Nomuraea Rileyi Research On Biological Control Of Orthosia Songi

Four nominated entomopathogenic fungis,which were isolated from the the Eucommia ulmoides artificial planting and demonstration base of dayi county,chengdu city,sichuan province,was investigated on its morphological and molecular identification,biological properties,Antagonistic effect of enzyme production,mass-produced method,indoor control effect.The results were as follows:Four strains were acquired by means of sample collection underwoods,tissue isolation and screen on PDA medium.Cultured on the PDA and SMAY medium,the Type I grew very fastly and its colonial diameter was dramaticlly 15-21mm in a week on SMAY medium,Colonies with a white,fawn on the back,surface with radial wrinkles,velvet texture,no stale and exudate.Seen from the microscope,the conidium was blue or green.So,based on these messages,the strain was initially identified as Penicillium.the Type II grewing well and its colonial diameter was dramaticlly 18-20 mm in a week on SMAY medium,Colony center changed from white to celadon,hyphae.Seen from the microscope,the conidium was green.So,based on these messages,the strain was initially identified as Penicillium.the Type III on the SMAY 25?grew very fastly with colonies on 3 day,convex,thicker colony,center,the early white,after gradient part,pale yellow on the back.Hyphae are cotton,loose,isolation,conidium dry loose,easy to spread,ovoid or oblong,light green.the strain was initially identified as Beauveria.the Type ? grew very slowly and the diameter of its colony is only 0.6~1.8 cm with short villous hyphae in a month on PDA medium,but grown on SMAY medium about 10 to 15 days later,the spores will be covered with new germination hyphae,no stale and exudate,cololess or light yellow on the back.Seen from the microscope,the conidium was green,oval or cylindrical which one end was subacute,Some times,some spores linked to string.Also,the nutrition hyphae of the strain was smooth,or a little color with transparent,no coremium with hyphae usually 2-3 ?m in diameter.So,based on these messages,the strain was initially identified as Nomuraea rileyi.Through the molecular biological method to identify its rDNA-ITS sequence and blasted in NCBI website Genbank database,results showed that Type I strains and Penicillium citrinum homology of 99%;Type II strains with Penicillium Chrysogenum homology of 99%;Type III strains and B.bassiana homology of 99%;Type IV strains and Nomuraea rileyi homology of 99%,its ITS sequence is 619bp.Comparing with the Nomuraea rileyi,the homology was 99%.So,the Type ? strain was finally identified as N.rileyi.The N.rileyi Cultured on different inoculumconcentration,temperature,pH,illumination and shaking revolution and analyzed using spss software,results were:under fluid medium,pH6 is the best for mycelium growth,when inoculumconcentration was l.0xl08 spore mL-1 and shaking revolution was 180 rmp/min,The dry weight reached a maximum at 25?,Diurnal sunlight(24L)can produce more hyphae.The N.rileyi Cultured on different culture substrates,carbon sourse,nitrogen sourse and vatamines,it demonstrated that for mycelium growth and sporulation,the best substrate for corn,the best carbon source was glucose,the best nitrogen source was acid hydrolysis of casein and tryptone.Add Ascorbic acid to the medium can stimulate the mycelium growth and sporulation.Using liquid-solid two-phase method to mass-produced sporulation of Nomuraea rileyi,the results were:with 1000g/L corn as the matrix and add 30g/L glucose,30g/L tryptone,10mg/L Ascorbic acid was the best choise.Using 3.70xl09spore·mL-1,3.70xl08spore mL-1,3.70xl07spore·mL-1,3.70x106spore·mL-1,3.70×105spore·mL-1 the N.rileyi conidial suspension to pathogencity tests on third instar larva Orthosia songi,the results were analyzed by SPSS software,the mortality rate was 73.33%,58.33%,36.67%,28.33%and 25.00%respectively,LCso was 1.07×108 spore mL-1 after 15 days and the regression equations was Y=0.254X+0.304.Production of chitinase,cellulose enzyme,and protease by the two strains was determined through the reaction and growth status of the two biocontrol strains(B.bassiana and N.rileyi strains)grown on chitin medium,cellulose medium,and casein medium.Measured their virulence against Songi larvae were investigated in laboratory by using two inoculating methods(Being dipped on larvae with conidia suspension of 107 spores·mL-1 and being sprayed with 107 spores mL-1).The results showed that the strain B.bassiana and N.rileyi could make cellulose hydrolysis medium's red color shallow in metabolic process,indicating that the two strains could produce Cellulase.The two strains had the ability to form hydrolyzed circle on casein medium,suggesting that the two strains had the ability to produce protease.The hydrolyzed circle and transparent circle were generated by the two strains in chitinmedium,inferring that the two strains were able to produce chitinase.According to the time-dose-mortality model analysis showed that:the Infection rate of the second instar larva Orthosia songi caused by the two strains was 61.11%?87.78%within 10 days,respectively,under the concentration of 1.0x107 spore mL-1.Inoculation by dipping method of B.bassiana and N.rileyi infection rate was higher,and the median lethal time was short,witch N.rileyi shows a high level of virulence,infection rate was 87.78%,LT 50=5.2 d;Spray inoculation of N.rileyi pathogenic strength is relatively weak,infection rate is less than 65%.Putting the powder firecracker compound with N.rileyi and B.bassiana to control Orthosia songi,experimental area of 500 mu,with the powder firecracker 2(250 g)per mu;Demonstration area of 4500 mu,with the powder firecracker 2(150 g)per mu.Sampling survey in test after 7days and l0days(compared to be not applied),results showed that the powder firecracker had a better control efficiency,population decline rate were 70%and rising after 7 days,to maintain a high level correction control effect were 85.1-90%.The demonstration area is consistent with the experimental area,and the control efficiency was higher than Beauveria bassiana high pollen bought on the market.So,in conclusion,makeing the powder firecracker compound with N.rileyi and B.bassiana had better development prosperity.