| Elaeagnus angustifolia is a kind of deciduous trees,belonging to the genus Elaeagnus of Elaeagnucea family.Elaeagnus angustifolia has the very high ecological and economic value,as greening tree species for breaking wind,fixing sands and urban construction.Especially as a salt-tolerant plant,it plays an important role for restoring saline-alkali soil.In previous studies,it found that Elaeagnus angustifolia rhizosphere exists nodular actinomycetes and arbuscular mycorrhizal fungi(AMF).At present,very few studies research the symbiotic relationship of Elaeagnus angustifolia,AMF and nodular actinomycetes.This study not only investigate microbial diversity of Elaeagnus angustifolia rhizosphere and soil physicochemical properties of Zhaodong test field but also separate nitrogen-fixing nodules actinomycetes and dominant AMF.Then it makes the actinomycetes and AMF inoculate into Elaeagnus angustifolia seedlings,deal with salinity stress and combine molecular method and physicochemical properties.According to the research that actinomycetes and AMF impacts on Elaeagnus angustifolia,it aims at revealing the mechanism of action that AMF and actinomycetes promote Elaeagnus angustifolia to resist saline-alkaline and filling the gap of domestic research on this field.So this research has certain innovative meanings.Main results for this study are as follows:1.Through surveying roots and rhizosphere soil of three different growth years of Elaeagnus angustifolia and measuring the rhizosphere infection rate and the number of actinomycetes,it found that physical-chemical indexes of rhizosphere soil has significant differences.Over time,available phosphorus,total nitrogen,soil moisture content,actinomycetes quantity and AMF spores quantity shows a significant increase.However,pH and conductivity decreased significantly.Through the correlation analysis between these indicators and microbial quantity of rhizosphere soil,it shows that the change has close correlation with the rhizosphere microorganisms,and actinomycetes quantity was significantly positively related with AMF spore density.It proves that actinomycetes and AMF can form a symbiotic relationship with Elaeagnus angustifolia,so as to improve soil fertility and restoration.2.The separated AMF spores are sieved from soil samples by sucrose-centrifugation method and used to calculate the relative abundance of species and separate frequency.According to a preliminary identification of AMF spore morphology structure,PCR on the LSU-ITS-SSU area and molecular identification,the study concluded that five kinds of AMF can form symbiosis with Elaeagnus angustifolia.Respectively they are:Septoglomus constrictum,Rihizophagus aggregatum,Rhizophagus intraradices,Funneliforrmis caledonius,Funneliforrmis mosseae.Among them,R.intraradices is the dominant fungus.3.Through separating and cultivating actinomycetes in Elaeagnus angustifolia rhizosphere soil,these actinomycetes belongs to 6 families,10 genera and 127 species.Using nitrogen free culture medium to culture 127 strains of actinomycetes,it found that 80 strains of actinomycetes are able to grow.In accordance with extracted the genome of these actinomycetes and using PCR to amplify for nifH and nodA gene,it found that 22 strains of actinomycetes have the function of nitrogen fixation and only one(14-1 strain)has nifH and nodA gene.Using the molecular identification of 16 srRNA,7 strains of actinomycetes have been identified.Respectively they are:Streptomyces massasporeus,Streptomyces iakyrus,Streptomyces scabies,Streptomyces lincolnensis,Streptomyces virginiae,Streptomyces olivoviridis,Streptomyces pratensis.Among them,Streptomyces pratensis is the dominant fungus.4.In 0 mm and 300 mm NaCl salt concentration,Rhizophagus intraradices and Streptomyces pratensis inoculated into the seedlings of Elaeagnus angustifolia by dual or single inoculation.After 30 days,by using photosynthesis system to measure photosynthetic rate,transpiration rate,stomatal conductance and intercellular CO2 concentration,it found that under salt stress,dual inoculation treatment can effectively improve the photosynthetic rate of the Elaeagnus angustifolia seedlings,decrease the stomatal conductance and reduce transpiration rate.5.This study measured aboveground and underground biomass of the Elaeagnus angustifolia seedlings,like basal diameter,plant height,dry weight,root length,root projected area,root volume,root area and so on.The result shows that under the condition of salt,root volume of AMF treatment is greater than the other,and other indicators are displayed as R.i+S.p.>R.i.or S.p.>CK.Under salt stress,the dual inoculation can significantly increase the biomass.6.Through measuring catalase,phosphatase,urease and sucrase of Elaeagnus angustifolia rhizosphere soil,it shows that under the both salt and non-salt stress,the enzyme activity of dual inoculation treatment is higher than any other treatments.Under the condition of salt stress,urease activity of actinomycetes treatment is higher than AMF treatment,but other enzyme activities were adverse.It follows that dual inoculation treatment can effectively improve enzyme activity of Elaeagnus angustifolia rhizosphere soil,and S.p.treatment can make the urease activity higher than R.i.treatment.7.By measuring PRO,MDA,CAT,SOD and POD,the result shows that under the condition of salt,dual inoculation treatment significantly improve SOD,POD,CAT and Pro,and effectively reduce the content of MDA.Also in contrast with actinomycetes treatment,the enzyme activity of AMF treatment is higher and the MDA content is lower.Thus under salt stress,dual inoculation treatment can effectively improve the oxidase activity,increase the content of Pro,reduce the content of MDA,and make plants grow normally.However,the effect of actinomycetes treatment is not better than AMF.8.Adopting FASS to measure the content of Na+and K+,and calculating K +/Na + ratio,it shows that under the salt stress,CK treatment has the highest concentration of Na+ and dual inoculation treatment is the lowest.Concentration of K+ and K+/Na +ratio is highest in the dual inoculation treatment,and the single inoculation treatments are the lowest.So under the salt stress,dual inoculation can effectively reduce the concentration of Na+ in the leaves and increase the K+/Na+ratio.9.According to mycorrhizal infection rate and actinomycetes nodulation rate of different treatments,author adopted factor analysis to deal with the obtained data and physiological and biochemical indexes by SPSS.It found that the AMF infection rate has significant correlation with plant height,and nodular rate has correlation with content of K +.10.Using RT-PCR to research relative expression of P5CS which is related with Pro and nifH Which is related with actinomycetes nitrogenase,the result shows that both of them are raised on transcription reaction,and the relative expression of dual inoculation treatment is higher than single treatments.P5CS expression is positively correlated with Pro content.nifH expression on transcription level is negatively related with urease activity in rhizosphere soil.11.The method of using Metagenomics sequencing to explore under salt stress inoculation R.i.and nitrogen-fixing actinomycetes S.p.processing of elaeagnus angustifolia rhizosphere microbial community composition and metabolic pathways related genes.The results show that:received 7932666 ORFs,the average sample 881407 ORFs;GC content is 62.69%,the complete genetic with 973472,accounting for 17.87%of the total number of all non redundant genes.Main microbial Phylum into deformation bacteria Proteobacteria?Acidobacteria)?Chloroflexi?Actinobacteria?Cyanbactiria?Gemmatimonadetes?Bacteroidetes?Verrucomicrobia?Nitrospirae and Chlorobi.To include all the metabolic pathways of pathways(metabolic pathways,synthesis of secondary metabolites,antibiotic biosynthesis,carbon metabolism,2-carboxylic acid metabolism,lipid metabolism,oxygen generation amino acid synthesis and degradation of aromatic compounds),carbohydrate metabolism pathways,amino acid metabolism,metabolism of cofactors and vitamins and plasma membrane has a close relationship.One of active defense under salt stress metabolic pathways for membrane,the passive defense the metabolic pathways of carbohydrate metabolism pathways,amino acid metabolism pathway,metabolism of cofactors and vitamins.Under salt stress conditions,adding of AM fungi and actinomyces can significantly change the soil microbial community structure and function,makes the soil microbial structure and function change,and change the structure and function of the law is consistent,and adding of AM fungi and actinomycetes would offset the salt treatment on narrow-leaved oleaster rhizosphere soil microbial structure and function,strengthen the resistance to salt stress in Elaeagnus angustifolia.Along with the longer transplanting date of Elaeagnus angustifolia,the effect of repairing saline soil is more obvious.The factors for repairing saline-alkali land are rhizosphere AMF and nodular actinomycetes.Under the condition of salt stress,two kinds of microorganism cooperate with each other can be used to improve the photosynthesis,biomass,soil enzyme activity and related enzymes activity for anti-saline of Elaeagnus angustifolia.These results of this article provide theoretical basis for ecological management of saline-alkali land,and lay a foundation for mycorrhizal cultivation of Elaeagnus angustifolia. |
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