â… .Study On Genetic Diversity And Virulence Variation Of Different Rhizoctonia Solani â…¡. Isolation And Cloning The CDNA Fragment Of Rhizoctonia Solani Against Jinggangmysin Using DDRT-PCR

Rhizoctonia solani AG1-IA is the predominat pathogenic strain of rice and maize in China. Under high temperature, high wet condition in the Yangtze River valley , they make the rice and maize drop in yield every year. At present, Jinggangmysin is still the main measure of prevention and cure rice sheath blight in China. Its action mechanism mainly is that it make the pathogenic hyphae unusal branch. But at the molecule level, how the antibiotic interfereing the gene, enzyme and metabolic process correlated with thallus normal growth is reported few.Because of the knowledge about the pathogen not enough and the characteristic of physiological race changing constantly in Sichuan province, we screened the isolates from rice and maize, expected to study the characteristic of the pathogenic bacterium distribute, the genetic variation, virulence in order to control the disease popular and make a base for allaying the harm to rice and maize through hyphal fusion , virulence variation and RAPD mark cluster's analysis. At the same time, cloned the cDNA fragment of Rhizoctonia solani RSCN9 against Jinggangmysin using DDRT-PCR, tried to illuminate at the molecular level the action mechanism of Jinggangmysin on pathogen.Fifteen isolates from different rice varieties and seven isolates from maize in Sichuan were analyzed for hyphal fusion, virulence variation and RAPD mark cluster's analysis. In addition, the Rhizoctonia solani RSCN9 was inoculated with the Jinggangmysin. Using DDRT-PCR technique to isolate differentially expressed cDNA fragment pathogenic hyphae induced by inoculum of Jinggangmysin. 7 differential cDNA fragment were obtained.After recovery and dot-blot confirm, 1 fragment was sequenced and named RSl.Blastn and Blastx were compared with the former sequences. The main results are summarized as below:1. The 22 isolates fused with 6 international standard isolates were identified as Rhizoctonia solani AG-1IA, what is more, in which the RSCN9, RSCN10 also can fuse with Rhizoctonia solani AG-1IC.2. The 22 isolates showed significiant virulence variation at 5% and 1% level. We also find that the result is different when use seedling stage short toothpick inoculating and sword leaf inoculating in room. For example, at 5% and 1% level the difference was not significant between RSCN8 and RSCN9, the disease degree were 4.78 and 3, respectively in room sword leaf inoculation, but was significant when using short toothpick inoculation, the disease degree were 2.33 and4.71, respectively.3. The genetic variation of isolates ofRhizoctonia solaniAG-HA were assessed with RAPD technique. In the dendrogram derived from RAPD data by UPGMA, the isolates could be divided into 5 subgroups at 0.92 similarity level, whereas the isolates from the same host clustered to the same genetic group or subgroup. It showed that the RAPD cluster groups were in line to some extent host origin of isolates, virulence variation were not related to host origin of isolates and RAPD groups. At 0.89 similarity level, the isolates except RSCN9 clustered to the same group with AG1 -IA, the AG1 -IB, AG-1IC, AG-3, AG-4, AG-5 became one group, respectively. The result is basically identical with hyphae fusion1.4. The RAPD data was analysed by NTSYS, and SPSS two software. The result got by Europe distance and similar coefficient which two research variable or sample close and distant quantitative index of degree is unanimous.5. The RSCN9 identified as Rhizoctonia solani AG-1 IA is unique. It can not cluster to Rhizoctonia solani AG-11A. We guess it is a " Bridging isolate " such as AG-B I (sometimes fuses with AG-2-1, AG-3, AG-6, often AG-2-2). It maybe belong to another group except the 6 international standard isolates used in our study because of more frequence. Or it maybe a new isolate unique in Sichuan.6. Using DDRT-PCR, we got a cDNA clone RS-1 which was expressed when Rhizoctonia solani RSCN9 was induced 0.5h by Jinggangmysin. Through BLASTX, RS-1 is homological with cDNA clone hags009xel9 (gi|44834292|gb|CK817367.1)at a 258 bp fragment in EST data, the identities is 85.6%. But the role of hags009xel9 is unknown. Through nr, RS-1 has 97% homology with small subunit ribosomal RNA gene of Ceratobasidium sp. GEL5602 (gi|32187461|gb|AY293223.1|) at 5 to 140 bp block. So RS-1 maybe an unknown gene fragment and make a role in hyphae unusal branch.