Growth Promotion And Salinity Tolerance Of Several Coastal Shelter Forest Tree Species Conferred By Root-associated Fungi And The Possible Underlying Mechanisms

Soil salinization is a major factor restricting the construction of coastal shelterbelts.Improving the productivity and adaptability,especially the salt tolerance of trees,is an important measure to promote the land use of the saline-alkali soil.Some achievements have been achieved in traditional breeding and genetic engineering,but it's not enough to solve the actual production problem.Rhizosphere symbiotic microbe are important to improve the productivity and adaptability of agriculture and forestry plants.Alough functions and molecular mechanisms of mycorrhiza and root nodule have been studied in depth,the rich genetic diversity of soil microbes suggests that there may be other symbiotic systems.Trees can establish a variety of symbiotic relationships with endophytic bacteria,endophyte,mycorrhizal fungi and other symbionts.However,there is a limited understanding of the application of tree rhizospheric symbiotic fungi and their mechanisms to assist in salt tolerance in the host forest.Using plant rhizosphere symbiosis fungi to establish symbiotic system with the root of coastal shelter forest tree species such as poplar and Liquidambar styraciflua,is helpful to explore tree symbiotic fungal resources,therefor obtain functional isolates for promoting growth and assisting salt tolerance in trees.In this research,the poplar rhizosphere symbiosis fungi in National Forest Farm in Xindeng Zhejiang,Sihong Jiangsu and Weixian Heibei are preliminarily investigated;a tree symbiotic fungi resource bank is established by isolates gained from poplar rhizosphere,root endophytic fungi of halophyte and broad-spectrum symbiotic fungi.Choosing isolates with potential application value as candidate isolates for experiment,inoculating the above isolates to the important coastal shelter forest tree plantlets?Populus tomentosa,P.× euramericana cv.‘Nanlin895',NL895 and L.styraciflua?root,screening isolates with the effect of growth promotion and salt tolerance by adding sea salt solution.The main research results are as follows:?1?Basic biological characteristics and preliminary screening of the tested isolates: 45 isolates of fungal isolates were selected?mainly isolated from halophytes rhizosphere,oak ectomycorrhiza and rhizosphere and buck soil of poplars?,in which,33 isolates belong to the Ascomycota,4 belong to the Basidiomycota and 8 belong to the lower fungi.The main groups include fungi belong to Fusarium spp.,Pleosporales spp.,Helotiales spp.and Mortierella spp..Establishing the symbiotic system of the above isolate with P.tomentosa and NL-895 poplar,preliminary screening of potential functional isolates by observing the phenotypic changes of poplar plantlets after inoculation and whether there is a special infection structure in the root cells,the physiological indexes of poplar plantlets of inoculation groups were also compared with those of the control group plantlets.?2?Effects of endophytic Curvularia isolate on the growth promotion and salt tolerance of P.tomentosa: Endophytic Curvularia isolate R2015-25 is isolated from healthy root of Suaeda salsa,can established symbiotic system with P.tomentosa successfully and promote the growth of poplar.Microscopic staining technique confirmed that the R2015-25 can penetrate the poplar roots after two week inoculation and readily form sclerotia-like structures and monilioid hyphal cells in root hair and/or cortex cells.This implied that this fungus can be referred to as a dark septate endophyte.Pot experiments revealed that R2015-25 significantly promoted the poplar growth and resulted in increased production of the antioxidant enzymes,particularly the superoxide dismutase?SOD?and ascorbate peroxidase?APX?under salinity stress condition.The presence of R2015-25 also enhanced chlorophyll a,b and proline contents in leaves,although not all differences were significant.Compared to the non-inoculated plants,the photosystem ?-based electron transport rate?ETR?,actual quantum yield in the light-adapted steady state(?_PS?)and photochemical quenching values?q P?was significantly higher in colonized plants.Collectively,our data supports the evidence of the ability of R2015-25 to alleviate the adverse effects of salinity stress on poplar growth and highlights the potential use of endophytes from extreme conditions as novel probiotics in improving salt tolerance of tree plantles.?3?Effects of three endophytic Fusarium isolates on the growth promotion and salt tolerance of Liquidambar styraciflua seedlings: To evaluate the potential use of endophytes from extreme conditions as novel probiotics in improving salt tolerance of tree seedlings,we investigated the effects of three endophytic Fusarium isolates,recovering from the halophytic Leymus mollis,on the growth and salt tolerance of Liquidambar styraciflua seedlings.The three isolates were identified through morphological description and molecular phylogeny.In vitro toxin production of the endophytic Fusarium isolates were analyzed.The co-culturing system was used to investigate binary interactions between gnotobiotic seedlings and Fusarium isolates under normal and saline conditions.Tissue staining method was applied to examine the fungal infection structures formed in roots.The two Fusarium isolates were identified to Fusarium culmorum?Class2-1B?and F.pseudograminearum?Class2-1C?,and the remaining one?Class2-3?was not currently identified to the species level.Class2-1B and Class2-1C can promoting the growth and assisting salt tolerance of Liquidambar formosana,but Class2-3 showed pathogenicity to Liquidambar formosana.?4?Analysis on whole genome resequencing and carbon metabolizing utilization of endophytic Fusarium isolates: Using the corresponding Fusarium genomes as reference genomes,we resequnenced 3 halophytes endophytic Fusarium isolates?Class2-1B?Class2-1C and SYC-Root-1?and analyzed the resequencing data preliminarily.To further reveal the physiological and ecological characteristics of 3 isolates,BIOLOG FF plan analysis was used to compare and analysis the carbon source utilization capacity of the 3 isolates.?5?Construction of GFP transformation system of endophytic Fusarium and Curvularia isolates: To learn details about the characteristics of infection and colonization of three isolates?Class2-1B?F.culmorum??Class2-1C?F.pseudograminearum?and R2015-25?Curvularia sp.??in tree roots,and track the actual infection efficiency of the strain in the field experiment.3 isolates were marked by green fluorescent protein?GFP?,through applied Agrobacterium Tumefaciens mediated transformation?ATMT?to the conidia of 3 isolates.Inoculating poplar plantlets with isolates maked by GFP and co-culture over 2 weeks.Fluorescence microscopic observation showed that the reproductive bodies of 3 functional isolates colonized in the poplar plantlets root successfully,what lays a technical foundation for the tracking of the ecological characteristics of the 3 isolates in the field experiment in the later period.