Effect Of Low Temperature On The Development Of Sclerotia In Villosiclava Virens And The Exploration Of Transformation System

Rice false smut is caused by Villosiclava virens,which attacked specifically rice stamen filaments and formed ball-like colonies on florets,and there is a layer of chlamydospores on the surface of every false smut ball.The sclerotia of Villosiclava virens were found commonly in the northern China and the southern China with high altitude in the later end of the autumn.Our previous studies indicated that sclerotia were most likely the exclusive source for primary infection of V.virens,and played an important role in the life cycle of Villosiclava virens.At the same time,the observation in fields for many years also found that there were more sclerotia if the mature stage of rice was late and the average temperature was relatively low in the autumn.To make sure if low temperature induce the sclerotial formation in V.virens,the inoculated rice panicles in laboratory and the diseased rice panicles cut from paddy fields were treated under different temperatures.The results as follows:1.After 15 days of artificial inoculation in rice,the day-and-night of 25?/15?and 20?/15? were applied to induce sclerotial formation,respectively.We found that the proportions of sclerotial formation at 3 days treatment reached to 20.0%and 16.2%,respectively.This implied that a low temperature,below 20?,is necessary for activating sclerotial development.But the low temperature for a long time will restrain the growth of rice and pathogen,and there was no sclerotium to be detected.In addition,we found that if the light intensity was not enough,there was no sclerotium to be detected.This indicatied that sufficient light is necessary for sclerotial formation.The sclerotia formed in the laboratory were much smaller in size than those collected from paddy fields,but they could germinate on sand surface and produce fruiting bodies and ascospores normally.2.For the diseased rice panicles in vitro,after 3 weeks of storing in the refrigerator at 10?,the sizes of false smut balls became bigger and similar to normal balls in fields,and there were few sclerotia.But the false smut balls in the panicles in the refrigerator at 1-20C grew slowly and did not produce sclerotium.These indicated that the temperature at 10? could allow the false smut balls to grow in some degree and induce a few of sclerotia.3.Low temperature treatment after the membrane of false smut balls breaking could not induce the differentiation of sclerotia.This indicated that sclerotial differentiation started at the early stage of false smut balls.To insure the sclerotial development,the transverse sections of false smut balls at different development stages were observed.We found that the young sclerotia began to form below the layer of chlamydospores.As the balls developed,the sclerotia grew in size and moved outward and emerged on the surfaces.These implied that the sclerotia formed originally from the central hyphae of the balls.4.The survey in fields displayed that if the mature stage of rice was late and the average temperature was relatively low in the autumn,the percentage of sclerotium-bearing balls was much higher.These supported the hypothesis that low temperature favors both the disease occurrence and the sclerotial formation.5.We explored the transformation system of Villosiclava virens and selected the UV8b-100 and UV8b-7336 to start our study,constructed knockout vector,we used the method of protoplast transformation to construct the transformation system.Eventually,we detected that the concentration of prepared protoplast is about 107/ml and the numbers of transformants were 342 and 382,respectively.After detection of the transformants,we could not find the knockout mutant,while we detected some mutants of translocation insertion,the percentages were 1.46%and 4.19%,respectively.And some of them had low growth rate and sporulation.The above results showed that low temperature is a key environmental factor of sclerotial formation in Villosiclava virens.In the late cultivars,the formation of sclerotia were more easily,it helped the pathogen to safely overwintering and become the primary inocula in the coming year.So breaking the life cycle of should become an important strategy on the prevention and control of Villosiclava virens.