Studies On Free Radical Scavenging Active Compounds And Metabolomics Of Several Strains Of Metarhizium Spp

Fungi of the genus Metarhizium are widely used entomopathogenic fungi. Metarhizium is also one of the first applied fungal genus for the biological control.Free radical scavenging substances have the potential to be applied, and relate to stress resistance ability, therefore, this study screened13strains of Metarhizium in the lab for free radical scavenging activity.This study focused on the virulence markers in Metarhizium with Metabonomics method for the first time.The results can provide a new scientific basis for screening and identification of high virulent strains of Metarhizium. Culture conditions of the strongest virulent strain of Metarhizium was also studied.The concrete research and results are as follows:Firstly,results of free radical scavenging activity comparison revealed that Metarhizium anisopliaee and M. Flavoviride have ralative stronger free radical scavenging activity. Higher polar and low polar free radical scavengers were existed at the same time in the M. Anisopliaee strain Ma12, for that all the methanol extract and ethyl acetate extract of the mycelia of strain had strong radical scavenging activity. Therefore, extraction process of the radical scavengers from the strain was optimized. Based on analysis of extraction rates with different extraction factors such as different solvent, different solid-liquid ratio, different ultrasonic power, different ultrasonic treat time, different extraction time and temperature etc., three main factors i.g. ultrasonic time, extracting time and methanol concentration were selected by method of range analysis. The extraction conditions were optimized through the Response Surface Analysis method as ultrasonic time51min, extracting time234min and the optimized solvent was90%methanol concentration. Free radical scavenging rate of the extract which extracted with the optimized conditions reached92.17%. Secondly, based on bioassay and analysis with DPS software, the virulence order of the tested strains of Metarhiziumagainst Galleria mellonella was: Mf01, Mc02, Ma09, Mvi02, Mt09. Virulence of the strain Mf01and Mc02was significantly higher than that of the other3strains.From LC-MS analysis and metabonomics analysis results of the tested strains one can see that there was significant difference between the metabolites of different strains. Onthe S-plot map one can see many scattered points which associated with the most related virulent metabolites.There were16kinds of different biomarkers identified. In PLS-DA model, parameters for R2Y to measure the classification model goodness-of-fit was0.973, for Q2parameters prediction ability to measure the classification model was0.924, which showed that the model was reliable. Therefore, the biomarks of virulence could be used as the index of a high virulent Metarhizium strain.Thirdly,studies on influence of different media, different nitrogen and carbon nutritions on growth of Metarhizium Flavoviride mycelium and sporulation of the strain revealed that among the PDA, PSA, SDAY and Czapek media, the PDA medium had the biggest colony diameter and the best grown mycelia, the followed medium was the SDAY medium, and the worst was Czapek medium. For sporulation, PDA was also the best medium, SDAY was the second, and Czapek was the worst. Results of different nitrogen nutrition tests showed that peptone was the best nitrogen source in mycelia growth ofMetarhizium Flavoviri deyeast powder was the second, and theammonium sulphate was the worstnitrogen source for mycelia growth. There were also great difference for sporulation between the6nitrogen nutrients. The potassium nitrate and peptone were the best nitrogen source for sporulation,, yeast and sodium nitrate were the second, and the ammonium sulfate was the worst.Of all the7carbon sources, maltose was the best for mycelia growth, sucrose and glucose were the second, and mannitol and glycerol were the worst. Different carbon sources had also great influence on sporulation. Maltose and mannitol were the best for sporulation, starch was the second, and glycerol was the worst.