Study On The Improvement Of The Tolerance Of Pinus Sylvestris Var. Mongolica To Heavy Metal Cadmium By Dark Septate Endophytic Fungi

Dark Septate Endophytes(DSE)are endophytic fungi with dark hyphae and distinct septa,which colonize plant roots to promote plant growth and improve its ability to resist pests and diseases and stress.In this study,seven dark septate endophytic fungi isolated from the roots of Pinus sylvestris var.mongolica were selected,the excellent strain with strong tolerance to heavy metal cadmium(Cd)was screened by plate and liquid fermentation methods,after optimizing culture conditions,physiological indicators of resistance of excellent strains under different concentrations of Cd stress were determined,and the changes of mycelial morphology were observed by scanning electron microscope to study the mechanism of Cd resistance.Potted experiments were carried out to determine the growth indexes,physiological indexes and rhizosphere soil indexes of 1-year-old pine seedlings cultivated in sterile soil and 3-yearold pine seedlings normally cultivated outdoors by inoculating DSE and not inoculating DSE under different concentrations of Cd.The effects of excellent resistant strains on the resistance to heavy metal Cd of P.sylvestris were analyzed.The main results were as follows:(1)Through morphological identification and phylogenetic tree analysis,seven DSE strains respectively belonged to Paraphoma chrysanthemicola(A041),Phialocephala forinii(A003,H051,J041),Phialocephala bamuru(A083)and Paraphoma radicina(H088,J016).Under heavy metal Cd stress,the growth rate and biomass of A041 were significantly higher than other strains,and it had the ability to dissolve phosphorus,produce cellulase and secrete indoleacetic acid(IAA).(2)The vitro pure culture conditions of resistant strain A041 were optimized by changing carbon source,nitrogen source,p H value and temperature on MMN medium.Under solid culture conditions,the optimum carbon source of A041 was soluble starch,the optimum nitrogen source was peptone,the optimum p H value was 6.5,and the optimum culture temperature was 25℃.Under liquid culture conditions,the optimum carbon source of A041 was fructose and soluble starch,the optimum nitrogen source was ammonium acetate,and the optimum p H value was 6.5.The optimum culture conditions of dark septate endophyte A041 in the roots of P.sylvestris were preliminarily determined,which laid a foundation for the research and application of dark septate endophytes.(3)The mechanism of DSE tolerance to heavy metal Cd was preliminarily explored,and the main physiological indicators of A041 under certain concentration gradient Cd stress were determined,including catalase(CAT),peroxidase(POD),superoxide dismutase(SOD),glutathione(GSH),malondialdehyde(MDA),soluble protein and melanin content,and the morphological changes of hyphae were observed by scanning electron microscope.The results showed that with the increase of Cd concentration,A041 resisted heavy metal stress by increasing antioxidant enzyme activity,GSH,melanin,soluble protein content,and decreasing MDA content,which reflected the degree of plant lipid peroxidation;under Cd stress,the hyphal morphology also changed significantly.With the increase of Cd concentration,mycelium diameter decreased and became more sparse.(4)Inoculation of A041 could effectively improve the ability of 1-year-old seedlings and3-year-old seedlings to resist heavy metal Cd stress.Under Cd stress,inoculation with A041 significantly increased the seedling height,ground diameter,dry weight and fresh weight of P.sylvestris one-year-old seedlings.Similarly,in terms of physiological characteristics of seedlings and rhizosphere soil indicators,inoculation of A041 also showed that the ability of seedlings to resist heavy metals was improved,compared with the non-inoculation treatment,under the same concentration of Cd stress,inoculation with A041 effectively increased the antioxidant enzyme activities,rhizosphere soil enzyme activities and soil nutrient content of 1-year-old and 3-year-old seedlings,and adjusted cell osmotic regulator substances to reduce the degree of cell damage.In conclusion,A041 has a significant effect in improving the Cd tolerance of P.sylvestris to heavy metals.It can improve the stress resistance of P.sylvestris by inoculating DSE,promote its growth,and improve the soil nutrient content in heavy metal polluted areas,which is of great significance in actual seedling cultivation and production.