Isolation And Characterization Of Entomopathogenic Fungus Aschersonia And Its Metabolites Activities

Aschersonia, which could infect whitefly and scale, is an important member of entomopathogenic fungi and a good model organism for biodiversity research and phylogenetic analysis for its strong ecological adaptability and wide distribution. Up till now, 78 species of Aschersonia have been reported, but they are only a fraction of the natural resources. To conserve and utilize the strains and gene pools is to obtain large scale of Aschersonia isolates firstly. That is, the isolation and identification of strains are the fundamental work for this fungus. However, some shortcomings (low toxicity speed and the restriction of natural factors) hampered the application of the fungus. The active metabolites of Aschersonia might be used to conquer the problems and develop new effective fungicide. Therefore, isolation of Aschersonia and identification of their metabolites play key roles in Aschersonia researches. Herein, this thesis focuses on these two important aspects.65 Aschersonia samples were collected from Wuyishan and Jianou of Fujian Province, China and primarily identified with morphologic data, including the morphological characteristics of stromata, the size of conidia and the length of paraphyses. Among them, 21 of all samples that differ from morphology were isolated and purified. Then they were stocked by three preservation methods using slant culture, liquid paraffin and liquid nitrogen, respectively. The last method was found to be the best for long time preservation of Aschersonia isolates. Through observation of morphology and data analysis of conidia and paraphyses, we could initially speculated that Wys20 and Wys29 were Aschersonia aleyrodis Webber; Jos009 was Aschersonia placeta Berk & Bvt; Jos56 and Jos57 were Aschersonia blumenaviensis P. Henn; Jos70 was Aschersonia badia Patouillard; Wys46, Wys47 and Wys48 were Aschersonia columnifera Petch. Two isolates (Wys23 and Jos78) with special morphological characteristic, which might be new species of Aschersonia, were also obtained.Partial LSU rDNA sequences were cloned and sequenced from the above 21 Aschersonia isolates, 4 Aschersonia isolates from foreign countries and 1 isolate of Verticillium sp. A dendrogram based on Neighbor-Joining analysis from the PHYLIP program was generated by comparing the LSU rDNA sequences with 11 previously determined LSU rDNA sequences of Aschersonia from GenBank and the genetic distances were calculated by MEGA program. The resulting phylogenetic tree showed that the above 21 Aschersonia isolates were in the same cluster with genetic distances ranged from 0.000-0.007 and closest with A. aleyrodis. The results also showed that the interspecies genetic distances were larger than the intraspecies genetic distances and the genetic diversity of Aschersonia was related to geographic regions. The LSU rDNA sequences could be used to identified different species of Aschersonia with quite different morphology and distinguish Aschersonia from other fungal genus, but might not be used for the identification of Aschersonia with nearly same morphology.Antimicrobial, insecticidal activities and antifungal mechanism of the metabolites of Aschersonia sp. Jos009, which showed strong drought-resistance ability, were investigated, and comparative studies on the bioactivities of the metabolites under different cultivation conditions were conducted. The results showed that liquid fermentation was better than solid fermentation for producing active metabolites of Aschersonia sp. Jos009. The active metabolites exhibited broad spectrum activities towards most of tested bacteria and pathogenic fungi. The minimal inhibitory concentration (MIC) values of the metabolites against sensitive bacteria ranged from 37.5 to 150μg mL^-1 . The active metabolites had inhibited a variety of plant pathogenic fungi, including Fusarium oxysporum f. sp. cucumerinum, Cunninghamella echinulata, Colletotrichum gloeosporioides, Aspergillus flavus and Fusarium oxysporum f. sp. cubense, which was the most sensitive fungus. The active metabolites also showed contact toxicity against Myzus persicae, with LC₅₀ values of 1.025 and 0.714 mg mL^-1 at 24 and 48 h, respectively. The antifungal mechanism of the metabolites against Fusarium oxysporum f. sp. cubense was further studied by scanning electron microscopy (SEM) technique. Considerable morphological alterations, including degradation, blistering and necrosis, were found in the hyphae exposed to active metabolites. A pure compound, 15α,22-dihydroxyhopane, was separated and purified by tracking the activities on the active metabolites. The results showed that this compound weakly inhibited Bacillus subtilis, Bacillus thuringiensis subsp. kurstaki 8010, Serratia marcescens and Salmonella typhimurium with MIC values of 500μg mL^-1.