Natural occurrence of phytoplasma associated with chickpea phyllody disease in Pakistan - a new record

During spring 2005-06 chickpea plants (cultivars Desi and Kabuli) were found to be affected by a previously undescribed disease in Pakistan. Symptoms consisted of proliferation of branches with smaller leaflets, giving a bushy appearance to the plants (Fig-1). Affected plants were scattered in the field and were more easily spotted at flowering and podding time. The flowers developed abnormal green structures (phyllody) instead of normal flowers (Figs. 2 and 3). At the time of crop maturity when the healthy plants were drying the diseased plants in the field were conspicuously green (Fig. 1).

Tissue samples from infected and uninfected plants were examined using a transmission electron microscope (TEM) to ascertain if the disease was associated with a phytoplasma. Typical pleomorphic bodies (phytoplasma) mostly spherical to oval of a size ranging from 200-600 nm were observed only in the sieve elements of affected samples. The bodies had opaque, low electron density cytoplasm that contained ribosome like granules, DNA-strand-like structures and lacked nuclear membranes (Fig. 4), similar to previously reported phytoplasmas (Ajayakumar et al., 2007). Such bodies were absent from healthy samples, from xylem cells, phloem parenchyma cells and companion cells of affected plants. In addition, DNA was extracted from affected plants and amplified using the universal phytoplasma PCR primers P1/P7 followed by R16F2n/R16R2. PCR products were characterized by RFLP analysis and partial sequencing. All samples from infected plants gave a 1250bp PCR product and the RFLP profile associated with 16SrII phytoplasmas, and partial sequencing confirmed that the phytoplasma had the greatest homology to 16SrII phytoplasmas. Koch's postulates were fulfilled in a greenhouse. Five week-old chickpea plants grown in pots were inoculated by grafting. A set of control plants was kept without grafting. Phyllody symptoms similar to those observed in the field started to develop after 25-35 days in graft-inoculated plants while no disease symptoms were observed on control plants.

Chickpea has been grown in Pakistan for many years without any reported incidence of phyllody. The presence of phytoplasma disease in chickpea presents a new threat to the chickpea in Pakistan. Previously the disease was found in Ethiopia, India, Myanmar (Ghanekar et al, 1988); Australia (Saqib et al. 2005) and Oman (Al-Saady et al., 2006). To the best of our knowledge this is the first report of chickpea phyllody disease in Pakistan.