Screening Of High Virulent Isolate Of Paecilomyces Cicadae, Culture And The Pathogenesis To Aphid

Paecilomyces cicadidae(Miquel) Samson.is an entomogenous fungus species widely distributed in China and a potential agent for biocontrol of insect pest.In the studies,a high virulent strain of Paecilomyces cicadae,LB strain,to aphid was screened out from the isolates collected in various areas of China.The studies of the strain with target aphid Lipaphis erysimi,a main pest of Chinese herbe Atractylocles macrocephala Koiolz,focus on the infection process, pathogenicity and factors related the biological characteristics,optimum fermentation conditions for liquid and solid culture medium,insecticidal activity of crude toxin,reciprocity of the fungal products mixed with pesticides.The studies aimed at establishing technical approach for producing of P.cicadae and revealing pathogenesis and potential of the fungus against the aphids. It may provide the fundamentals for the application of the fungus in the integrated control of the aphids and set a new exemplum for the research on interaction between insect host and pathogens.1.The screening of superior strains.The pathogenicity of strains of Paecilomyces and Beauveria from different host insects,were tested with pahid.The result showed that there were obvious differences in pathogenicity among the strains,of which some strains of P.cicadae showed higher pathogenicity than others.By comprehensive comparing of 12 P.cicadae,LB strains was.recognized as the most effective because of its higher pathogenicity,higher sporulation yield and higher tolerance.The corrected mortality was 80.97%,LT₅₀ was 4.44days,and LC₅₀ was 0.87×10⁶/mL in the aphids.2.The pathogenic mechanisms to aphid.Scanning electron microscopy was employed to investigate the infection process of P. cicadae on aphid.It was discovered that conidia of LB strain could germinate on the cuticle of aphid,the germ tube or by means of an appressorium of conidia might penetrate into the cuticle. Intersegmental membranes were preference sites of infection.Mycelia,which developed from the tube after penetration,might grow out of the integument and then produce new conidia for reinfecting if it was under heavy humid condition The relationship between extracellular protease and virulence in I2 strains of P.cicadae were studied by gelatin-agar plate methods.The result showed that productions of extracellar protease were significantly different among the strains,and there was a liner relationship between the protease activity level and the virulence of P.cicadae to the aphid.This indicated that activity of extracellular protease could be regarded as a main element consisting of the virulence of P. cicadae.3.Biological characteristics with emphasis on relationship with pathogenity.Biological characteristics were observed with cultures under series conditions of temperature and humidity.The results suggested that the temperature,pH values,RH have significantly effects on the mycelial growth,sporulation and conidial germination.Temperature of 25℃is optimum for the mycelial growth,sporulation and conidial germination.The pH 6.0 is optimum values for the mycelial growth and pH 6-7 for the sporulation and germination.Illumination had no effect on the growth of mycelium and the germination of conidia,but could increase spore yield.The RH for the conidial germination was 90～100%and the optimum of RH 100%.The fungus could cause disease of aphid within temperature 20-30℃and RH 90-100%, with the optimum of 25℃and RH 100%.Although temperature and pH had markable effect on the conidial yield,the spore cultured under different temperatures or pH values,had no differences in pathogenic capability to the aphids at same conditions.Temperature and humidity were the principle factors influencing pathogenicity,but the later played a key role in primary infection of aphid.4.Optimization of submerged fermentation conditions.Submerged culture ofp.cicadae was made With different sources of carbon and nitrogen,and inorganic compounds in order to selecting favorite cultural conditions.The results showed that the optimum carbon source was glucose,the optimum nitrogen source peptone and the optimum inorganic salt K₂HPO₄,MgSO₄ for liquid culture medium.The orthogonal test showed that the optimum compositions of a fermentative medium are as follows:3%glucose,1.5%peptone,0.1% K₂HPO₄,0.2%MgSO₄·7H₂O,pH 6～7.The highest yield of mycelia was obtained after fermenting for 7days under the conditions with the shaker rotation speed at 150 r/min,the temperature at 25～27℃,the inoculum amount 6%in volum,the medium 100mL in 500mL flask.5.Metabolism characters in liquid culture. The contents of reducing sugar,amino nitrogen,pH values and mycelia biomass in nutritive medium changed obviously with the time increasing during process of inoculation.The mycelial biomass increased rapidly in early 48-144h and reacbed 22.45mg/ml after 168h.The pH value,the contents of reducing sugar and amino nitrogen decreased gradually in the course of submerged culture ofp.cicadae.After 72h inoculated,pH value of the medium dropped from 5.63,the initial value,to 4.42.The acid degree should therefore be regulated reasonably for well development of the mycelia after 72h inoculated.6.Optimization of solid culture condition.The fermentation components of solid medium optimized by mono-factor,Multi-factor combination and orthogonal experiments are corn flour+wheat bran+soybean powder+chaff (6:4:1:1).The optimal fermentation conditions were suggested as follows:the amount of inoculation 6%,initial content of water 120%,natural pH6～7,culture temperature 24℃, fermentation time 22 days,the thickness of medium about 2cm,and illumination 24h/d.ZnSO₄ or BaCl₂ can improve sporulation of the fungus.The conidia production could be a constant yield about 45×10⁸ spores per gram and the maximum yield reached to 52.32×10⁸ spores per gram in optimized medium under the optimal conditions.7.Insecticidal activity of crude toxins to aphid.Fermented broth caused higher correction mortality(81.11%) of aphids.Crude extracts of P. cicadae endotoxins and exotoxins were obtained from submerged culture with organic solution and then used in an insecticidal assay against aphid.Investigation of insecticidal characteristics of the fungal toxins revealed that the aphid was susceptible to the exotoxins and had higher mortality rate after 2 days treatment.Moreover,mortality rate of the aphid increased with prolongation of treatment duration or increasing concentration of the toxins.It showed that the aphid was also susceptible to the endotoxins,but of which insecticidal activity was not as strong as that of the exotoxins.It is suggested that the exotoxins are the active secondary metabolites which represent a new source of toxins ofp.cicadae.8.The compatibility and synergism of pesticide with P.cicadaeTwenty widely used pesticides were assessed for compatibility with P.cicadae.The affects of these pesticides on mycelial growth,sporulation yield and spore germination were quantified.The results showed that Imidacloprid has the lowest restraining rate to the myceli.al growth and insignificantly affect on the spore germination.Imidacloprid was thereby thought as the best pesticide to be blend with P.cicadae.The results of bioassay showed that there was significant synergism against the aphids using the mixture of P.cicadae(concentration of 5×10⁷ spore/mL) with Imidacloprid(1:15000 or 1:30000),which made LT₅₀(0.37days,and 0.3 days respectively) shorter and corrected mortality rate higher than those using P.cicadae(5×10⁷ spore/mL) alone.