2.2.99
GENETIC DIVERSITY AMONG PHYTOPLASMAS ASSOCIATED WITH SUGARCANE WHITE LEAF, SUGARCANE GRASSY SHOOT, GRAMINEOUS WEED WHITE LEAF DISEASES AND POTENTIAL INSECT VECTORS

P WONGKAEW1, Y HANBOONSONGl, P SIRTHORNl, C CHOOSAIl, S BOONKRONGl, T. TINNANGWATTANA2, S DAMAK3

lFaculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand; 2Northeastern Sugarcane Agricultural Centre, Office of the Cane and Sugar Board, Ministry of Industry, Thailand; 3Centre for Molecular Biology, Lincoln University, Canterbury, NewZealand

Background and objectives
Sugarcane white leaf disease (SWL) is one of the most destructive diseases of sugarcane in Thailand. This disease is caused by phytoplasma which produces typical symptoms of total leaf chlorosis and tiller proliferation. Losses due to SWL disease are estimated to be over $20 million each year to the Thai sugar industry. Another phytoplasma disease of sugarcane, the sugarcane grassy shoot (SGS), has recently spread to cane growing area and causes severe loss, especially in central part of Thailand. Plants with grassy shoot disease are dwarfed and develop a large number of tillers. Several kinds of gramineous weeds such as Bermuda grass, crowfoot grass and brachiaria grass also show the symptoms of white leaf disease associated with phytoplasma infection. The leaf hopper, Matsumuratettix hiroghyphicus is one of the insect vectors known to transmit SWL phytoplasma, while the information on the other vectors is still lacking. Relationship among these phytoplasmal diseases are also unclear. In this study, we have developed molecular methods such as serological procedures and DNA techniques to analyse the inter-relation and diversity among these phytoplasmas in plants and some potential insect vectors collected from sugarcane field.

Results and conclusions
The two probes, Sl and S28 from randomly cloned extrachromosomal and chromosomal DNA fragment of SWL phytoplasma, respectively, showed specific and strong hybridization signals with DNA from sugarcane and gramineous weeds white leaf and grassy shoot diseases. Positive hybridization of the probes was also evident with total DNA from Matsumuratettix hiroglyphicus, Bhatia olivacea Exitinus indicus and Recilia distinctus /i>, which might be the potential phytoplasmal insect vectors among 100 species of leafhoppers collected from sugarcane fields. Differences among SWL, SGS and gramineous weeds white leaf phytoplasmas were reviewed by ELISA test using SWL plyclonal antibody. Analysis of RFLP patterns by digestion of PCR amplified 16S RDNA with nine restriction endonucleases showed that the diseases investigated in sugarcane and gramineous weeds are probably caused by different phytoplasmas. Diversity among these phytoplasmas in plants and insects was then confirmed by sequencing a DNA fragment containing the 3' end of the 16S RRNA and the spacer region between the 16S RRNA and the TRNA. Sequence alignment showed that all phytoplasmas infecting the various plants were different. The sequence showed that phytoplasmas from sugarcane white leaf and sugarcane grassy shoot diseases were closely related and clustered in one group and those from the gramineous weeds in another. Amplification of 16S/23S RRNA gene spacer of phytoplasmas in known insect vector, M. hiroglyphicus and three new potential insect vectors revealed approximately 210 ;bp fragment. Sequencing of these PCR products is in progress.