Screening Of Zinc Resistant Fungal Strains From Dark Septate Endophytes (DSE) And Studies On Their Resistance Mechanism

With the rapid development of industry and mining, a large quantity of containing heavy metals wastes are released into environment. Heavy metal has posed great threat on environmental safety. The reduction and removal of heavy metal is the key for the detoxification of heavy metal. Many tolerance microorganisms have been found to be able to bioaccumulation of heavy metal and bioremediation has been regarded as a promising approach for the treatment of heavy metal pollution.Dark septate endophytes (DSE) comprise a heterogeneous assemblage of fungi that inhabits in the roots of healthy plants commonly occurred in stressed environment and is a kind of endophytic fungi which may have a similar function of ecology as mycorrhizal fungi. In this thesis, the indigenous DSE strains were isolated from different habitats of Yunnan Province China. First of all, resistant and sensitive strains were screened from all the strains by limited screening method. And then, the fungus was identified based on their morphology and internal transcript space (ITS) sequence and their EC50(median effect concentration) were determined. The biosorption, bioaccumulation characteristics and antioxidant system response of zinc by growing cells were studied in detail. The main achievements are as follows:1. A resistant DSE strain H93and a sensitive one B40-3have been screened out from225strains that isolated from different habitats in Yunnan Province China by the limited screening method. Based on the morphological and molecular characters, the strain H93which came from Huize was identified as Exophiala pisciphila. The Zn-sensitive strain B40-3culture identified as Exophiala sp. H93showed50%growth inhibition at1010mg/L Zn(II)(EC50for H93), whereas the biomass of B40-3was reduced by50%at26mg/L of Zn(II)(EC50for B40-3). There was a39-fold between them. Meanwhile, the two fungal strains showed significant differences in their zinc tolerance. 2. The factors that affected the biosorption efficiency by the two strains were comparatively investigated, such as initial pH value of liquid medium, initial Zn(II) concentration, biosorbent dose and contact time. The results showed that the biosorption of the two strains was a physical process of cell wall. Optimum conditions for biosorption were pH5.5, initial Zn(II) concentrations of100mg/L, at a dose of5g/L and contact time at60min. Under the same conditions, the biosorption efficiency of the two strains was no significant differences. The intracellular Zn(II) accumulation of H93was5.24-fold higher than that of B40-3at their EC50; however, the biosorption by H93was only2.52-fold than that of B40-3. This suggests that resistant strain has higher accumulation ability.3. It was observed that high concentration of Zn(II) could restrict the growth of H93and B40-3obviously, caused decrease the soluble protein content and increase in glutathion (GSH) levels, which could alleviate the oxidation stress of Zn(II). The activities of superoxide dismutase (SOD) and peroxidase (POD) in cells of H93and B40-3increased obviously with the concentration of Zn(II) increasing, which was one of the attributes of two stains'resisting peroxidation. The enzyme activities of H93increased highly, so the resistance to Zn(II) of H93were stronger than B40-3. The malondialdehyde (MDA) levels enhanced in the cells of H93and B40-3under the stress of Zn(II). It showed that the mechanisms for Zn(II) to damage H93and B40-3by inducing reactive oxygen species (ROS) and lipid peroxidation.The results of the study showed that there was significant difference of Zn(II) resistance between two strains of the same genus of DSE, including resistant strains and sensitive strains. The resistant strains and sensitive strains obtained for further research on the resistant mechanism and provided strain security. Zinc can be accumulated in DSE cell wall and the process was a simple physical process, at the same time, resistant strains can also accumulated Zn(II) within cells. Excessive Zn caused the contents of soluble protein in cells decreased, and increased the content of MDA (cause damage to cells). But the content of antioxidant compounds (GSH) and anti-oxidant enzymes (SOD, POD) activities increased could be an important way of DSE to physiological detoxification. These research results and conclusions would provide basic informations for the further research on DSE strains resistant to zinc.