Ectomycorrhizae Of Populus Yunnanensis In Huangmao Mountain And Their Application In Restoration Of Heavy Metal Mine Area

The irrational exploitation of open-pit non-ferrous metal mines leads to serious soil heavy metal pollution,which is one of the urgent problems in Yunnan province of China,and even in the whole world.In this thesis,Populus yunnanensis,which planted in an open-pit non-ferrous metal mine of Huangmao mountain in Gejiu city of Yunnan Province,China,was collected and investigated the diversity of macrofungi.The morphological and molecular diversity of ectomycorrhiza（ECM）in the roots of P.yunnanensis were analyzed.A successfully subcultured ectogenetic ECM fungus（ECMF）was isolated from the dominant macrofungal fruiting bodies in the forest of P.yunnanensis,and its taxonomic status,heavy metal（Cd）absorption and accumulation characteristics and the mechanism of Cd tolerance were systematically studied under experimental conditions.On this basis,the strain was successfully inoculated to P.yunnanensis.And the effect of ECMF inoculation on P.yunnanensis under Cd stress and the mechanism of enhancing Cd tolerance were discussed.The model of P.yunnanensis-ECMF was established to remediate heavy metal contaminated soil in mining area.The main results are as follows:（1）Our results revealed that the roots of P.yunnanensis was dominantly colonized by diverse ECM fungi according to the field investigation.And the colonization rate of ECMs was 65%.Based on the morphological observation of ECM,a total of 23morphological ECM with different size and/or color were found.According to the phylogenetic analyses of ITS1-5.8S-ITS2 genes,a total of 18 molecular sequences were obtained.And fungi belonging to Helotiales and Geopora were the dominant ECMF.（2）There were 9 species of macrofungus,whose fruiting bodies distributed in P.yunnanensis forest.They were identified as Hygrocybe conica,Inocybe sp.,Hebeloma sp.,Omphalina pyxidata,Hygrocybe acutoconica,Bovista limosa and Inocybe tjallingiorum by the phylogenetics of ITS1-5.8S-ITS2 genes.（3）An ectomycorrhizal fungi（PY5）was isolated from the dominant macrofungi fruiting body in the forest of P.yunnanensis.The optimum synthetic medium and natural medium were obtained to sustain the growth of hyphae.PY5 was identified as Bovista limosa by the molecular phylogeny and morphological analysis of fruiting bodies and mycelia.（4）We investigated the accumulation characteristics of heavy metals（Pb,Zn and Cd）in the fungal fruiting bodies and hyphae,which were the major pollutants in soil.It was found that both fruiting bodies and hyphal bioconcentration factor（BCF）of Zn and Cd were above 1,enriched Cd and Zn.However,the BCF of Pb was less than 1,excluded Pb accumulation.The curve fitting equation was used to measure the EC₅₀ of PY5 to Cd,and it was 77.85 mg/L under liquid condition.We found that each gram of dried hyphae accumulated 7.67 mg Cd and it’s BCF was high as 98.52.The strong Cd resistance of PY5 might be related to the morphological variation of mycelium and the intriggered antioxidant response system.（5）PY5 successfully colonized the root of P.yunnanensis and formed a typical ECM structure in the experimental conditions.ECM significantly reduced the concentrations of Cd in roots and leaves of P.yunnanensis under 50 mg/kg Cd treatments（p<0.05）.Regardless of Cd stress,the inoculation of PY5 could enhance the photosynthetic efficiency of P.yunnanensis and significantly increased plant height and net biomass,compared with their non-inoculated controls.The inoculation of PY5 also enhanced the response of the antioxidant system of P.yunnanensis with the significantly increased content of antioxidants,including glutathione（GSH）,metallothionins（MTs）,phytochelatins（PCs）and the enhanced activity of antioxidant enzymes,superoxide dismutase（SOD）and catalase（CAT）.The inoculation of PY5 also changed the subcellular distribution and the chemical forms of Cd in the roots and leaves of P.yunnanensis.In other words,PY5 enhanced the tolerance to Cd by stimulating the host plant’s antioxidant system and changing the subcellular distribution and chemical forms of Cd in P.yunnanensis.