| Pinus sylvestris var.mongolica is an evergreen tree species with drought resistance,cold and barren tolerance,strong adaptability,and fast growth rate properties.It is one of the major afforestation tree speciesin thenorthern of China.At present,chemical control is the main method in nursery disease control.Over reliance on chemical pesticides will destroy soil ecosystem,lead to drug resistance of harmful microorganisms and pollute soil ecological environment.In contrast,biological control is an effective measure to control plant diseases by using probiotics that can promote the growth of host plants and improve the resistance to disease and stress of host plants.It has many advantages includingno environmental pollution,no residual effect,and no harm to natural enemy.Ectomycorrhizal fungi and Trichoderma spp.are two important soil microbial groups,which could promoting plant growth and enhance the host stress resistance,and were widely used in agriculture and forestry production.In this study,the microbial community structure in rhizosphere soil of Pinus sylvestris var.mongolica plantation was studied by high-throughput sequencing.Probiotic microorganisms with plant growth promotion and disease resistance were isolated and screened.T.harzianumn E15 which can potentially promote the growth of the annual P.sylvestris var.mongolica seedlings,was screened by pot experiment.The antagonistic effect and mechanism of T.harzianum E15 against Rhizoctonia solani were researched by in vitro antagonistic experiments and transcriptome sequencing.Suillus luteus N94 and T.harzianum E15 were co-inoculated to study the improvement of damping-off resistance of P.sylvestris var.mongolica seedlings and its effects on seedling growth,nutrients,soil enzyme activities,and soil microbial community structure.The main results include:1)The physical and chemical properties of shoot blight non-susceptible P.Sylvestris var.mongolicasoil were significantly higher than those of susceptible P.sylvestris var.mongolica soil,and the soil nutrient content was also significantly different in different seasons.There was no significant difference in the absolute content of fungi in the rhizosphere soil with susceptible and non-susceptible P.sylvestris var.mongolica,at the level of phylum.At the level of genus,there were 28 genera with significant differences,the absolute content of fungi in the rhizosphere soil of non-susceptible P.sylvestris var.mongolica was significantly higher than that of susceptible groups.The different genera included Trichoderma,mycorrhizal fungi,and dark septate fungi.The environmental factors such as pH,OM,TN,NH4+,NO3-,AP,TP,AK,and TK were the main factors affecting the fungal community structure in rhizosphere soil of P.sylvestris var.mongolica.The proteobacteria,actinobacteria,and acidobacteria were the dominant bacteria in the rhizosphere soil of P.sylvestris var.mongolica.At the level of genus classification,there were 19 genera with a significant difference.The relative abundance of bacteria with high abundance in rhizosphere soil of non-susceptible P.sylvestris var.mongolica was significantly positively correlated with the soil nutrient content,while that of susceptible rhizosphere soil was negatively correlated with the soil nutrient content.2)T.harzianum E15(introduced from UK)and T.virens ZT05(isolated from the Zhanggutai Experimental Forest Farm of Liaoning Province,China)were used to study the effects of Trichoderma spp.on the growth and root structure,the physical and chemical properties of rhizosphere soil,and the fungi community structure of P.sylvestris var.Mongolica annual seedlings.Single inoculation of T.harzianum E15 and T.virens ZT05 could promoted the growth of pine seedlings and changed the root structure and rhizosphere microenvironment.The promoting efficiency of T.harzianum E15 was significantly higher than T.virens ZT05,at the same time,T.harzianum E15 inoculation increased the contents of organic matter and total phosphorus in rhizosphere soil.High throughput sequencing results showed that the colonization rate of T.harzianum in the rhizosphere of annual P.sylvestris var.mongolica seedlings was significantly higher than T.virens.3)T.harzianum E1 5 had a significant inhibition effect on Rhizoctonia solani J.G.Kiihn.In the paired culture experiment,T.harzianum and R.solani could formed an obvious antagonistic line,and finally,T harzianum E15 colonies covered the pathogen colonies.The non-volatile metabolites of T.harzianum E15 could inhibited the growth of R.solani and decreased the activity of protective enzymes,which led to the death of pathogenic mycelium.The transcriptome sequencing analysis results showed that the expression of genes related to the mycoparasitism and antibiosis in T.harzianum E15 was significantly up-regulated during the paired culture with R.solani,and 70 and 181 genes related to mycoparasitism and antibiosis were screened.4)The co-inoculation of S.luteus N94 and T.harzianum E15 could significantly enhanced the damping off disease resistance of P.sylvestris var.mongolica seedlings.The activity of CAT,PAL,POD,SOD,?-1,3-chitinase,and glucanase enzymeand the content of proline,soluble protein,and sugar were significantly higher than the control treatment.And 9884 different expressed genes of P.sylvestris var.mongolica seedlings were screened out through the transcriptome sequencing analysis,Among the turquoise,pink,and green co-expression modules obtained by WGCNA analysis,the differential gene expression levels of S.luteus N94 and T.harzianum E15 were higher than the control treatment,and in the riched module included the disease resistance functional gene and metabolic pathway.5)The co-inoculation of S.luteus N94 and T.harzianum E15could significantly improved the seedlings' growth,nutrient content,rhizosphere soil nutrient content and soil enzyme activity of annual and biennial seedlings of P.sylvestris var.Mongolica.And with the annual change,seedling growth,nutrient content,rhizosphere soil nutrient content and soil enzyme activity increased significantly.The co-inoculation of S.luteus N94 and T.harzianum E15 had a significant impact on the fungal community structure in the rhizosphere soil of the P.sylvestris var.mongolica seedlings.Trichoderma was the dominant genus in the rhizosphere soil of annual seedlings under the combined inoculation treatment,while the fungal community structure was relatively rich in the non-inoculation treatment,including some plant pathogens.The relative abundance of the Trichoderma genus decreased in the rhizosphere soil of biennial seedlings,whereas,the relative abundance of ectomycorrhizal fungi increased,and the relative abundance of probiotics in the symbiotic environment also increased.The correlation network analysis results between the relative abundance of fungal and environmental factors showed that Trichoderma and Suillus in rhizosphere soil of annual seedlings significantly promoted the transformation of total phosphorus and organic matter,and Suillus in rhizosphere soil of biennial seedlings significantly promoted the transformation of K,P,N,and organic matter. |
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