Anastomosis Groups Identification And Specific Detection Of The Major Anastomosis Group Of Rhizoctonia Spp. From Cotton In Northern China

Cotton damping-off is one of the most important soil spread fungus diseases, the disease generally occurre in the cotton seedling, especially in the replanted cotton fields, and the incidence rate as high as50%～60%. It directly affect the cotton high density uniform strains, indirect cause mid-and late diseases and cotton boll disease, and affect the yield and quality of cotton seriously. China is one of the major cotton producing countries in the world, the output of cotton reached6.7814millon tons one year during2004～2008, about27.91%of the total output cotton of the world. It is very important to control the cotton damping-off effectively.Northern cotton-growing area include Shandong, Hebei and Henan provinces is one of the main cotton producing areas in China, perennial cultivated area and total production are more than25percent of the country. The harm of cotton damping-off in the region is extremely serious. So in this study, we carried out the systematic identification on the anastomosis group of Rhizoctonia spp. and did further investigation on the genetic diversity of the AG4-HG-Ⅰ groups. The main results are summarized as follows:1. More than one hundred of the diseased cotton samples and soil were obtained from Shandong, Hebei and Henan provinces separately, using the infected soil to cultivate cotton in greenhouse, and separate pathogenic fungus as a supplement to amount less of strains from cotton samples. One hundred and ninty-eight isolates were obtained, Anastomosis group identification showed that the isolates belonged to multinucleate Rhizoctonia groups AG4-HG-Ⅰ、AG4-HG-Ⅲ and binucleate Rhizoctonia groups AG-A. AG-F, AG-Fb. AG4-HG-I was the major anastomosis group (AG)(88.38%of total isolates), followed by it was AG4-HG-Ⅲ(10.10%). Binucleate Rhizoctonia groups AG-A, AG-F and AG-Fb only have one strain separately. Binucleate Rhizoctonia AG-A, AG-F, AG-Fb were isolated for the first time from cotton in China.2. The nuclear phase observation showed that:Binucleate Rhizoctonia isolates have two nuclei, multinucleate Rhizoctonia isolates have more than three nuclei. The numbers of nucleus were significantly different between the two subgroups of AG-4:the isolates of AG4-HG-Ⅰ subgroup have six to ten nuclei, while the AG4-HG-Ⅲ subgroup isolates are three to five. 3. Phylogenetic analysis among isolates belonging to different AGs was studied based upon5.8S rDNA-internal transcribed spacer (ITS) sequence. The results revealed that the isolates of the same AGs (or sub-AG) had a high sequence similarity (98～100%) and different AGs were significant different. So using5.8S rDNA-ITS analysis, we could easily identify the AGs of unknown isolate. Based on the phylogenetic tree which was constructed with16representative measured isolates and13isolates belonging to different fusion groups from GenBank, we found that the29isolates could be divided into3large branches. The two subgroups of AG-4(AG4-HG-I and AG4-HG-Ⅲ) constituted the first branch; the second branch included AG-F and AG-Fb isolates; AG-A isolates constituted the third branch. From the further analysis of the genetic relationship among the three branches, we could find that binucleate Rhizoctonia AG-F isolates and AG-Fb isolates have close genetic relationship to AG-4isolates, but Rhizoctonia AG-A isolates have the relatively far relationship.4. The pathogenicity of AG4-HG-I and binucleate Rhizoctonia groups were determined by soil inoculation. The results showed that all AG4-HG-I isolates had strong pathogenicity, and the average disease index was about55to100, but the three binnucleate Rhizoctonia solani isolates had an average disease index of10.85.5. Biocontrol effect against cotton damping-off dominate pathogenic group AG4-HG-Ⅰ with three binucleate Rhizoctonia were determined. Use the DPS sofeware to test the Student t of the disease indexes, the results show that three binucleate Rhizoctonia isolates had unconspicuous biocontrol effect.6. Thirty five ISSR primers were used to analyze the strains of AG4-HG-Ⅰ group (eight) and the strains of other groups. We chose eight pairs of the primers which could amplified the AG4-HG-Ⅰ group, to amplify the representative strains of every groups by ISSR-PCR. Specific segments of AG4-HG-Ⅰ group were recoveried, sequenced and then designed the specific primers of AG4-HG-Ⅰ group according to the result.7. PCR determination showed that the primers of J6-S and J6-A can be used to the amplification of AG4-HG-Ⅰ group strains, and the fragment was363bp, but the strains of other groups could not amplified. Use the specific primers to dectet the DNA that extracted from soil which was infected by AG4-HG-Ⅰ, replanted cotton soil, crop soil and sterilized soil. The result of once PCR shows that the specific fragment was amplified from the soil infected by AG4-HG-Ⅰ only, no fragments was amplificed from other soils. But, the specific fragment was amplified from replanted cotton soil by second time PCR, no fragments was amplificed from the sterilized soil. This means that this specific primers works for Rhizoctonia solani AG4-HG-Ⅰ group, but the detection sensitivity should be further improved and enhanced.