Isolation,Identification And Application Of Isaria,An Entomogenous Fungus

In this paper,an entomogenous fungus was isolated and purified from a diseased larva of Suana divisa,which taxonomic status was determined based on morphological characteristics and multi-gene analysis,which biological characteristics were determined by various biological indexes such as colony growth,spore yield and germination rate,which host range was tested by inoculating a variety of insect pests,which spores were inoculated on locusta migratoria manilensis,dendrolimus punctatus and Solenopsis invicta to determine its virulence,which cultural condition was tested and which was applied to control eucalyptus pests in field.The main results were as follows:1.A diseased larva of Suana divisa was collected from the Guangxi State-owned Qinlian Forest Farm.An entomogenous fungus was isolated from the diseased larva and purified,which was further inoculated on larvae of Suana divisa and successfully reisolated to determine its pathogenicity.The entomogenous fungus was identified as Isaria javanica,which belonged to Ascomycota,Sordariomycetes,Hypocreales,Cordycipitaceae,Isaria based on morphological characteristics and a combined phylogenical tree generated with r DNA-ITS、EF1-α、β-tublin gene sequences.2.Six well-growing strains were obtained from the entomogenous fungus wth single-spore isolation and biological characteristics of two strains（DMC01and MCC02）with the strongest virulence were tested.The results showed that their suitable temperature of mycelial growth and sporulation ranged from 15°C to 30°C and the optimum temperature of mycelial growth and sporulation was26°C;full light culture was the best for mycelial growth and sporulation;the optimum p H for mycelial growth and sporulation was 7;starch was the most favorable carbon source for mycelial growth and sucrose was the most favorable carbon source for sporulation;yeast extract powder and beef paste were the most favorable nitrogen sources for mycelial growth and sporulation;The lethal temperatures of hyphae and conidia were 60°C and 52°C,respectively.3.Nineteen species of insect pests were inoculated with 1×10⁸spores/m L of strain DMC01 suspended with 0.05%Tween-80 and results showed that 17species of insect pests in 5 orders[Lepidoptera′s Dendrolimus punctatus（Walker）,Spodoptera frugiperda（Smith）,Parasa sp.,Trabala vishnou（Lefebure）,Acanthopsyche subferalbata（Hampson）,Buzura suppressatia（Guenee）,Milionia basalis（Walker）,Jankowskia fuscaria（Leech）,Chalcosia pictinicornis auxo（Linnaeus）,Orthaga achatina（Butler）and Chilo suppressalis（Walker）,Coleoptera′s Monochamus alternatus（Hope）and Araecetus fasciculatus（De Geer）,Hymenoptera′s Solenopsis invicta（Buren）,Isoptera′s Coptotermes formosanus（Shiraki）as well as Orthoptera′s Locusta migratoria manilensis（Meyen）and Tettigonia sp.]were infected except 2 species[Endoclyta signifier（Walker）and Perina nuda（Fabricius）]of insect pests.4.The 4^thinstar larvae of locusta migratoria manilensis,dendrolimus punctatus and adult of Solenopsis invicta were inoculated with spores of strain DMC01 suspended with 0.05%Tween-80 ranged different concentration gradients and results showed that the calibrated mortality of the 4^thinstar larvae of locusta migratoria manilensis,dendrolimus punctatus and adult of Solenopsis invicta reached 100%.5.The cultural condition of Isaria javanica strain DMC01 were determined.Under the condition of liquid fermentation,the temperature was 26°C,p H value was 7,the rotating speed of shaking table was 150 r/min,the liquid volume was1/4 of the bottle capacity,and the initial bacterial concentration was 1×10⁸spores/m L,the mycelial growth reached the maximum.Under the condition of solid fermentation,it was showed that conidial yield reached the maximum in case of the proportion of wheat bran:chaff was 7:3,the water content of solid substrate was 55%～60%,inoculation amount was 15%of the solid substrate,and cultural time was longer than 11 day.6.The conidial powder produced by strain DMC01 was sprayed on the annual eucalyptus forest to control pests,with 1 kg of bacterial powder per mu.The control effect was investigated 10-15 d after spraying.The average control effect was 51.9%in 2020 and 79.1%in 2021.This study proved that Isaria javanica has strong virulence and wide host range,and the preliminary study of fermentation technology laid a foundation for production of Isaria javanica spore powder being applying on field control in the future.