Study On Screen For High Virulence Strain Of Entomopathogenic Fungus Against Nilaparvata Lugens Stal And Its Application

The brown planthopper (BPH) Nilaparvata lugens St l is an important rice pest thatcan cause serious damage to the rice production in China and Southeast Asia. it isnecessary to change passive situation, change control strategy and find new biologicalcontrol method to manage the pest. In this paper, a number of entomopathogenic fungistrains were further tested on the basis of existing data and a new strain of Metarhiziumflavoviride (Mf), isolated from N. lugens with higher virulence to BPH was selected. Thebiological characteristics of Mf82and its verulence to different stages of N. lugens werecompleted studied. On the basis of bioassay, and its pathogenesis to BPH was also sudied,using its emulsifiable formulation. the components of solid-state fermentation medium forMf82spore production was optimized in order to lay foundation for large scal productionof fungi insectcide which is used to control N. lugens. The primary results are as follows:1. The new strain of M. flavoviride Mf, isolated from N. lugens and fiveentomogenous fungi strains of two Beauveria bassiana and three Metarhizium kept in theLaboratory were tested for their cultural characteristics on SDAY and PDA and virulenceto the adults of N.lugens. Results indicated that6strains showed obvious difference onSDAY. Mf grew faster and produced more spores than other strains on SDAY. Its colonydiameter increasing per day was2.59mm and sporulation was17.34×10~7conidia/cm~2.SDAY was more suitable for Mf to grow than PDA. Mf obviously controlled N. lugens. ItsLT50value to the adults of N. lugens was4.47d and adjusted accumulative mortality was83.8%when concentration was1.0×10~8conidia/mL.2. one isolated strain of M. flavoviride (Mf) which was high virulence to N. Lugenswas identified by Molecular biology method, three different methods were respectivelyused to extract genomic DNA from this fungus: benzyl chloride, CTAB and crackingsolution. Its rDNA ITS sequence was amplified with ITS1and ITS4as primers, and theamplified ITS sequence was detected by agarose gel electrophoresis and was analyzed inGenebank．The results of the experiments showed that the cracking solution method couldobtain the highest quantity of the purest DNA, benzyl chloride method took second place，but CTAB method was not suitable. Homologous sequence contrast was conducted in theDNA sequence database and the result showed that this fungus was M. flavoviride.Identifying the entomogenous fungus species which infects N. lugens would play animportant role for further studying on its biological characteristics and pathogenicity, andcontrolling N. lugens with it. 3. Nine strains of different species of Metarhizium, including strain of M. flavoviride(Mf82) newly isolated from N. lugens, and eight M. anisopliae strains from lab collection,were tested against the adults of N. lugens. The chitinase activities of these strains werealso assayed. The results showed that both the cumulative mortality (82.1%) and thechitinase activity (9.78U/mg) of strain Mf82were the highest among all tested strains.Scanning electron microscope (SEM) observations showed the germinating conidia of thestrain Mf82penetrated the cuticle not only via the intersegmental membranes and foldedregions, but also through the pronotum which contained a large quantity of chitin. Thebiological characteristics of these strains, including growth rate, sporulation, germinationrate and timing of sporulation, were further investigated. The results showed Mf82was thebest strain having strong pathogenicity of N. lugens.4. In this study, laboratory bioassays were carried out on isolate of theentomopathogenic fungal specie, M. flavoviride82for the virulence to different stages of N.lugens at a series of spore concentration of5296-5368,1055-1121,585-623,126-144and22-31conidia/mm~2respectively and gravid female, female, male and eggs laid wereinfected by suspension of1100conidia/mm~2. The results showed that the pathogenicity ofMf82to adults, nymphs at different instars and egg stage were assessed at a series ofconidia concentration. This strain was most virulent to adults, moderate to old nymphs, andleast to young nymphs. The susceptible sequence was gravid female> female> male. Itwas also found that Mf82could infect both oviposition marks and eggs in rice seedlings,and the infection rate reached66.7%and51.2%respectively at10d after treatment. Theyounger the eggs, the higher the infection rate, with the0.5d-old eggs to be most easilyinfected.5. The infection process of M. flavoviride conidia to N. lugens was observed usingscanning electron microscope (SEM).The results showed that most of conidia weredeposited on intersegmental membranes, folded regions on cuticle. The germinated conidiaof the isolate invaded the cuticle not only directly with germ tube but by producingappressorium during the course of infection. Then hypha and spores grew on cuticle of N.lugens. Thalluses entered into hemocoel and then made use of nutrition to reproduce inlarge quantities, diffused and infected eggmass of N. lugens. Even though covered withhumoral, hypha infecting eggmass still continued to grow and sporulate, inactivated eggsand inhibited the number of next generation. This phenomenon can show the infectiveability and invasive approach by infection process in vitro and in vivo. It also providesevidence that evaluate insecticidal effect and application prospect of M. flavoviride. 6. Ten strains of entomogenous fungi emulsifiable formulation; a new strain of M.flavoviride Mf82, isolated from N. lugens and six strains of Beauveria bassiana and four ofMetarhizium were bioassayed for its virulence against the adults of N. lugens and theirvirulence compared. The results showed that cumulative mortality (81.7%) of strain Mf82was the highest among all tested strains, the LT50value was4.6d at a concentration of1.0×10~8conidia/mL. On this basis, a kind of emulsifiable formulation of Mf82was studiedin the laboratory, and the bioassay of virulence of Mf82against the N. lugens was carriedout. The results showed that the daily cumulative mortality of N. lugens increased with theinfect concentration of conidial suspension, the highest concentration (1048conidia/mm~2)treatment caused85.0%. A time dose mortality model was used to analyze the bioassaydata and the model fitted the data well, resulting in parameters for estimating the time anddose effects. The estimated LC50values on day7and9after treatment were2.1×103,9.9×102conidia/mm~2, respectively; while LC90were7.8×104，3.7×104conidia/mm~2,respectively. The median lethal time of the emulsifiable formulation of M. flavoviride tothe N. lugens differed at various logarithmic dose, values of LT50of tested agents wereshortened with logarithmic dose, logarithmic dose increased from7.0to8.0, the LT50values reduced from8.9days to5.7days. The results showed that the emulsifiableformulation of M. flavoviride to N. lugens with high virulence.7. The medium is filled with rice flour, pupa shell of Tenebrio molitor, shell ofgroundnut and shell of sun-flower seed, then summing up the best preservation program. Insolid-phase fermentation conditions, use Design-Expert software analyzing the factors forspores of significant，Optimize M. flavoviride by response surface. As a result, pupa shellof Tenebrio molitor is the significant influence factors. The best preservation program isthat the medium is filled with0.25g rice flour,0.14g pupa shell of Tenebrio molitor,1.00g shell of groundnut,0.25g shell of sun-flower seed. The greatest spore productionquantity is100.013×10~8conidia/mLevery. Using this optimization of the medium afterformula, reduce the production cost, produced higher yield spores. There was no pollutionto environment, so it is beneficial to mass production in future.To sum up: The Mf82strain which had high pathogenecity to adults、nymphs andeggs of BPH, and what’s more, it was easy to yield and use in this paper is a promisingapplication biocontrol agent.