Screening And Identification Of Antimicrobial Active Strains Of Endophytic Fungi From F.Unibracteata And Activities Of Their Secondary Metabolites

Fungal endophyte is a microorganism that exists in the host plant tissue without causing plant diseases, which formed a mutually symbiotic relationship in the long-term co-evolution of host plants. Endophyte is highly diverse microbial resources can produce a variety of secondary metabolites with different efficacy value, which is an important resource for a new type of natural anti-bacterial and anti-tumor drugs. Many endophytic fungal strains or their secondary metabolites have antimicrobial activity, and is expected to become an important source of microbial pesticide formulations, which have a major significance for the biological control of agricultural pests and diseases.In this paper, we studied the endophytic fungi from Fritillaria unibracteata, and screened the antimicrobial activity of strains and their secondary metabolites. The efficient strains with higher and broad-spectrum antibacterial activities have been identified by morphological and molecular biology methods, and optimized its fermentation conditions. The main study results included the following aspects:1. The methods of fungus block, double agar plate and plate agar diffusion were used to screen the inhibitory activities of fifty strains isolated from F.unibracteata. First, the four kinds of indicator bacteria (Gram-positive and Gram-negative bacteria) and one indication of fungi as indicator bacteria were screened. All of these 50 endophytic fungi strains had different inhibitions to those indicator bacteria. There are 41 endophytic fungi, which accounted for 82.00% of the total strains, could inhibit at least one kinds of indicator bacteria, and 23 endophytic fungi, which accounted for 46.00% of the total isolates, could suppressed three or more kinds indicator bacteria.Double agar plate method was used to rescreen the inhibitory activities of 23 strains. The results showed that 15 strains, which occupied 65.22%, could significantly inhibit at least one kindof indicator bacteria. Four efficient strains (WBS001, WBS002, WBS008 and 7WBY2) could strongly inhibit the four kinds of indicator bacteria.10 strains were used as the next object of study, which inhibited more than two kinds of indicator bacteria or strongly suppressed one of indicator bacteria (W21 and 1WBY2).Next, plate agar diffusion was used to screen the inhibitory activities of 10 endophytic fungi strains agaist plant pathogenic fungi. It was found that 8 endophytic fungi strains has significant antibacterial activity of one or more kinds of pathogenic fungi, which occupied 80.00%. The inhibition rate of WBS008 against Helminthosporium maydis reached 57.35% and WBS002 reached 50%. Similarly, the inhibition rate of WBS001 against Sclerotinia sclerotiorum was as high as 52.44%.Three endophytic fungi strains (WBS001,WBS002,WBS008) were used for the next antibacterial test., which expanded the type and quantity of plant pathogens. The results showed that three strains were have significant inhibitions to more than three kinds of pathogenic fungi.2. With petroleum ether, ethyl acetate, methylene chloride and n-butanol extraction to keep track of the active site, it was finally discovered the antibacterial active ingredients of WBS001 and WBS008 strains in the ethyl acetate extracts, while the antibacterial activity of strain WBS002 mainly in the petroleum ether extract.3. Through the morphological characteristics and molecular methods identified the active strains WBS001 and WBS002. The results showed that these two strains belonged to different species. The strain WBS001 was Aspergillus strains of Aspergillus fumigates, and strain WBS002 was Colletotrichum of colletotrichum circinans.4. The fermentation conditions of strain WBS008 was optimized, and initially established a fermentation system that the endophytic fungus could stably secrete antibacterial active ingredients, that is, its inhibitory rate reached the highest value of 24.74%, and improved 16.37% than before optimization, when the optimum inoculation amount was 10%, the optimum cultivation temperature was 28℃, the optimum pH was 8.0, and the incubation time was 6d.