Isolation Of Pathogenic Fungi From Spodoptera Frugiperda And Preliminary Study On Their Pathogenic Mechanism

The fall armyworm（FAW）,Spodoptera frugiperda,is a new,dangerous,invasive pest in many countries.In recent years,chemical pesticides have been widely used abroad to control S.frugiperda,leading to the enhancement of its resistance,so biological control is particularly important.Entomopathogenic fungi（EPF）isolates have been considered as potential biological control agents against the FAW in newly invaded areas.Fungal insecticides have the characteristics of host specificity,non-target organisms and environmental safety.In this study,we isolated and purified a dominant pathogenic fungu of S.frugiperda,studied its biocontrol potential and infection process,and preliminarized its pathogenic mechanism,in order to provide a new theoretical basis for the control of S.frugiperda.The afflicted larvae of S.frugiperda were collected from corn fields corn fields near Danzhai County,in the Guizhou Province of China,and 8 isolate strains were isolated from the body surface of the insect,numbered as EFNl-1～EFNL-8.In this study,the virulence of 8 isolate strains against the 3^rd instar larvae of S.frugiperda was determined.The results showed that when the concentration of spores was 1×10⁷spores/m L,the corrected mortality of isolate EFNL-8 strain was greater than 85%at12 d after treatment,which was significantly higher than that of other isolates.Comparing LT₅₀ values of different isolates,it was found that the LT₅₀ of isolate EFNL-8 strain was the lowest,4.75 d.Isolate EFNL-8 was selected as the dominant pathogenic fungus of S.frugiperda by using corrected mortality and LT₅₀ value.According to the morphological characteristics and ITS sequence analysis,it was identified as Metarhizium rileyi,numbered EFNL-8.We investigated the biological activities of M.rileyi EFNL-8 against different stages of S.frugiperda under laboratory cnditions.The results indicated that M.rileyi EFNL-8 had larvicidal activity against S.frugiperda young instars(1^st-3^rd).The corrected mortality of 1^st larvae was 75.54%post inoculation of M.rileyi EFNL-8（15d）.With the increase of larvae instar,the efficacy of this strain decreased.M.rileyi EFNL-8 had no insecticidal activities to pupal and adults and did not affect female oviposition,but significantly inhibited egg hatching.The hatching rate of eggs inoculated with M.rileyi EFNL-8 was 22.67%,significantly lower than that of the control,and most of the hatched larvae died,and the survival rate of the first hatched larvae was only 8.67%.Scanning electron microscopy was used to monitor morphological aspects of the infection process.The following stages were observed;conidia adhered on the cuticle of M.rileyi EFNL-08 and began to germinate within 6h of inoculation,appressoria development after 12h,germ tube penetration within 24h,emergent hyphae within48h,mass hyphal proliferation occurring on cadavers within 60h with secondary conidiogenesis within 72h.The results in vivo study indicated that when hyphal bodies（Hbs）of M.rileyi were injected into host hemocoel,they would not be recognized by the host’s immune system as invaders.There were no significant differences in the hemocytes and phenoloxidase activity between the infected and control larvae at the initial 30h,indicated that the fungus was able to successfully avoid the attack from the cellular and humoral immune systems,therefore,it could multiply freely in the hemocoe.However,it would inhibit the enzyme activity at the later stage when the Hbs reached a high density;the population of hemocytes fluctuated naturally as same as the control in the whole process of Hb reproduction.The pathogenicity of M.rileyi EFNL-8 on S.frugiperda was preliminarily explored.When M.rileyi EFNL-8 infected S.frugiperda larvae,the activity of SOD in S.frugiperda larvae increased firstly,then decreased rapidly,then increased slightly and then decreased again;the activity POD increased at first,then decreased rapidly,then showed an upward trend and finally decreased lower than that of the control group.During the observation period,CAT activity was higher than that of the control group,and showed a general trend of first increasing and then decreasing.When M.rileyi EFNL-8 infected S.frugiperda larvae,the detoxification enzyme GST activity showed an upward trend;the activity of AchE increased firstly and then decreased.