Study On The Mechanisms Of Ectomycorrhizal Fungi And Dark Septate Endophytes Improving Resistance To Pine Wilt Disease Of Pinus Tabulaeformis

In this study,the influence of pine wilt disease（PWD）on community structure of root associated fungi and rhizosphere microbe of Pinus tabulaeformis Car.were examined through Illumina Miseq sequencing in Zhashui county,Shaanxi province and the relationship between PWD and soil characters were analyzed.Based on pots experiment,we screened and selected PWD resistant ectomycorrhizal fungi（ECMF）and dark septate endophyte（DSE）strains,and their disease resistance mechanisms were revealed from the following aspects: the resistance-related enzymes,resistance-related substances and the effects of pinewood nematode（PWN）infection on the dynamic changes of root associated fungi and rhizosphere microbial community.The main results were as follows:1.The relationship between PWD and soil charactersThree P.tabulaeformis stands,Undisturbed（UD）,Middle Disturbed（MD）and High Disturbed（HD）by PWD,were selected for this study.The effects of the disturbance degree of PWD and soil depth on soil factors were studied.Principal component analysis（PCA）of soil characters of three soil layers in three stands showed that the soil properties were more easily be affected in 5³0 cm than 30⁴5 cm by PWD.The correlation analysis showed that the PWN infection rate was negatively correlated with mycelium density,soil p H and soil total P content,and positively correlated soil invertase.The mortality of pine tree was negatively correlated with mycelium density and soil p H,and positively correlated with soil total N,soil nitrate nitrogen and soil oganic carbon.2.Distribution of fungi and bacteria in rhizosphere of P.tabulaeformis in different disturbance pine standsThe effects of PWD on distribution of fungi and bacteria in rhizosphere of P.tabulaeformis were studied by high-throughput sequencing.The results indicated that the main fungal groups of rizosphere of P.tabulaeformis were Basidiomycetes and Ascomycetes fungi,and the main bacterial groups were Proteobacteria,Acidobacteria,and Actinomycetes.The relative abundance of Basidiomycota decreased sharply from 81.45% in UD to 46.85%and 44.45% in MD and HD respectively,while Ascomycota increased from 17.59% in UD to50.74% and 52.66% in MD and HD respectively.The change of rhizosphere bacteria caused by PWD was not obvious among different pine stands.Cluster analysis showed that the taxon similarities between PWD disturbed and not disturbed pine stands were different.Followed by the increased severity of PWD disturbed,the fungal abundance increased first and then decreased,diversity gradually increased while bacteria rich and diversity gradually reduced.The mycelium density of P.tabulaeformis stands was significantly decreased from 1334.6mm g^-1 to 606.1 mm g^-1 by PWD disturbed.3.Relationship between root-associated fungal colonization,community structure and PWDHigh performance liquid chromatography（HPLC）analysis showed that the infection of PWN significantly reduced the fungal biomass of P.tabulaeformis root,from 0.272 mg g^-1 to0.182 mg g^-1.And ECMF colonization rate was also reduced from 84.4%（UD）to 64.1%（HD）.While the colonization rate of DSE was increased followed by decreased,were 52%（UD）,45.3%（MD）and 64.7%（HD）,respectively.The correlation analysis showed that colonization rates of ECMF and DSE were significant correlated with PWD.High-throughput sequencing indicated that the infection of PWN decreased the relative abundance of ECMF and community diversity.The OTU counts of UD,MD and HD stands were 69,70 and 55,including 9,8 and 3 peculiar OTUs respectively.The relative abundance of Tuber,Russula,Agaricomycetes,Tricholoma and Trichophaea were higher than others,occupied more than80% sequences,but they have a different abundance distribution among the study sites.Followed by increasing of disturbance degree,the abundance of Tricholoma was increased,Tuber and Russula were increased then decreasing while Agaricomycetes and Trichophaea were in opposite.Principal component analysis（PCA）showed that fungal community composition was different among different PWD disturbed degree sites.4.PWD resistance ECMF and DSE strains screeningThe effects of different inoculation treatments on physiological indexes and root morphology of P.tabulaeformis seedlings were different.PCA results showed that,inoculation treatment affected the growth characteristics of P.tabulaeformis on the whole.There was no significant difference in the inoculation effect of ECMF and DSE on P.tabulaeformis.The correlation analysis showed that the seedling morbility caused by PWN was negatively correlated with root length,root fork number and root surface area,and positively correlated with carotenoid content.And the tissue water content of P.tabulaeformis was negatively correlated with seedling mortality caused by PWN infection.P.tabulaeformis inoculated with ECMF/DSE in natural soil condition,two strains of ECMF（Amanita vaginata and Suillus bovinus）and DSE（Gaeumannomyces cylindrosporusand Paraphoma chrysanthemicola）were obtained to improve the resistance to PWD of P.tabulaeformis.Three months after PWN infection,morbility and mortality of P.tabulaeformis seedlings inoculated with G.cylindrosporus,A.vaginata,S.bovines and P.chrysanthemicola were 16.67%,26.67%,20%,6.7% and 20%,26.67%,23.33%,10% lower than control group,respectivily.5.Responses of disease resistance-related enzymes and substances to the infection of PWN of P.tabulaeformis seedlings inoculated with ECMF/DSEAccording to the result of PWD resistance fungi screening,the dynamic changes of the activities of pathogen resistance-related enzymes such as phenylalanine ammonia lyase（PAL）,lipoxygenase（LOX）,chitinase and β-1,3-glucanase,and pathogen resistance-related substances such as lignin,soluble phenol,malondialdehyde（MDA）and free proline in different ECMF/DSE treatments P.tabulaeformis seedlings were studied.The results showed that,after PWN infection,A.vaginata and G.cylindrosporus could induce the rapid increase in PAL activity of P.tabulaeformis seedlings compare to control group.It was suggested that the inoculation of A.vaginata and G.cylindrosporus could activate Salicylic Acid（SA）pathway of P.tabulaeformis.After inoculation of PWN,the LOX activities of A.vaginata,S.bovinus,G.cylindrosporus and P.chrysanthemicola treatment P.tabulaeformis seedlings decreased by 19.59%,40.98%,17.08% and 30.50%,respectively.Thus inoculation could reduce the allergic reactions caused by PWN infection.The content of soluble phenol in P.chrysanthemicola and G.cylindrosporus treatments increased by 43.04% and 6.93%,respectively,while that of A.vaginata and S.bovines treatments decreased by 2.02% and9.73%.The lignin content of A.vaginata,S.bovinus and P.chrysanthemicola treatments increased by 37.95%,3.64% and 85.97%,respectively,while that of G.cylindrosporus treatments decreased by 4.14%.The content of free proline in A.vaginata and S.bovines treatments was 140.79% and 84.44% higher than that in the control group after PWN infection.However,β-1,3-glucanase and chitinase activity were not significantly different from those in the control group.Thus indicated that ECMF/DSE could improve P.tabulaeformis resistance to PWD by regulate disease resistance-related enzymes activities and substances contents.6.Responses of root associated fungi and rizosphere microbe to the infection of PWN of P.tabulaeformis seedlings inoculated with ECMF/DSEWe studied the community structure of root associated fungi and rizosphere microbe of P.tabulaeformis seedlings inoculated with ECMF/DSE after 3,6 and 9 months PWN infection.Microbial carbon biomass and soil enzymatic activity were also analyzed among different treatments after 6 and 9 months PWN infection.The results showed that,under PWN infection condition,the content of microbial carbon in the rhizosphere of P.tabulaeformis inoculated with A.vaginata,S.bovines,G.cylindrosporus and P.chrysanthemicola were38.16%,49.67%,42.11% and 96.05% higher than that of the control group,respectively. Inoculation of ECMF and DSE can reduce the decrease of rhizosphere microbial biomass caused by PWN infection.Both ECMF/DSE inoculation treatments and PWN infection could affect pot soil enzymatic activity.Under PWN infection condition,the effects of different ECMF/DSE inoculation treatments on soil enzyme activities were different.The effects of PWN infection on the community structure of P.tabulaeformis root associated fungi,rhizosphere fungi and bacteria were analyzed by DGGE.The results showed that the infection of PWN reduced the abundance and diversity of P.tabulaeformis root associated fungi.The exogenous fungi（ECMF/DSE）maintained the stability of fungal community and reduced the influence of PWN in roots fungal community of P.tabulaeformis.The effects of different ECMF/DSE inoculation treatments on rhizosphere microbial abundance and diversity of P.tabulaeformis were different.Inoculation of ECMF/DSE can increase beneficial bacterial groups of P.tabulaeformis rhizosphere,such as Burkholderiales,Rhizobacter sp.,Mesorhizobium sp.