Mycoviruses Diversity In Fusarium Pseudograminearum And The Hypovirulence Effect Of FpgMBV1

Wheat crown rot caused by Fusarium pseudograminearum is a worldwide soil-borne disease,which seriously affects the yield and quality of wheat.The main control methods of wheat crown rot include breeding resistant varieties,agricultural control,chemical control and biological control.Mycovirus is a new resource for biological control of plant diseases,which has attracted more and more attention.In this study,high-throughput sequencing method was used to analyze the potential resources of mycoviruses in 200 strains of F.pseudograminearum.The hypovirulence effect of Fusarium pseudograminearum megabirnavirus 1(FpgMBVl)on F.pseudograminearum and its biocontrol effect in laboratory were studied by the methods of preparation of virus-free strains,horizontal transmission and indoor pathogenicity test,which laid a theoretical foundation for the use of FpgMBVl to control wheat crown rot.The main results are as follows:A total of 261 Fusarium strains were isolated from 10 wheat growing areas in Henan Province.Among which 200 strains of Fusarium pseudograminearum were obtained,accounting for 76.63%of the total strains.The results showed that F.pseudograminearum had become the dominant pathogen of wheat crown rot in many areas of Henan Province.High throughput genome sequencing of 200 strains of F.pseudogram inearum showed that there were abundant fungal viruses in F.pseudograminearum,and 15 virus species were obtained.In the tested strains,47 double-stranded RNA viruses,accounting for 31.5%,80 positive single-stranded RNA viruses,accounting for 31.5%,and 20 negative single-stranded RNA viruses,accounting for 1.3%of the total number of viruses were found.Among them,positive single-stranded RNA viruses accounted for the highest proportion,mainly including viruses of the family Narnaviridea,Hypoviridae,Potyviridae,Potyviridae,etc.Negative single-stranded RNA viruses mainly include suspected viruses of Bunyaviridae and some unassigned viruses.Double-stranded RNA viruses mainly include the families of Totiviridae,Megabirnaviridae,Partitiviridae,and Reoviridae.Double-stranded DNA virus is a member of Phycodnaviridae.High throughput sequencing results enriched the diversity resources of virusesin F.pseudograminearum.FpgMBV1 is a megabirnavirus found in F.pseudograminearum strain FC136-2A.A virus-free strain FC136-2A-V-with the same genetic background as the strain FC136-2A was successfully obtained by ribavirin assisted hyphae tip virus-removingmethod.There is no obvious difference in colony morphology,mycelial growth rate,spore morphology and sporulation between FC136-2A and FC136-2A-V-.Butsignificant differences were found between the pathogenicity of these two strains on wheat stem base,which indicated that the hypovirulence of FC136-2A on wheat stem base is caused by FpgMBV1,although it did not affect the growth phenotype of FC136-2A.Results showed that FpgMBV1 could be horizontally transmitted from the original host strain FC136-2A to different mating types of F.pseudograminearum WZ-8A-HygR-V-and Flag-MAT1-2-V-through plate confrontation experiment.No significant difference in the growth phenotype of the strains carrying FpgMBVl obtained by horizontal transmission,but their pathogenicity to wheat was significantly reduced.The above results showed that FpgMBV1 did not affect the growth of the strain,but had obvious attenuated effect,which could significantly reduce the pathogenicity of F.pseudograminearum to wheat.It could be a potential new biological resource for the control of wheat crown rot disease.The biological control effect of FpgMBV1 against highly virulent strain was analyzed by laboratory virulence test on wheat seedlings.The disease index was carried out after inoculation with the strain carrying FpgMBV1 as a microbial inoculum and a virulent strain of PY20-20 without virus.Results showed that the three different strains carrying FpgMBV1,namely FC136-2A,WZ-8A-HygR-V+and Flag-MAT1-2-V+,all presented significant control effects on PY20-20 with control rates of 57.30%,52.42%,and 64.37%,respectively.These results indicated that FpgMBV1 could be expected to have good potential in the application on the control of wheat crown rot caused by F.pseudograminearum.In order to clarify the biological control mechanism of FpgMBV1,Fusarium species were re-isolated from the stem base of wheat seedlings simultaneously infectedwith WZ-8A-HygR-V+and PY20-20 or Flag-MAT1-2-V+and PY20-20.By PCR verification with specific primers,it was confirmed that the dominant Fusdrium species colonized in the stem base of infected wheat seedlings after mix-inoculation were the FpgMBV1 carrying progeny of PY20-20.It’s interesting to find that some newly isolated FpgMBV1 carrying progeny of PY20-20 strains from mix-inoculated wheat seedlings carrying only dsRNA1 of FpgMBV1.Among them,12 strains isolated from the wheat seedlings mix-inoculated with??WZ-8A-HygR-V+and PY20-20 were all carrying only dsRNA1 of FpgMBV1.While 9 out of 12 strains isolated from the wheat seedlings mix-inoculated with WZ-8A-HygR-V+and PY20-20 were carrying only dsRNA1 of FpgMBV1.That means dsRNA2 of FpgMBV1 could be lost during the mix-inoculation process and how this lost related to the pathogenicity of F.pseudograminearum on wheat stem base and the replication cycle of FpgMBV1 is yet unknown,which is worthy of further study.