Diversity And Heavy Metal Tolerance Of DSE From Eupatorium Adenophorum

In recent years,it has been found that root-associated endophytic fungi are closely related to their host plants' resistance to stress.Some root-associated endophytic fungi can enhance the tolerance of host plants to the heavy metal stress,and in the combination of plant and microbial remediation,thus it shows obvious advantages in the phytoremediation of heavy metal-contaminated soil.In this study,we take the invasive plants of Eupatorium adeophorum growing in Gejiu Sn slag heap and Kunming non-mining area of Yunnan province as research object,the colonization of dark septate endophytes(DSE)and arbuscular mycorrhizal fungi(AMF)in the roots of E.adenophorum were studied.DSEs isolated ed from the two plots were identified based on morphologic and molecular characters,and their tolerance to heavy metal(cadmium)were investigated.We selected four root samples with high colonization intensity from Gejiu Sn slag heap,the molecular diversity of endophytic fungi in E.adenophorum was explored by high-throughput sequencing.Further more,5 DSE representative strains were inoculated to maize under greenhouse conditions to evaluate the inoculation effects of DSEs on their host plant under heavy metal stress.The major results of the thesis are as followings:(1)In the 50 root samples of E.adenophorum(30 from mining and 20 from non-mining areas),98%of the samples were infected by DSE and AMF simultaneously.The colonization intensity of DSE and AMF were 14.15 ± 2.34%and 20.84 ± 2.70%from Gejiu Sn slag heap,respectively,and the colonization intensity of DSE and AMF were 42.26 ± 5.07%and 38.48 ± 4.08%from non-mining area,respectively.(2)In total,145 DSE strains were isolated from the root of E.adenophorum by PDA and MEA medium.Among the isolated strains,101 strains were isolated from Gejiu,which can be clustered into 18 groups,and 8 groups were clustered from 44 strains in Kunming,there were 6 cluster groups in the two plots in common.Alternaria spp.was the most dominant with a proportion of 33.10%.,and followed by Paraphoma spp.strains(29.66%)and Herpotrichiellaceae(29.60%).(3)Using the ITS sequence of fungal as the molecular marker,high-throughput sequencing was hired to analyze the fungal diversity in the root of the E.adenophorum from Gejiu.There are relatively abundant root endophytic fungi in the roots of E.adenophorum,suggesting the fungi composition of the samples is stable relatively.The roots of E.adenophorum dominantly colonized by the members of Ascomycota,among which Cladosporium spp.take the absolute dominance,followed by Microidium spp.and Monographella spp.,Compared with the method of laboratory plate separation,it can be more comprehensive to discover community structure and composition ratio of E.adenophorum root-associated endophytic fungi by high-throughput sequencing.(4)The DSE strains isolated from the mining and non-mining areas were cultured in different concentrations of cadmium and the tolerance to heavy metals was evaluated.There were 37 DSE strains from mining area could be grown in the mediun containing 500 mg/L Cd2+,showing higher tolerance to Cd2+,but 21.59%(19 strains)could not grow in the mediun containing 100 mg/L Cd2+.Whereas,51.06%(24 strains)from the non-mining area were able to grow well in the mediun containing 500 mg/L Cd2+,while 29.79%(14 strains)could not grow in the mediun containing 100 mg/L Cd2+.(5)Five representative DSE strains with strong resistance to Cd2+were selected to inoculated to maize grown in green house under Cd stress It was showed that these strains could successfully colonized in the roots of maize under the condition of heavy metal Cd2+ concentration of 25 mg/kg,While,DSE inoculation did not significantly increase the biomass of maize.