Studies On Effect Of Oxidative Stress On Physiology And Metabolism Of Beauveria Bassiana

Beauveria bassiana is one of the most widely used entomopathogenic fungi in research and application.However.its insecticidal effect is restricted by various stressors in practice,including a significant impact on the effect of fungal infection caused by active oxygen radicals damage.In order to resist oxidative stress,entomopathogenic fungi own superoxide dismutase?SOD?,catalase?CAT?,glutlthione perolidlse?GPX?,other antioxidases and small molecule metabolites with radical scavenging activity,which are universal in other living bodies,responding to the stress as a whole antioxidative system against active oxygen radical damage.In this paper,B.bassiana was selected as research object and different kinds and levels of radical reagents were used to study the influence of oxidative stress on strain growth and cell development of B.bassiana and to comprehensively research some relevant antioxidases activity and the dynamic change process of transcription level under stress.At the same time mycelia metabolite differences of B.bassiana under H₂O₂ stress was also studied on the basis of metabonomics.The research also explored a variety of biological effects and relative mechanisms in terms of changes in cell development and antioxidase activity as well as adjustments of zymoprotein synthesis transcription and metabolites when it's under the oxidative stress.The study includes the following aspects:Activity of SOD and DPPH scavenging capability were used as screening index to evaluate antioxidative capability of different strains of B.bassiana.The results indicated that the total SOD activity levels of various strains have a significant difference,and the DPPH scavenging capability of the tested strains were also different significantly.For B.bassiana strain Bb202,the research used two types of radical reagents-H₂O₂ and K₂NO?SO₃?₂ to investigate the strain growth and morphology as well as physiological and biochemical effects of B.bassiana under the stress of different concentration of oxygen radicals.In the range of experimental investigation,it turned out that 12 h after stress,different concentrations of H₂O₂ and K₂NO?SO₃?₂ were not conducive to the growth of cells,and1mmol/L and 2mmol/L H₂O₂ could stimulate intracellular SOD increase although less effect on bacteria growth.Conversely,higher concentrations of H₂O₂ stress had significant effects on the cell growth,while intracellular SOD activity decreased greatly.Under low concentrationsof K₂NO?SO₃?₂ stress,there was no obvious difference in biomass compared to control,but SOD activity changed significantly which was obviously higher than that of the control.A high concentration of K₂NO?SO₃?₂ also inhibited the growth of cells and enzyme activity.Microscopic observation analysis showed that,different concentrations of H₂O₂ stress has caused the mycelium cell damages,morphological significant differences and high concentrations of H₂O₂ may result in mycelium advance into the aging state.K₂NO?SO₃?₂stress effect on mycelium cell was relatively gentle,damage effect was later compared with that of H₂O₂ and the degree of cell damage was less than H₂O₂ stress.Exogenous and endogenous reactive oxidative stress had great differences on the effect among three antioxidant enzymes of B.bassiana cells,and those kinds of cells had different response degrees under oxidative stress.Further more,the stress of exogenous H₂O₂ could quickly cause the rise of intracellular CAT activity?12h stress?and relative enzyme activity increases by 136.5% compared with control,with prominent difference.The elevation of GPX activity was stimulated under the stress of exogenous H₂O₂ and reached the highest in stress condition for 24 h.The relative enzyme activity was 112% to the control,which was a dramatic difference.Although SOD activity reached the maximum in the stress of 24 h,it was very low?1h stress?in the beginning,only 81.9% of the control group.K₂NO?SO₃?₂ promoted the rise of three kinds of antioxidant enzyme activity more strongly so that cells respond more rapidly,and 12 h after treatment in stress,SOD enzyme activity was 123.8% to the control group,while the CAT and GPX comparison of 125.9% and 125.9% respectively,but the degree of emergency response exhibited by GPX was higher than CAT,whose activity was accompanied with intracellular SOD activity increase so both enzyme activity increased synchronously.Quantitative analysis of gene expression demonstrated that H₂O₂ and K₂NO?SO₃?₂ stress had a significant impact on three kinds of antioxidant enzymes mRNA expression quantity.The expression level of CAT mRNA performed the most active under the stress of exogenous H₂O₂.After oxidative stress of 1h and 12 h,mRNA expression quantity compared with a control group enhanced 1.57 and 3.72 fold at the same time period;while the response degree of expression level of SOD and GPX was slightly lower than that of CAT.Change of relative expression in Cu/Zn-SOD and GPX mRNA was not clear after 1h oxidative stress of H₂O₂,whereas the expression rised 5.4 and 2.88 fold than the control at the same time period after12 h.Under the stress of K₂NO?SO₃?₂,the transcription level of Cu/Zn-SOD,GPX and CATincreased rapidly,and its mRNA expression quantity was of 1.49,2.8 and 2.41 fold respectively to the control at the same time period after stress of 1h.However,the expression of Cu/Zn-SOD and CAT mRNA increased 2.5 and 2.4 fold at the same time period than in the control group.Under the stress of H₂O₂,there were distinct differences among mycelium extraction by MeOH,DPPH radical scavenging rate and hydroxyl radical scavenging rate in different cultivation time.Within the scope of stress time from 12 h to 36 h,the rate of hydroxyl free radical scavenged by mycelium extraction was observably higher than that of control group,nevertheless the inhibition of the DPPH was significantly higher than that of control only in 36 h stress treatment,was 49.6%.The GSH content in mycelium changed greatly,which was generally lower than the control group.On the contrary,stress caused by K₂NO?SO₃?₂ gived rise to GSH content increase in B.bassiana.H₂O₂ stress had greater damage on B.bassiana mycelial biomembrane,MDA content in the B.bassiana mycelium were higher than control group at the same time,while at the time of 12 h,its content had a significant difference compared with control.Both VC/FeS04 model and NADH model system could cause lipid peroxidation to B.bassiana mitochondria,however certain range concentrations of GSH may has good resistance to mitochondrial lipid peroxidation.By using high-resolution liquid chromatography-mass spectrometry combined with multivariate statistical analysis of metabolomics methods,the research analyzed B.bassiana mycelium metabolites under stress of H₂O₂ PLS-DA analysis results revealed that oxidative stress distinctly affected the metabolism of B.bassiana,while multivariate analysis and cluster analysis showed that there was a significant difference in mycelium metabolites between the control and the H₂O₂-treated group.Spectral library search methods respectively and accurately characterized 43 kinds of metabolites were differences.Further cluster analysis of oxidative stress towards B.bassiana mycelium metabolite showed that the treatment and control group clearly separated,and metabolite variations associated with their respective gathered together as a class.Cluster analysis results further confirmed that the metabolism of H₂O₂-treated group and control group had significant differences.