Studies On Biological, Infection Characteristic And Genetic Diversity Of Ustilaginoidea Virens Causing Rice False Smut

Rice false smut is a worldwide disease caused by Ustilaginoidea virens(Cooke) Tak. In recently years rice false smut become a important disease with the planting of new hybrid variety and the change of cultivation molde. The disease outbreaked in 2004～2005 in china and caused about 20%³0% yield loss. The false smut balls growing on panicle of rice plant could produces toxins poisoning to human beings, domestic animals and affecting rice production and food safety. In the present paper the pathogen's biological characteristics, Infection Characteristic and genetic diversity were systematically studied.Test of asexual spore germination showed that the patohgen's chlamydospore and conidia couldn't germination within 3h in distill water. primary-conidia and chlamydospore could germinate and produce secondary-conidia and conidia at 6h, respectively. The gemination rate of young chlamydospore at 24h in 2% sucrose solution and distilled water reached 63% and 93% respectively, but the old chlamydospore's germination rate in two medium was only 54% and 76.6%, respectively. Among the solid culture medium tested, PSA medium supported the fast growth of the mycelium, the growth rate was 2.54mm·d^-1. Incubated in liquid culture medium, the mycelium dry weight was highest in the PDB medium with 2.4467g·100mL^-1. the amount of conidia was the largest in the rice extract medium and PSB medium with 2.63×10⁷个/mL and 2.28×10⁷个/mL respectively. however, both solid and liquid medium of Czapek and wakimoto toceshi were not suitable for pathogen's growth. The result of Fungicides screening indicated that 20.67% Flusilazole-famoxadone and 40% Flusilazole had the greatest inhibition against asexual-spores germination among ones tested. the 50% effective concentration (EC₅₀) value is 2.217ug/mL, 2.234ug/mL and 3.633ug/mL, 2.656ug/mL to chlamydospore and conidia, respectively. while 10% Difenoconazole and 43% Tabuconazole has lower inhibition to sexual-spore's germination. Rough ustiloxins produced by culture filtrate of the pathogen had obvious inhibition on seed germination and root growth of gramineous crops and leguminous crops, the highest inhibition rate against bud and root growth was 49.15% and 46.43%, respectively.Artificial inoculation cultured was carried out by injecting different development stage of rice with conidia and chlamydospore suspension of U. virens. The result indicated that the rice booting stage and heading stage were the optimum infection stage. Liangyoupeijiu, honglianyou-6hao and guohaozayou-1hao inoculated at booting stage and heading stage with conidia suspension manifested symptom at late stage in 2005 to 2006, but the rice inoculated at tillering stage, elongation stage and flowering stage with conidia suspension didn't manifest symptom in 2005. The disease incidence of same rice variety was different in different years.The rice panicle was inoculated by injecting 1～2mL of conidia and chlamydospore suspension of Ustilaginoidea virens at the concentration of 10⁶/mL, respectively. Samplings collected from different timing interval after inoculation(AI) were decolored, stained for observation infection process of U. virens on rice glume with microscope. The results demonstrate that the primary-conidia could germinate and produce secondary-conidia within 6h AI on glume, and the mycelium produced by conidia was observed outside and inside epidermal cells at 24h AI. The chlamydospore didn't produce hyphae but produced germling tube or conidia at 6h AI. The germination rate reached 37% within 24h, one to two conidia was observed on end of germling tube withing 36h AI. This result showed that, both chlamydospore and primary-conidia germinated and produced conidia or secondary-conidia at first, then hypha from germination conidia and sub-conidia infected the rice glume.The infection characteristic was studied used specific primer of Ustilaginoidea virens. The result showed that, samples collected from buding stage inoculated with chlamydospore, root of three-leaf soaked with conidia, elongation stage and booting stage inoculated with conidia were used for PCR detection by specific primer of U.virens. a 230bp specific band was amplified from collected different stage of samples. DNA sequencing result showed that the 230bp band was apart of pathogen's ITS. The result demonstrated the pathogen could infect rice at buding stage, seedling stage, elongation stage and booting stage, and then moved up from the infection site. The infection is systematic and latent. The seed without symptom could carry pathogen.The studies of overwintering of chlamydospore revealed that chlamydospore under 3cm depth of soil layer with natural condition and wet condition could survive 10 months and 9 months, respectively. while the survival time of chlamydospore was only 8 and 7 months under 20cm depth of soil layer. With the soil depth and water content increasing, the viability and survival time became lower and shorter.The genetic diversity of 66 strains of U.virens collected from Hubei province in 2005 were assessed with RAPD (random amplified polymorphic DNA). A total of 97 bands were amplified with 12 primer selected from 80 random primers. The similarity coefficient of 66 strains of U.virens was over 0.72, and among most of the strains, the coefficient was over 0.79. The coefficient of 20 strains collected from sam plot in Huazhong Agricultural University was over 0.73, and all the strains were divided two sub-group under this level, one of the sub-group included 15 strains. The preliminary analysis showed that the genetic diversity of U.virens in Hubei province had no significant difference. The location and variety had little influence on genetic differentiation of U.virens. The strains from same field have little difference in genetic variability and population differentiation, and there was no specific interaction between rice variety and U. virens.