Screening And Identification Of Endophytic Fungus YD-25 From Glycine Soja And Its Activity In Promoting Growth And Resistance To Heavy Metals

Endophytic fungi are abundant in resources,and there are many kinds of secondary metabolites and active substances with a wide range of functions including promoting plant growth,enhancing plant stress resistance,inhibiting pathogens,resisting cancer and degrading environmental pollutants.Glycine soja,a near-source species of soybean,is a leguminous plant with strong adaptability and stress resistance.Therefore,it is of great significance for enriching endophytic fungi resources,the interaction mechanism between endophytic fungi and host,and exploring the utilization and potential value in Glycine soja to study the diversity of endophytic fungi,exploring the growth-promoting characteristics of secondary metabolites and the anti-heavy metal characteristics of endophytic fungi.In this study,the endophytic fungi of wild soybean were isolated by tissue block method,and the isolated strains were identified by morphological and molecular biology methods.The strains with fine growth-promoting effects were screened by hydroponic soybean plants.The intracellular and extracellular products of the strains were obtained by filtration,ultrasonic fragmentation and centrifugation,and their effects on the growth of hydroponic soybean seedlings were compared.The optimum medium and culture conditions of the strains were determined by single factor experiment and orthogonal experiment.The resistance of the strains to heavy metals was determined by mycelial dry weight method under different stress concentrations of three heavy metals including copper,lead and cadmium.The antioxidant enzymes of the strains under heavy metal stress were obtained by grinding and centrifugation,and the changes of antioxidant enzyme activities were determined.Main results of the research are as follows:1.Isolation and identification of Endophytic Fungi from wild soybean.A total of122 endophytic fungi were isolated from fresh healthy wild soybean plants.Among them,the numbers of species derived from leaves,stems,roots and seeds are 37,46,29 and 10.They could be divided into 9 genus by morphology and molecular biology,which were Fusarium,Colletotrichum,Ceratobasidium sp,Acremonium,Penicillium,Discellaceae,Epicoccum,Alternaria Nees and Pestalotiopsis.2.Screening of strains with growth promoting activity.The characteristic was tested by hydroponic method.Among all strains,strain YD-25 performed the most obvious promoting effect on soybean seedlings and was non-pathogenic,which was identified as Epicoccum nigrum.The extracellular product increased the plant height of soybean seedlings at 4 and 6 days,respectively,by 15.4%and 18.1%.3.Optimization of culture conditions of strains.The single factor and orthogonal test showed that the optimal culture condition of strain YD-25 was as below:based on potato liquid medium,soluble starch 4%,yeast dip powder 0.5%,KH₂PO₄ 0.15%,MgSO₄ 0.05%,pH 3.0,culture for 8 days.4.The study of the resistance of strains to three heavy metals.The YD-25 strain had good resistance to heavy metal Cu²⁺.When the concentration of Cu²⁺in the solution reached to 2.5 mmol·L^-1,the tolerance of the strain was best.When the concentration of Cu²⁺in the solution was 5 mmol·L^-1,the mycelium could still grow slowly.As the concentration of Cd²⁺increased,the dry weight of mycelium gradually decreased.When the Cd²⁺concentration reached to 7.5 mmol·L^-1,the mycelium grew slowly.The strain was resistant to heavy metal Pb²⁺.When the concentration of lead was 3 mmol·L^-1,the tolerance of the strain was best.When the concentration of Pb²⁺was 10 mmol·L^-1,the mycelium could grow slowly.5.Detection of antioxidant enzyme activity of strains under heavy metal stress.With the increase of Cd²⁺concentration,the SOD value of YD-25 strain decreased.The SOD activity of YD-25 strain was the lowest at 1 mmol·L^-11 Pb²⁺,and it was significantly different from other groups.the SOD activity of YD-25 strain was the highest when Cu²⁺was 3 mmol·L^-1.