The Mechanisms Of Maize Phosphorus Absorption Enhanced By A Dark Septate Endophyte (Exophiala Pisciphild) Colonization

Dark Septate Endophytes(DSE)are a group of multifunctional fungi that ubiquitously colonize in the roots of diverse plants around the globe,benefiting the nutrition and adaptation of hosts to various stressed environments.However,it is unclear whether DSE are beneficial to phosphorus absorption of their host plants.In this thesis,the capacity of 90 DSE strains to solubilize sparingly soluble inorganic phosphorus(Tricalcium phosphate,TCP)into phosphate(Pi)was tested in liquid culture.Then,about 34 strains with relatively strong P-solubilizing activity were selected to further explore the P-solubilizing mechanism of DSE.One P-solubilizing representative DSE strain(Exophiala pisciphila,H93)was inoculated to maize in pot culture with different P sources(low Pi and/or sparingly soluble P)in green house.The influences of DSE on phosphorus absorption of maize were assessed.Physiological and molecular mechanism of DSE(H93)contribution to phosphorus utilization of maize was also studied in the thesis.The major results of the thesis are as follows:1)The capacity of the 90 DSE strains to solubilize sparingly soluble inorganic phosphorus(TCP)was confirmed in PVK liquid culture in vitro.Thirty-four strains with the top TCP solubilizing ability were further selected for analyzingP-solubilizing mechanisms.It was found that the organic phosphorus(lecithin)was more easily than TCP for DSE to solubilize into soluble phosphorus in the liquid culture.Phosphorus solubilization of DSE relied mainly on secreting acid phophatase(ACP),as well as the decreased pH of the environment.In the process of transforming the sparingly soluble organic and inorganic phosphorus into soluble forms,DSE got soluble phosphorus and grown well.2)In the pot cultures,all Pi-deficient treatments decreased maize biomass and total P content,increased the root/shoot ratio of maize,and inhibited photosynthesis and enzyme activities in the rhizosphere,in comparison to the normal Pi condition.However,DSE(H93)promoted the biomass,P absorption and photosynthesis of the inoculated maize by the enhanced phosphorus enzymes activities in the rhizosphere,as well as the decreased pH of the rhizosphere compared with the non-inoculated controls.H93 improved both lecithin and TCP utilization of maize.Moreover,H93 showed more efficiency in TCP utilization when colonized in maize,as the biomass(89.6%)and total P contents(222.9%)of maize were obviously increased comparing to the non-inoculated maizes.3)The influences of Pi-deficiency and H93 inoculation on the expression of 9 maize genes involved in phosphorus metabolism were analysized by q-PCR.These genes included 4 phosphate transporter genes(ZmPht1),3 acid phosphatase genes(ZmPAPs),one alkaline phosphatase gene(ZmALP)and one phytase gene(ZmPHY).In comparison to the normal Pi treatment,all genes were up-regulated under low Pi stress in non-inoculated maize.In comparison to the non-inoculated treatments,the expression of these maize genes were up-regulated by H93-inoculation in normal Pi treatments,but were down-regulated by H93 in the low Pi treatments.Interestingly,a distinct expression patterns of these genes in response to H93 colonization occurred in the deficient Pi conditions supplemented with different sparingly soluble P.All genes were up-regulated by H93-inoculation under low Pi condition with TCP supplement,but were down-regulated by H93 under Pi condition with lecithin supplement.It was suggested that DSE(H93)might promote maize phosphorus absorption by enhancing the expression of these genes under normal Pi condition,by partially replacing the host to transport Pi under low Pi condition.When there was only sparingly soluble P source(no sufficient Pi),DSE might enhance the release of mass acid and alkaline phosphase from the DSE-maize association to dissolve lecithin and TCP,as well as H+,which is helpful for Pi transportation.4)H93-maize association model has also been established in a five-compartment system in laboratory.The enhancement of maize phosphorus absorption by H93 was confirmed in this model system,and it indicated that H93 not only dissolved the sparingly soluble phosphorus into Pi,which was available for plant direct uptake,but also involved in Pi transportation via the fungal hyphae(DSE pathway).The above results suggested that all investigated DSE strains shown the ability of dissolving phosphorus,but their capacity varied a lot among the strains.The enhancement of maize phosphorus absorption by DSE(H93)was based on the inherent mechanism of maize phosphorus adaptation.The combination of the absorption mechanism of maize and the survival strategy of DSE guaranteed the maize-DSE association more efficiently in phosphorus utilization.The potential mechanisms were:DSE may up-regulate plant genes involved in phosphorus absorption under normal Pi supplying;Under Pi-deficient condition,DSE-plant association may absorb phosphorus via two pathways:One is the plant pathway,through which plant directly transports Pi released from sparingly soluble organic and/or inorganic P by DSE dissolving;the other is that DSE transforms P by external hyphae and transports P via hyphae into roots,and consequently exchange nutrients with host cells,that could be defined as DSE pathway.