| Eucommia ulmoides Oliv.,a unique multi-functional tree species in China,distributed in Shanxi,Gansu,Henan and other provinces.It has medicinal and edible value,and also has extremely high industrial value.Multi-purpose tree species.However,the cultivation of Eucommia is difficult,the yield is low,and the bark with the most medicinal value is difficult to regenerate,which restricts the industrialization of Eucommia.For many years,Eucommia cultivation has relied on chemical fertilizers in order to obtain the highest possible yield per unit area,but excessive use of chemical fertilizers has led to soil salinization,heavy metal accumulation,water eutrophication and nitrate accumulation.At present,the important trend of increasing production and improving quality of forestry and agriculture is to reduce the use of chemical fertilizers and explore environmentally friendly biological agents.It is a feasible strategy to replace chemical fertilizers with biofertilizers made from plant growth-promoting fungus,which have the advantages of safety,effectiveness,and environmental protection.At present,the endophytic fungal resources of Eucommia have not been fully exploited.This study aims to isolate and identify the endophytic fungus of Eucommia ulmoides,screen out functional strains with strong growth-promoting activities,conduct in-depth research on their growth-promoting functions,and provide scientific basis for the development of efficient biological agents.The specific research results are as follows:Separation and purification of endophytic fungus in Eucommia ulmoides leaves,bark,stems and branches obtained 37 strains of endophytic fungus from Eucommia ulmoides.Among them,14 strains were isolated from leaves,10 strains were obtained from bark,3strains were obtained from stems,and 10 strains were obtained from branches.By observing the morphology of the isolated strains and observing their spore morphology using an optical microscope,combined with r DNA-ITS gene sequence and phylogenetic tree analysis,it was shown that 37 strains were distributed in 8 species,namely Fusarium sp.,Phomopsis sp.,Trichoderma sp.,Alternaria sp.,Aspergillus sp.,Curvularia sp.,Tobacillus sp.,Bjerkandera sp.,and Epicoccum sp.Screen the isolated endophytic fungus for nitrogen fixation,phosphorus dissolution,ammonia production,IAA production,hydrolase production(amylase,protease,pectinase),inhibition of growth of pathogenic bacteria and pathogenic fungus,and other in vitro growth-promoting activities.The results showed that 9 strains had nitrogen-fixing activity,3 strains had activity of dissolving phosphorus,10 strains could produce ammonia,7 strains could produce IAA,and 11 strains have one or more direct in vitro growth-promoting activities;7strains had amylase-producing activity,6 strains had protease-producing activity,and 8 strains had pectinase-producing activity;15 strains had antibacterial effect.Through comprehensive analysis of in vitro growth-promoting activity,Y232 is a functional strain with strong activity-promoting activity among endophytic fungus of Eucommia ulmoides.In order to clarify the basis of Y232 growth-promoting substances,UPLC-QqQ-MS/MS was used to extract,identify,analyze and detect the endogenous plant hormones in the fermentation broth extract of growth-promoting fungi Y232.The results showed that Y232could secrete zeatin(ZT),indole acetic acid(IAA),and salicylic acid(SA),the contents of which were 0.14μg/m L,0.88μg/m L,and 0.53μg/m L,respectively.In order to clarify the molecular basis of Y232’s antibacterial activity,the gene fragments of non-ribosomal peptide synthase(NRPS)and polyketide synthase(PKS)were amplified by PCR,and the genes of NRPS and PKS were successfully amplified in Y232,indicating that non-ribosomal peptides and polyketide antibiotics in Y232 metabolites may be the main components of its antibacterial effect.Using the pot experiment method,taking Eucommia ulmoides and corn as experimental materials,using the concentration of 1×10~6 CFU/m L Y232 fungi suspension to treat Eucommia ulmoides seeds,biennial potted Eucommia ulmoides,corn seeds,and corn potted seedlings,respectively,and evaluate their effects on plant growth development and quality.The results showed that compared with the control,the emergence rate,bud length,root length,and seedling fresh weight and dry weight of the Eucommia ulmoides seeds treated with the Y232fungi suspension were significantly increased after 14 days(P<0.05),respectively,59.41%,47.37%,67.54%,50.79%,and 78.09%.The plant height and stem diameter of the biennial potted Eucommia ulmoides treated with the Y232 fungi suspension were significantly increased after 40 days of culture compared with the control group(P<0.05),which increased by 18.69%and 31.03%respectively.The spore peduncle of Y232 was found in the intercellular spaces of the root of Eucommia by staining with 0.05%Trypan Blue,and examined by light microscope,which indicated that Y232 had successfully colonized in the root of Eucommia ulmoides.The biocontrol effect of Y232 on corn strains infected with maize leaf spot was further evaluated.The results showed that the suspension of growth-promoting fungi Y232 inhibited the harm of Setosphaeria turcica to corn plants.The germination rate of corn seeds treated with Y232 fungi suspension increased by 59.75%,and the germination rate,bud length,rooting rate,and root length after three days increased by 67.35%,58.62%,6.90%,and 80.63%respectively.The germination rate,shoot length,rooting rate,and root length after five days increased by 11.15%,146.72%,2.05%,and 106.90%respectively(P<0.05).After five days of cultivation,the fresh weight and dry weight of 100 seeds in the experimental group increased by 49.64%and 6.25%respectively compared with the control group(P<0.05).The potted corn seedlings treated with Y232 fungi suspension had significantly increased plant height,fresh weight,dry weight and root length after 20 years of growth(P<0.05),which increased by 60.44%,97.70%,132.61%and 67.57%.In order to evaluate the effect of Y232 treatment on the quality of Eucommia ulmoides medicinal material and its possible molecular mechanism,14 kinds of marker compounds in Eucommia ulmoides leaves inoculated with Y232 fungi suspension for 0 days,10 day,20 days,30 days and 40 days were detected by UPLC-QqQ-MS/MS detection method.The transcriptional expression levels of four enzyme genes(PAL,4CL,CHI,HCT)involved in the biosynthesis of phenylpropanoids were detected by q RT-PCR technology.The results showed that 12 compounds in the leaves of Eucommia ulmoides(aucubin,geniposidic acid,protocatechuic acid,chlorogenic acid,caffeic acid,geniposide,pinesterol diglucoside,liriodendrin,rutin,syringaresinol,quercetin,and kaempferol)were significantly increased,and the expression levels of genes encoded by PAL,4CL,CHI,and HCT were up-regulated in the inoculation group,indicating that Y232 can regulate the expression of key coding genes for biosynthesis of phenylpropanoids,which can improve the synthesis of Eucommia’s symbolic compounds,thereby improving the quality of Eucommia. |
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