First report on the association of a 16SrII phytoplasma with sesame phyllody in Pakistan

Khalid P. Akhtar^1*, M. Dickinson², G. Sarwar¹, F. F. Jamil¹ and M. A. Haq¹

¹ Nuclear Institute for Agriculture and Biology, Faisalabad, Pakistan
² School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK

*kpervaiz_mbd@yahoo.com

Accepted for publication 31/01/08

Sesame phyllody disease has been recorded on sesame in Pakistan for a number of years, and is characterized by virescence, phyllody, yellowing, floral sterility and stem proliferation of infected plants (Figs. 1 & 2). The disease causes significant losses in Pakistan (Sarwar et al., 2006) but prior to this report the causal agent had not been identified. However, in Oman, phytoplasmas of the 16SrII group have been reported as the causal agent of sesame phyllody (Al-Sakeiti et al., 2005).

Figure 1: Sesame inflorescence showing floral virescence (phyllody) without pod formation	Figure 2: Sesame witches’ broom symptoms due to the result of flower proliferation

Tissue samples from infected and uninfected plants were examined using a light microscope using Dienes’ stain. Regularly distributed dark blue areas were observed in the phloem cells of stem (Fig. 3), leaf and stalk sections of infected sesame plants but these areas were absent from phloem cells of healthy samples, confirming that a phytoplasma is associated with the disease in sesame. To identify the phytoplasma associated with the disease, DNA was extracted from affected plants and amplified using the universal phytoplasma PCR primers P1/P7 (Deng & Hiruki,1991) followed by R16F2n/R16R2 (Gundersen & Lee, 1996). All samples from infected plants gave a 1250bp PCR product and the RFLP profile associated with 16SrII phytoplasmas when digested with HaeIII, RsaI and AluI and compared with Faba bean phyllody (GenBank Acc. No. EF193355) and Australian tomato big bud (Acc. No. EF193359). Partial sequencing (500 bp from each end using primers R16F2n and R16R2) confirmed that the phytoplasma had >99% sequence identity with sesame phyllody from Oman (Acc. No. EU072505). The disease was shown to be graft transmissible in a greenhouse experiment. Six week-old sesame 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 50-60 days in the graft-inoculated plants whilst no disease symptoms were observed on control plants.

 
Figure 3: Light micrograph of transverse section of phyllody infected sesame
stem treated with Dienes’ stain showing dark blue areas in phloem region

To our knowledge this is the first molecular evidence for the association of a phytoplasma of the 16SrII group with phyllody disease in Pakistan and its sequence is essentially identical to that of the phytoplasma causing sesame phyllody in Oman.

References

Al-Sakeiti MA, Al-Subhi AM, Al-Saady NA, Deadman ML, 2005. First report of witches’ broom disease of sesame (Sesamum indicum) in Oman. Plant Disease 89, 530.

Deng S, Hiruki D, 1991. Amplification of 16S rRNA genes from culturable and nonculturable mollicutes. Journal of Microbiological Methods 14, 53-61.

Gundersen DE, Lee IM, 1996. Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer pairs. Phytopathologia Mediterranea 35, 144-151.

Sarwar G, Akhtar KP, Haq MA, Jamil FF, Alam SS. 2006. Prevalence of phyllody and sesame leaf curl virus disease in sesame and their impact on seed yield. Pakistan Journal of Phytopathology18, 1-10.