by electrophoresis using a 1.5% agarose gel, excised from the gel and eluted using the QIAquick gel extraction kit (Qiagen, Hilden, Germany). The DNA fragments were then cloned and five recombinant clones from each grass species were sequenced. Sequences were then assembled and edited using DNASTAR’s Laser Gene software (DNASTAR) and consensus sequences generated. Sequence alignments were performed by using CLUSTAL version 5, of the same software. The sequences obtained in the present work have been deposited in GenBank database (http://www.ncbi.nlm.nih.gov/Genbank) under the accession numbers GQ403690 (O. burmannii isolate) and GQ403689 (D. sanguinalis isolate).

Phylogenetic and molecular evolutionary analyses were conducted using the neighbour-joining program of the genetic analysis software Molecular Evolutionary Genetics Analysis (MEGA), version 4 (Tamura et al. 2007). Acholeplasma laidlawii was included in the analyses as an outgroup. Nucleotide sequence comparisons revealed that the phytoplasmas detected in diseased O. burmannii and D. sanguinalis plants shared 98% 16S rDNA sequence similarities to each other and 99% with members of the Bermuda grass white leaf (BGWL) phytoplasma group or 16SrXIV group which includes ‘Ca. Phytoplasma cynodontis’ (GenBank accession numbers AJ550984 and EF444485), and 97% sequence similarities with sugarcane white leaf (SCWL) phytoplasma (GenBank accession numbers FM208259 and AB052874). Also, the mentioned phytoplasmas clustered together with members of the BGWL group (Fig. 2). Therefore, the phytoplasmas infecting O. burmannii and D. sanguinalis plants in India were identified as isolates of ‘Ca. Phytoplasma cynodontis’ (Marcone et al. 2004). This taxon has previously been reported from India in Bermuda grass (Rao et al. 2007; Snehi et al. 2008) and Dichanthium annulatum (Rao et al. 2009). To our knowledge this is the first report on the occurrence of ‘Ca. Phytoplasma cynodontis’ in O. burmannii and D. sanguinalis grasses. Recognition of weeds as hosts of 16SrXIV group phytoplasmas in India has epidemiological significance and therefore we suggest that emphasis should be given for weed control in and around agricultural fields to minimise phytoplasma disease incidence.