The Potential Primary Inocula Of Villosiclava Virens And Its Infection Mechanism On Rice Stamen Filaments

For a long time,occurrence of false smut disease hadn't been enough attention,because historically,false smut was a minor disease.However,in recent years,false smut has become one of the most devastating grain diseases in the middle and lower reaches of the Yangtze River valley in China,it causes quantitative and qualitative losses in rice industre worldwide.So,the researchers of China,Japan and other countries strengthen the study of false smut.Recent reports have been made some important progress,mainly in pathogen biology,inoculation technique,mycotoxin,genetic diversity and molecular biology,etc.But in many ways such as the life cycle of Villosiclava virens(Vv)and infection mechanism,are still controversial.Based on the above issues,this paper has carried on the related research,the main results as followed.1.Through improved the method of measuring chlamydospore germination rate,we found that light and temperature difference can obviously promote the different color chlamydospore germination.The dormant chlamydospores need 5 to 30 days to germinate,so the traditional germination determination method underestimated severity germination abilities of chlamydospores.Further study found germination abilities of three different color chlamydospores could keep the long time under the condition of indoor dry and low temperature,the cumulative germination rate of black chlamydospores which were stored for 21 months under low temperature can reach 72.3%.The germination rate of earth surface chlamydospores is higher than that of chlamydospores in soil.The chlamydospores in rice field were still able to germinate in Mar-July next year.Simulated field flooding tests showed that chlamydospores were susceptible to microbial degradation in the soil and could not germinate.These results indicated that the chlamydospore in field and indoor storage could safely overwinter in the dry conditions,and the number was much larger than that reported in previous studies.But the chlamydospore can only overwinter in a relatively dry environment,paddy field irrigation is not conducive to chlamydospore overwintering.So the role of chlamydospores in the disease epidemic remains to be further research.2.In recent years,the role of sclerotium in the epidemic of rice false smut in sub-tropical areas of China has been studied in our laboratory.It was also found that a large number of sclerotia were formed in subtropical zone of China.When the temperature was low in late autumn,the number of sclerotium significantly increased,and the number of sclerotium per unit area was much higher than that of temperate zone.The maturity of sclerotia formed in the subtropical zone is lower than that of in the temperate zone.Under the condition of the field,?3%of sclerotia can overwinter and germinate in the following year.The basic number of sclerotia is large,and a single sclerotium can produce many ascospores,and further produce a lot of conidia.Therefore,in the subtropical rice growing zone the sclerotia played an important role;a wide range of planting the rice cultivars with high yield and long growth period lead to have a relatively low temperature at the later stage of rice growth,which increased the occurrence of rice false smut and the formation of sclerotium,this may be the reason that the false smut continuously extend.3.By comparative analysis of parameters such as different development stage of rice,inoculant source,light after inoculation,for rice cultivar Yongyou 12,we find that using intelligent climate box to cultivate rice,and the liquid culture of 5-6 d isolate ZjO10 as inoculum.About one week before heading,inoculum was injected into the mid-upper point of panicles.After inoculation,all the rice plants were kept at 20/26?(night/day),maintain the 100%relative humidity for 6 d.Using the inoculation method,incidence of disease can reach 100%.We found that the pathogenicity difference between different isolates was significant.The isolates which are subcultured for more than 3 years have low pathogenicity.After inoculation of three rice cultivars,we found when the distance between pulvinus is 4-7 cm,incidence of disease and number of disease grain are the highest.4.After Vv infected the stamen filaments of rice and barley,we found that Vv attacks dominantly rice stamen filaments and extends intercellularly.The experimental results showed that the fungus could penetrate quickly into barley stamen filaments and extends both intracellularly and intercellularly.It also attacked young barley lodicules and grew intercellularly by chance.The light microscopic observations found that the epidermal and cortex cells in barley stamen filaments arranged loosely with very thick cell walls and large cell gaps.The stamen filaments of rice and barley have the ability to elongate quickly by many folds during anthesis.Cellulose microfibrils in barley stamen filament cell walls arranged very sparsely so that the cell walls looked like transparent.The cell walls were very soft and flexible,and often folded.Vv extended dominantly in the non-cellulose regions and seemed never to degrade microfibrils in barley and rice cell walls.Based on cytology,Vv mainly infects host plant tissues that have the capacity of high elongation,the elongation properties of the cell wall is one of the important reasons cause Vv infection.5.Our laboratory using GFP-tagged transformant C6 and wild type isolate ZjO10 to inoculate the rice root.The ultrastructural results showed that Vv could only infect root epidermis layer,and hyphae grew intercellularly.Vv could not penetrate root thick endodermis cell and enter the internal organization of the root.Occasionally,Vv could invade epidermal cells of individual coleoptile and not infect rice leaf.Real-time PCR showed that Vv proliferated rapidly at 1-10 d after inoculation,grew slowly from 10 d to 20 d,after 20 d growth of fungus began to decline.After inoculation,plant height and root length of rice were inhibited to some extent.The above results indicte that Vv can infect the elongation ability stronger organization such as young rice root in a short time,but the infection of Vv is not systemic infection.6.The interaction between rice and Vv was analyzed by using Illumina Hisep2000/2500 system to detect transcripts of rice florets and healthy uninoculated floral organs.After Vv infects the florets,1859 genes of floret express significant difference,of which 870 genes for up-regulation,and 989 genes for down-regulation.Differentially expressed genes involved in the main pathways such as apoptosis,Toll-like receptor signaling,tryptophan metabolism,retinol metabolism,pentose and glucuronic acid ester interconvert,etc.There are 48 resistance genes appeared significant difference expression,including 15 pathogenesis-related genes for up-regulation significantly.The transcription level of CNGC,JAZ,NBS-LRR,RPM1 and transcription factors such as ERF,DREB,WRKY,NAC and Myb change.There are 106 sugar metabolism related genes change significantly.The expressions of sucrose transporter gene SUT3,HXK5,hexose transporter gene(HWH2)and fructokinase gene(FRK)were significantly down-regulated.But monosaccharide transporter MST3,MST4 and SWEET11,SWEET 14,SWEET 15 and hexose carrier gene(HEX6)were significantly up-regulated expression.The starch synthase genes such as GBSS1,ISA3,Os02g0456100,SBE?b,?-amylase and OsPUL were down-regulated and the genes related to grain filling were also down-regulated.The anther and pollen related genes were suppressed in Vv-infected florets.This indicated that the pathogens of Vv may mainly use monosaccharide as the nutrient.