Studies On Virus DsRNA Rapid Detection And Virus-eliminated Methods Of Flammunlina Velutipes

Flammunlina velutipes is a kind of edible fungi with widely and large cultivation.Prevention and control of disease is the important technical difficulties during cultivation and production of F.velutipes.Virus disease is one of numerous diseases,which has a long incubation period.Disease symptom is not stable and obvious.If it's going to happen,it will cause huge economic losses.Rapid detection of virus and virus-elimination treatment with carrying virus strains provide virus-free strains for research and production,which has great significance to optimizing market germplasm resources,breeding materials selection and cultivation production39 strains of F.velutipes were used as the research material,including 21 main cultivated varieties in the domestic market.We detected and identified virus by molecular biological and virology techniques,sush as dsRNA technology,transmission electron microscope,reverse transcription PCR(RT-PCR),and so on.This research compared the kits with Redzol method to search out the better way.Effect of different processing are researched to establish rapid detection system on dsRNA.Six virus-eliminated methods,i.e.Hyphae tip isolation,tissue isolation(primordium,small fruit body,lamella),protoplast regeneration,protoplast monokaryotization,sexual reproduction and nuclei migration were used.Hyphal growth rate,biomass,pre bottle yield and other biological characteristics and cultivation characters of different elimination treatment strains were determined.The results showed as follows:1.2.0kb dsRNA can be detected in the market main variety F-4889 and F-4891 from 39 varieties of F.velutipes.The virus particle diamete was 25nm found by TEM,which was similar to the reported FvBV.NCBI alignment of partial sequence of detected dsRNA with virus genome of FvBV showed that the sequence had 99%similarity.These results indicate the virus is FvBV.2.Comparison results of two dsRNA detection methods are given to show that Redzol is a kind of low cost,quicker and easy operation method.Especially the testing cost is 1/10 times of the kit.Redzol method is suitable for general laboratory and edible fungus factory.Rapid detection system was established.3.Effect results of 6 virus-elimination methods indicated as follows:Tissue isolation and protoplast regeneration method failed to eliminate the virus.Hyphae tip isolation,protoplast monokaryotization,sexual reproduction and nuclei migration methods can be used to get virus-free strains.Virus-free rate are 25.0%,7.5%,57.5%and 100%,respectively.The above virus-free strains fully proved that FvBV exists in host cytoplasm,rather than the nucleus.FvBV of virus-containing strain may spread vertically to basidiospore by sexual reproduction.In hybridization process,FvBV of virus-containing strains are not migrated to virus-free hypha with the host cell nucleus by horizontal transmission.4.Mycelium growth rate,biomass and laccase activity of different elimination treatment strains detection results proved that TIPS,PMH and NM strains are obviously superior to the original strain and virus-carrying strains of TIPS and PMH.The biological characteristics of PMH and NM strains reached significant level,but had no significant effect on TIPS strains.Cultivation experiment showed that spawnruning time,formation primordium time and fruit body development period of virus-eliminated strains was shorter than that of virus-carrying strains.Per bottle yield and stipe length of virus-eliminated strains was significantly greater than virus-carrying strains.There is no significant difference between virus-free strains of different methods.These results indicate FvBV has certain inhibitory effect on growth and development of F.velutipes.Through sexual reproduction and nuclei migration method virus-free applying,8,107 detoxification strains were successfully obtained,respectively.From these strains,7 strains were bred as material of virus-free excellent strains.In conclusion,we detected FvBV in the domestic main F.velutipes strains.Redzol method is used to massively test market varieties in order to avoid potential hazards as soon as possible.Virus-carrying strains are timely eliminated.Different virus-elimination methods are used for virus-carrying and excellent strains by different purposes and application to provide virus-eliminated and superior strains for production.The research promotes healthy and stable development of F.velutipes industry in China.