Studies On The Genetic Differentiation, Infection Characteristics And Pathogenic Mechanism Of Rhizoctonia Solani

Tobacco target spot is a newly-found and seriously harmful leaf disease of tobacco in China in recent years, and quality and yield of tobacco leaves are seriously affected, which runs short of references. Given that, the genetic differentiation, infection characteristics and pathogenic mechanism of Rhizoctonia solani were systematically studied. The results were as follows:1. Anastomosis group of R. solani isolates from tobacco target spot was first defined to AG-3, and the isolates could be classified into three pathogenic types, including strong pathogenic type, moderate pathogenic type and weak pathogenic type. Fifty-eight isolates were isolated from tobacco target spot disease samples which were collected from Liaoning tobacco productive areas. The test of anastomosis groups (AGs) indicated that all the isolates belong to AG-3. The rDNA ITS sequence of the R. solani strain YC-9 was PCR amplified and sequenced. And then, the ITS sequence was compared and analyzed with the nucleic acid sequences of GenBank database. The result showed that the ITS sequence was 653bp in length, and the homology of ITS sequence of YC-9 and that of the R. solani(AG-3) isolate from tomato foliar blight was 100 percent. The analysis result of rDNA ITS sequence was consistent with that of morphology. Select 16 representative isolates for the analysis of pathogenic type. The result showed that there were significantly different in pathogenicity among isolates by inoculation of the four tobacco varieties, including NC89, K326, Yunyan 85 and G80. They could be classified into three pathogenic types. The result suggested that the R. solani isolates of tobacco target spot had clear pathogenicity differentiation and there was no significant correlation between the distributions of the pathogenic types and geographic locations of R. solani isolates.2. Genetic diversity of 16 R. solani isolates of tobacco target spot was analyzed by RAPD and ISSR. Heredity variation and evolution of the pathogen were explored on molecular level. The correlation of RAPD or ISSR grouping and geographic location or pathogenicity of the isolates was systematically analyzed.9 RAPD primers and 7 ISSR primers were screened to amplify 16 isolates using optimal reaction system. The results showed that the 9 RAPD primers and 7 ISSR primers obtained 111 and 83 scored bands respectively, and the ratio of polymorphic bands was 64.86% and 68.67% respectively. Similarity coefficient of RAPD among the isolates was from 0.63 to 0.93, and when similarity coefficient was 0.74, the isolates were divided into three groups. Similarity coefficient of ISSR among the isolates was from 0.61 to 0.94, and when similarity coefficient was 0.75, the isolates were divided into three groups. The analysis results of RAPD and ISSR all suggested that there were rich genetic diversity and remarkable genetic differentiation among 16 R. solani isolates of tobacco target spot, and genetic cluster groups had certain correlation with geographic location, but no significant correlation with pathogenicity of the isolates. Genetic diversity of 16 R. solani isolates of tobacco target spot and R. solani isolates of other plant diseases was analyzed by RAPD and ISSR. The results indicated that there was closest genetic relationship among 16 R. solani isolates of tobacco target spot, but they showed remote affinity to R. solani isolates of other plant diseases, and there were greater genetic differences between them.3. Study on the infection characteristics of R. solani of tobacco target spot, and first define the relationship of interactive pathogenicity between R. solani isolate of tobacco target spot and R. solani isolates of corn sheath blight and rice sheath blight. The results showed that mycelium of R. solani or mycelium with host residue overwintered in the soil, or mycelium in host residue overwintered. R. solani invaded through the stoma and wound of tobacco leaves, and wound was beneficial to the invasion. R. solani could infect tobacco stems of seedling and adult plant stage, and main and lateral veins of tobacco leaves. The study of infection process indicated that R. solani invaded tobacco leaves after inoculation for 24h, hyphae of R. solani expanded rapidly between and within cells after inoculation for 24-48h, and infected cells were damaged and died finally. Optimum infection conditions of R. solani were determined, the optimum temperature was 25-30℃, the longer moist and inoculation time, the more benefit to the infection of R. solani,12h illumination 12h darkness and continuous darkness were good for the infection, but continuous illumination had significant inhibition. The twenty tested tobacco varieties had different resistance to R. solani, VRG2 had stronger disease resistance and the morbidity was 25 percent. The test result of the host range of R. solani demonstrated that R. solani could infect tobacco, tomato, eggplant and capsicum, cucumber, wax gourd and chenopodium amaranticolor, and eggplant had the highest disease rate (97.5 percent). The test result of interactive pathogenicity demonstrated that R. solani of tobacco target spot could not infect rice, corn and cabbage, but R. solani of rice sheath blight, corn sheath blight and cabbage Rhizoctonia leaf rot could infect tobacco leaves. Field experiment was conducted to investigate damage assessment of the disease, and the statistical result showed that there was significantly positive relationship between the loss rate of yield and value and the disease degree. The higher the disease degree, the greater the yield and value loss, and the relational mode was established. The fungistatic actions of ten fungicides to R. solani were determined. The toxicity regression equations of the fungicides were established and EC50 was computed. The results showed that Diniconazole, dimethachlon, Mitaijunjing, Tuzet and Mancozeb could strongly inhibit the colony growth, so they could be used to control the disease.4. The study defined the basic feature and pathogenic mechanism of toxin of R. solani. The tan crude toxin was obtained from Richard culture filtrate of R. solani by the methods of activated carbon adsorption, methanol elution and rotating evaporation. The biological activity assay of the crude toxin showed that it could induce the characteristic symptom of tobacco target spot, inhibit seed germination and radicle growth, and make seeding wilting. The optimum conditions of producing toxin were as follows:Richard culture filtrate, pH value 6 to 7,25℃,15 to 20 days under dark and vibration 2 times every day. The test result of basic feature of toxin showed that toxin was of thermostability, photostability and reservation, and it was a kind of nonprotein polar substance. Toxin had effect on physiological and biochemical indexes related with resistance of tobacco leaves. The results demonstrated that toxin activated POD, PPO, CAT and PAL, but inhibited activity of SOD. The contents of chlorophyll and soluble sugar of injured tobacco leaves declined remarkably, but the contents of soluble protein, MDA and free proline increased significantly, and the relative conductivity of the leaching solution of leaf tissue also had a sharp increase. The changes of those indexes illustrated that toxin had poisonous effect on tobacco. Meanwhile, it also showed that toxin could trigger defensive reactions of tobacco. R. solani isolates of different pathogenicity also had different abilities of producing toxin. The ability of strong pathogenic isolate YC-9 was stronger than weak pathogenic isolate LF-1.5. The study defined pathogenic mechanism of cell wall degrading enzyme of R. solani, and the ability of producing enzyme of strong pathogenic isolate was significantly stronger than weak pathogenic isolate. R. solani could produce pectinase (PG, PMG, PGTE and PMTE) and cellulase (Cx andβ-glucosidase) in Marcus culture solution, and the activity of PG was the highest. The optimum conditions of producing enzyme were determined. The optimum culture time of Cx andβ-glucosidase was 10d, and that of PG, PMG, PGTE and PMTE was 12d; the optimum culture temperature was 25℃; the optimum pH of Cx,β-glucosidase, PG and PMG was 5, and that of PGTE and PMTE was 6; and static culture and continuous darkness were good for the production of enzyme. The enzyme activities of different disease spot parts were also different, and the activities of enzymes in the junction parts of disease and health were the highest. Activity of cell wall degrading enzyme was determined during interaction process of R. solani and tobacco. The result showed that the activities of PG, PMG, PGTE, PMTE, Cx andβ-glucosidase of tobacco leaves infected by R. solani increased markedly. Among these enzymes, the activities of Cx andβ-glucosidase were the highest, followed by PG and PMG, but the activities of PMTE and PGTE were the lowest. Cell wall degrading enzyme of R. solani could make tobacco leaf produce disease spot, the damage effect of mixed enzymes was notably higher than single enzyme, and the damage effect of pectinase was higher than cellulase. Moreover, the abilities of producing enzyme of isolates of different pathogenicity were compared. The result showed that the ability of strong pathogenic isolate YC-9 was stronger than weak pathogenic isolate LF-1.6. A homologous cDNA fragment encoding the endo-polygalacturonase gene from R. solani was obtained. The method of RACE was used to clone and generate full-length cDNA. The cDNA full length of endo-PG gene of R. solani was 1399 bp. The length of CDS was 1086 bp and it encoded 361 amino acids.