Inhibitory Effect On The Disease Caused By Meloidogyne Incongnita With RNAi

Root-knot nematoes (Meloidogyne spp.),one of the most severe diseases, are able to infest more than5000plant species. Infective second stage juvenile (J2) of RNNs infest plant roots and induce galls, which dramatically limits water and nutrient uptake, and causing causes considerable quantitative and qualitative losses in crop plants. As a result, PKNs bring about an estimated$100billion annual loss worldwide. So it is urgent to find a new path to control RKNs. On the base of publshed genome data of Southern root-knot nem&todes(Meloidogyne incongnita) and Northern root-knot nematodes(Meloidogyne hapla), the research screened out RNAi tagert genes relative to growth and development of M. incongnita, and suppressed the expression level of these tagert genes by using RNAi technique, finally resulting in reduced damage to host plants, the main results are folio wings:1. Using bioinformatics method,50potential RNAi target candidate genes were predicted on the base of the published genomes of M. incongnita, M. hapla and Caenorhabditis elegans, and the RNAi data in the wormbase library.30target candidate genes with the highest synthesis scores were selected and cloned using M. incongnita as material with the special primers designed accroding to the predicted target genes. The results showed a total of25genes related with growth and development of RKNs in M. incongnita genome were obtained.2. On the base of the gene prediction in the previous charpter, Mitochondrial ATP synthase b subunit in M. incongnita(MiASB) gene was cloned. After analysing the sequence characters, a instantaneous expression vector TRV2-MiASB was constructed and was introduced into tomoto plants using virus induced gene silencing (VIGS) technique. The tomato plants with TRV2-MiASB were inculated with M. incongnita to identify the relationship between MiASB gene and nematode disease. The results showed the cloned MiASB gene and the predicted MiASB gene shared100%similarity.60days after inoculation, the expression level of MiASB gene in M. incongnita colleted from the TRV2-MiASB treated tomato plants were reduced. The number of galls on the TRV2-MiASB treated tomato plants was reduced64.3%and64.1%compared to that on the empty vector and the water control. The finding revealed that MiASB gene silence exerted a significant inhibtiroy effect on the disease cuased by nematodes, and also showed MiASB gene was involved in process of the nemotode pathogenicity.3. On the base of research of dsRNA instantaneous expression by VIGS, a plant expression vevtor pCAGC1341-dsASBi was constructed and transferred into Agrobacterium tumefaciens. Then it was introduced into tomato plants(Moneymaker).5plants was identified to be transgenetic seedlings, froming5transgenic lines after propagation. The TO generation positive plants form the5transgenic lines was inculated with M. incongnita to determine the inhibitory effects of MiASB gene RNAi mediated by plants. The results showed the number of galls onthe transgenetic tomato plants with MiASB RNAi was greatly reduced. The reduction percentages were51%～75.4%for transgenetic plants from the5lines. This revealed that MiASB RNAi mediated by plants significanlty control the disease caused by M. incongnita.4. On the base of the previous studies, a fungus expression dsRN A vector pHDT-dsAsbi mediated by Agrobacterium tumefaciens was constructed. Then the pHDT-dsAsbi was introduced into fusarium oxysporum CS-20,resulted in the biocontrol fungus pHDT-dsAsbi/CS-20. pHDT-dsAsbi/CS-20was inculated to tomato and cucumber plants to evaluate the inhibitroy effects of MiASB RNAi on the namotode disease. The results from the potted and field experiemts showed the disease symptom of the treated plants was slightly and the number of galls also significantly reduced, revealed the MiASB RNAi biocontrol fungus mediated by fusarium oxysporum CS-20had a significant inhibitory effect on nematode disease.