The Mechanism And Effect Of Arsenophonus On The Virulence Of Metarhizium Flavoviride To Nilaparvata Lugens

The brown planthopper?BPH?,Nilaparvata lugens?St?l?is a primary important rice pest in China.Metarhizium flavoviride is a kind of important entomopathogenic fungi in BPHs.In our preliminary studies,we found that symbiotic bacteria Arsenophonus could inhibit the virulence of M.flavoviride to BPHs.Published results have shown that many factors could affect the virulence of M.flavoviride.In addtion to its virulence,environmental temperature and preservation methods are the ones of those factors.Furthermore,the effects of resistant rice varieties on virulence of M.flavoviride to BPHs should be focused,due to their negative effects on the growth and development of BPHs.Therefore,based on the screening of M.flavoviride strains and exploration of preservation methods,the influence of Arsenophonus on the virulence of M.flavoviride to BPHs was studied,along with the relationship between Arsenophonus and rice varieties,and Arsenophonus and environmental temperature.After observating hemocytes types and their numbers in BPHs,the mechanism that Arsenophonus affects on the virulence of M.flavoviride to BPHs was preliminarily explored.1.Isolation,identification,toxicity determination and preservation of M.flavoviride:A M.flavoviride strain was isolated from individuals that have been infected with bacterias.One sequence was cloned using one pari of primers,ITS4/ITS5.The sequence alignment showed that this sequence shared 99%identity with that from M.flavoviride ARSEF 1764.According to these,the M.flavoviride strain were considered as M.flavoviride ARSEF 1764.After exposure to M.flavoviride ARSEF 1764for 9 d,the incidence rate of BPHs was 73.5%,revealing its high virulence.Furthmore,the effect of the filter paper preservation method on virulence of M.flavoviride was compared with that of PDA culture dish preservation.The results showed that the difference virulences of M.flavoviride F₄ generation were found between the two methods.The incidence rates of BPHs that were from two methods were 63%and 23%,respectively.These results showed that the filter paper method was better than PDA culture dish preservation.2.Effect of Arsenophonus on the virulence of M.flavoviride to BPHs and its relationship with rice varieties and temperatures:After exposure to the spore suspensions of M.flavoviride,the incidence rates of BPHs with Arsenophonus?BPH++?to M.flavoviride were lower than BPHs without Arsenophonus?BPH--?for all tested rice varieties.Out of tested temperatures,only at 25°C,27°C and29°C,BPH++had lower incidence rates than BPH--at 3 d and 5 d.In contrast,there was no significant difference among other temperatures.The two-way ANOVA between Arsenophonus and rice varieties also indicated that the each incidence rate of BPH++was lower than that of BPH--.However,two-way ANOVA between Arsenophonus and temperatures only indicated the incidence rates of BPH++were lower than that of BPH--at 3 d and 5 d.The effect of temperature on the virulence of M.flavoviride to BPHs was significant.The suitable temperature range of M.flavoviride infected with BPH was23°C-29°C,among which 27°C was the best one.The virulence of M.flavoviride to BPHs was low at31°C or 21°C.The temperature also affects the LT₅₀ of BPHs.The LT₅₀ of BPH++was longer than that of BPH--at 27°C and 29°C,respectively.However,the LT₅₀ of BPH++was equal or even shorter than that of BPH--at other temperatures.In summary,Arsenophonus significantly suppresses the virulence of M.flavoviride to BPHs which is affected by temperature.The temperature also affects on the incidence rate of BPHs to Metarhizium flavoviride,significantly.However,the effects of rice varieties and interactions of Arsenophonus and rice varieties and Arsenophonus and temperatures on the incidence rate of BPHs were not significant,respectively.3.Effect of Arsenophonus and M.flavoviride on hemocytes in BPH:The results showed that the three types of hemocytes in BPHs were found including plamatocyte,granulocyte and adipocyte.Among these hemocytes,the number of plamatocytes were the highest,followed by granulocytes and adipocytes.The types of hemocytes were the same between BPH++and BPH--.The numbers of plamatocytes were the highest,followed by granulocytes and adipocytes in BPHs.After exposure to M.flavoviride for 1-5 d,the numbers of plamatocytes in M.flavoviride-treated BPHs were increased significatly,and the numbers of plamatocytes in BPH++were higher than that of BPH--at different times,and there was a significant difference between two species of brown planthopper population at 2d.The differences within granulocyte and adipocyte were found at the early stage of M.flavoviride exposure,and the numbers of granulocytes and adipocytes in BPH--were higher than that of BPH++at0 d and 1 d.It is inferred that plamatocyte plays important rols in the process of M.flavoviride,and Arsenophonus can promote its effect.The effects of granulocyte and adipocyte on this process were not significant.In summary,M.flavoviride ARSEF 1764 that has high virulence to BPH was isolated and identified.The filter paper preservation method is suitable for long-term preservation of M.flavoviride ARSEF 1764.Arsenophonus suppresses the virulence of M.flavoviride to BPH.The suppression is affected by temperatures,but is not relative with rice varieties.Plamatocyte of BPH may participate in the inhibition of Arsenophonus on the virulence of M.flavoviride to BPH.These results provide an fundemtnal materials for revealing the roles and its mechanism of Arsenophonus in the process of M.flavoviride,and for using of M.flavoviride to control BPH.