Screening Of Antagonistic Bacteria Of Isaria Farinosa And Study On Its Antifungal Active Substance

Chinese caterpillar fungus is complex formed by Ophiocordyceps sinensisparasitizing on Thitarodes armoricanus larvae.As a precious traditional Chinese medicinal material,it has the functions of tonifying kidney and lungs,hemostasis and phlegm.It is mainly distributed in alpine meadows with an altitude of 3～5km above sea level.The producing area of wild Chinese caterpillar fungus in China accounts for more than 90% of the world’s total producting area.The price of wild Chinese caterpillar fungus is expensive,and it has been over-mined by people,the ecological environment has been seriously damaged,meanwhile,the contradiction between supply and demand of the Chinese caterpillar fungus market is becoming increasingly prominent,so large-scale artificial cultivation of Chinese caterpillar fungus is urgent.Therefore,in order to protect the ecological environment of the plateau,Chinese caterpillar fungus resources and promote the healthy development of the Chinese caterpillar fungus industry,it is urgent to establish a thorough system of artificial cultivation of Chinese caterpillar fungus.The key technologies of artificial cultivation of Chinese caterpillar fungus are selection and breeding of high-quality Thitarodes armoricanus,selection of high-quality Ophiocordyceps sinensis,scientific infection methods,and optimization of fruiting body development conditions.Among them,rearing Thitarodes armoricanus larvae have become a problem due to severe environmental conditions,long growth and development cycles,and vulnerability to pathogens.When Thitarodes armoricanus larvae are artificially reared,they are often attacked by Isaria farinosa,Hexamermis,Cephalosporiur,Mucor,etc.As a result,the survival rate of Thitarodes armoricanus larvae is greatly reduced,which seriously restricts the development of the industry.Among them,Isaria farinosais the most harmful to Thitarodes armoricanus larvae.Physical control and chemical control are often used for the prevention and control of Isaria farinosa.The deficiency is time-consuming,laborious and environmental pollution,prevention and control effect is poor.Therefore,seeking a time-saving and labor-saving,environment-friendly and efficient and durable biological control method has a positive effect on the artificial breeding of Thitarodes armoricanus larvae and the development of the entire Chinese caterpillar fungus industry.In this study,through the screening of antagonistic strains of Isaria farinosa,optimization of fermentation conditions of the antagonistic strains,we separated the bacteriostatic active substances,and finally detected the antagonistic effect of antagonistic strains on Isaria farinosa in soil environment,with the aim of lay the foundation for prevention and control Isaria farinosa.The main results are as follows:1.Screening of antagonistic strains of Isaria farinosaUsing the method of inhibition zone method,4 strains with antagonistic activity were screened.Strain S868 and strain F31 were derived from Kangding soil samples.Serratia plymuthica 11N2 and Streptomyces malaysiensis F913 were derived from laboratory deposited strains.Using the 16 Sr RNA sequence molecular identification method,the strain S868 and Pseudomonas brassicacearum subsp.Neoaurantiaca aggregated in a branch,and it is initially identified as Pseudomonas;the strain F31 belongs to the same branch as Streptomyces olivochromogenes,preliminary identified as Streptomyces.Further analysis of the range of antifungal spectrum of 4 antagonistic strains,the results showed that the fermentation supernatant of strain F913 was effective against filamentous fungi(Isaria farinosa,Beauveria bassiana,Metarhizium anisopliae,Penicillium ochrochloron,Penicillium verticillium),yeast and Bacillus;However,the fermentation supernatants of strains F31,S868,and11N2 only have antagonistic activity against Isaria farinosa and yeast,no antagonistic activity against the tested bacteria.To verify the potential pathogenicity of the four antagonistic strains,the toxicity tests were conducted on silkworm larvae.The experimental results showed that the four antagonistic strains had no effect on the growth and development of silkworm after feeding on them.Addition of crude extract of the fermentation broth on silkworm do not affect growth and development,but there is a lethal phenomenon after addition of concentrated liquid.2.Optimization of fermentation conditions for antagonistic strain F913The fermentation process of actinomycetes is complicated and is affected by many factors,such as the culture medium,the initial p H of the fermentation broth,the fermentation time,and the fermentation temperature.Taking the antifungal effect of the fermentation broth of strain F913 as an index,the fermentation conditions of the fermentation medium,initial p H,fermentation time,and fermentation temperature were optimized.Finally,it was determined that the fermentation medium of strain F913 was ISP medium 1 and ISP medium 2;the fermentation time was 7 days;the fermentation temperature was 30-35°C;and the initial p H of the fermentation broth was 7.3.Purification and identification of antifungal active substance of antagonistic strain F913The results of the physical and chemical properties of the antifungal active substance of strain F913 showed that the antifungal active substance is able to withstand 60℃ for 30 min,and the antifungal activity decreased after treatment at 80～100℃ for 30 min;The results of common organic solvent extraction tests show that the bacteriostatic active substance can be extracted by dichloromethane,ethyl acetate,and n-butanol,but the extraction effect of petroleum ether is poor,indicating that the bacteriostatic active substance is less polar.The crude extract of the obtained fermentation broth was concentrated and subjected to preliminary separation through a silica gel chromatography column.The results showed that the eluent had antifungal activity when the ratio of methylene chloride to methanol was 95:5,90:10,30:70 and 0:100.The high-performance liquid chromatography(HPLC)18C column was used to further separate and purify the above four components,and each step was tested for activity by the zone of inhibition.The results showed that strain F913 produced at least 7 types of metabolites with Isaria farinosa antagonism.Three high-purity samples have been obtained,S1-1,S2-3,and S2-4,while samples S3-8-2、S3-8-3、S3-8-4 and S3-9 still require further purification.4.Application of antagonistic strain F913Through the activity test,it was found that both thetransgenic Isaria farinosa and wild type Isaria farinosa could be inhibited by the strain F913.Colonizing the strain F913 and Isaria farinosa in the soil,the results showed that the strain F913 can successfully colonize in soil,but the Isaria farinosa colonization effect is poor.In the soil environment,if the strain F913 is colonized first,then Isaria farinosa can be inhibited,but if Isaria farinosa is colonized first,the strain F913 cannot be inhibited growth of Isaria farinosaIn summary,in this study,four antagonistic strains of Isaria farinosa were screened.Among them,Streptomyces malaysiensis F913 has a wide range of bacteriostasisand strong antagonistic activity.The screening of the fermentation medium and fermentation conditions of strain F913 has been completed.The isolation and purification of antifungal substances were accomplished by silica gel chromatography column and HPLC,etc.At the same time,it was confirmed that strain F913 could also play a certain antagonistic effect on the Isaria farinosa in the soil.