| Ormosia hosiei Hensl.et Wils.tree is a semi-evergreen tree in Ormosia of Leguminosae.As the endemic in China,O.hosiei are merited in the second-class rare protected plant,which integrates material,medicinal,and humanistic values.The seedlings prefer shady,cold,and moisture during growth.Thus the high requirement of precipitation makes it challenging to raise O.hosiei on the dry hillside and the top of the hill.Hence,the improvement of drought tolerance of its seedlings will display an important application for the protection and utility of O.hosiei tree.Dark septate endophyte(DSE)is a class of endophytic fungi,which is characterized by dark and septal hyphae and could form microbacterial nucleus.The fact that it will not have an adverse effect on plants root systems after colonization makes it an attractive candidate for plant protection.The distribution of DSE is pervasive,and it has many potential ecological functions as mycorrhizal fungi,which show important application values in improving plant growth and plant drought resistance.In this study we isolated DSE strains from the roots of O.hosiei tree.,and identified the type of strain in the molecular level.With the potted water control experiment,we did research on the effects of DSE inoculation in the growth,photosynthetic physiology,cell submicroscopic structure,and leaf proteome of O.hosiei seedlings under drought stress.Besides,the comprehensive analysis was made on DSE promotion effect on the growth on drought tolerance response in O.hosiei seedlings,to provide theoretical basis and technical support for DSE's ecological function,as well as introduction and seedling raising theories for O.hosiei.The main findings of this study are as follows:1.We separated five DSE strains candidate from wild O.hosiei tree roots,and identified two strains of them had the DSE infection characteristics supported by back-infection experiment.These two strains were identified as Acrocalymma vagum and Leptosphaeria sp.AS7-1,according to the morphological characteristics of the strains and the results of molecular biological identification with r DNA-ITS.Both of these two DSE strains improved the growth of O.hosiei seedlings,but the effect of growth-promoting ability was different.A.vagum showed a better growth-promoting fiction than that of Leptosphaeria sp.AS7-1.These two DES strains had different optimum carbon and nitrogen source.The optimum carbon and nitrogen sources were glucose and sodium nitrate for A.vagum,and the most suitable carbon source was lactose for Leptosphaeria sp.AS7-1,while the most suitable nitrogen source was sodium nitrate and hydrolyzed casein.2.Inoculation with A.vagum significantly alleviated the influence of drought stress on the growth of O.hosiei seedlings.Under low water condition,seedling height,ground diameter,leaf area,root volume,root surface area,and biomass of inoculated seedlings increased by 30.49%,28.32%,21.36%,182.77%,107.02%,and 82.72%,respectively,compared with non-inoculated seedlings.A.vagum promotes the growth of O.hosiei seedlings under drought stress by increasing the root absorption area,changing the root morphology and topology,and promoting the absorption of nitrogen and phosphorus nutrients.3.The effect of A.vagum on the anatomical structure and cell submicroscopic structure of O.hosiei seedlings under drought stress.By observing the ultrastructure and anatomical structure of leaves and roots of inoculated and non-inoculated seedlings,we found that the drought stress destroyed the ultrastructure of leaves and root cells of O.hosiei seedlings,reduced the thickness of palisade tissue,destroyed the arrangement of palisade tissue and sponge tissue,and affected the vascular bundle area and the number of ducts in stems,roots,and leaves.The inoculation with A.vagum improved the structural stability of leaf and root cells of O.hosiei seedlings,maintained the integrity of organelles such as cell membranes,mitochondria and chloroplasts,and increased the thickness of leaves,palisade tissues and the density of arrangement of palisade tissues and sponge tissues,the area of vascular bundle,and the number of ducts in leaf and root systems.Thus,the seedlings could obtain more water and nutrition,effectively alleviate the damage caused by drought stress on O.hosiei seedlings and improve the tolerance of O.hosiei seedlings in the arid environment.4.A.vagum inoculation obviously alleviated the influence of drought stress on physiology and biochemistry of O.hosiei seedlings.The leaf water holding capacity,osmotic adjustment capacity,antioxidant enzyme activity and photosynthetic capacity of A.vagum inoculated seedlings were significantly higher than those of non-inoculated seedlings,but themembrane lipid peroxidation products were lower than those of non-inoculated seedlings.Under low water condition,the relative water content,soluble sugar content and soluble protein content of inoculated seedlings were 10.55%,70.39% and 69.20% higher than those of non-inoculated seedlings.The activities of SOD,POD and CAT were increased by 16.52%,207.84% and 14.27%,respectively,while the content of MDA was decreased by 43.85%.The total chlorophyll content,photosynthetic rate and transpiration rate of the inoculated seedlings were 15.88%,15.88% and 170.09% higher than those of the non-inoculated seedlings.The maximum quantum yield,electron transfer rate and photochemical fluorescence quenching coefficient in inoculated seedlings were also higher than those in non-inoculated seedlings.Moreover,inoculation with A.vagum increased the accumulation of abscisic acid(ABA),indole acetic acid(IAA),and IAA/ABA ratio,while decreased the contents of gibberellin(GA)and zeatin riboside(ZR),as well as GA/ABA ratio and ZR/IAA ratio.Thus,DSE can improve the drought tolerance of seedlings by enhancing the water holding ability of O.hosiei.seedlings,improving the photosynthetic efficiency and gas exchange of seedlings,enhancing cell osmotic regulation and scavenging of reactive oxygen species to cells,and regulating hormone balance.5.The effect of the inoculation with DSE on leaf proteomics of O.hosiei seedling.We analyzed the protein expression characteristics of inoculated and non-inoculated seedlings with i TRAQ technique.The results showed that the different proteins in inoculated seedlings were 62,among which 35 were up-regulated,and 27 were down-regulated.Functional analysis showed that the key experiment of DSE's helping-respond to drought was the maintenance of photosynthesis and the removal of reactive oxygen species.Furthermore,the related protein expression of glucose metabolism and energy metabolism in mitochondria was also up-regulated,indicating that DSE could help host plants cope with drought stress through synergistic changes of different proteins of various organelles in cells. |
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