Physiological Mechanism Of Epichlo(?) Endophyte Infection To Enhance Salt Tolerance Of Wild Barley

Soil salinity is a major constraint to agriculture production.Improve plant salt tolerance and breed new salt tolerance cultivars are one of the main strategies to develop and utilize saline-alkali land,use of these strategies must be based on the overall understanding the physiological mechanism of salt tolerance in plants.Symbiotic microorganisms infection play a positive key role in enhancing biotic and abiotic resistance of host plants.Species of the fungal genus Epichlo???are a special group of biotrophic endophyte that form symbioses with temperate grasses in the subfamily Pooideae.Certain species Epichlo???endophytes are toxic to mammalian herbivores by producing several classes of biologically active alkaloids,leading huge economic losses to grassland animal husbandry production.Many Epichlo???endophytes forms mutualistic symbiotic associations with many cool-season grasses and can also endow hosts with increased stress tolerance.In this study,important forage grass-wild barley?Hordeum brevisubulatum?which can be commonly found in saline grassland in northern China,was selected as study material,we identified their Epichlo???endophyte,studied biological,physiological,alkaloid biosynthesis characteristics of the endophyte.Further we built genetically comparable endophyte infected?E+?and endophyte free?E-?H.brevisubulatum populations,and studied 1)The effects of the endophyte infection on root,stem,leaf structures.2)Several metabolites?amino acid,total phenols,flavonoid,free fatty acid,total soluble sugar and polyamines?.3)Photosynthesis,physiologic adjustment,ion(sodium?Na⁺?,potassium?K⁺?,calcium(Ca²⁺)),and nutrient absorption?nitrogen?N?,phosphorus?P?and carbon?C??of H.brevisubulatum under salt stress.4)Effects of salt stress on fungal concentration and peramine concentration in basal stem of H.brevisubulatum.5)Effects of the interaction between endophyte infection and exogenous spermidine treatment on salt tolerance of H.brevisubulatum.The main objective of this study was to gain insights as to the mechanism by which the systemic Epichlo???endophyte enhances growth of H.brevisubulatum in high salinity environments,expect to provide theoretical basis and basic data for the use of fungi to salt tolerance varieties innovation and breeding,and for the sustainable development of grassland animal husbandry,ecological conservation.Results showed as follows:1.We acquired 22 endophyte isolates from seeds of Hordeum bogdanii collected from Xinjiang,Inner Mongolia,Ningxia province,these isolates showed different characteristics in culture.Based on tefA,actG and tubB gene sequences,these isolates were identified as Epichlo???bromicola,Epichlo???sp.and a new interspecific hybrid species with three copies,named as Epichlo???sp.HboTG-4?H.bogdanii Taxonomic Group 4:E.bromicola×E.typhina×E.sp.?.These isolates showed the different alkaloid gene profiles.Based on taxonomic status and alkaloid biosynthetic genes?mating-type genes,perA gene,EAS cluster genes,LOL cluster genes and LTM/IDT cluster genes?,these isolates can be divided into 7 different genotypes.The new interspecific hybrid species with three copies represent a mammalian-friendly characteristic that retains the positive benefits of insect pest resistance.2.Epichlo???endophyte isolated from seeds of H.brevisubulatum collected from Gansu province,Linze county,were identified as E.bromicola based on morphological characteristics and tefA and actG gene sequences.These isolates contained peramine biosynthetic gene and chanoclavine I biosynthetic genes.Five out of 11 LTM/IDT cluster genes were present,but no idtG,indicating an inability to produce even early intermediates in the lolitrem B pathway.Only one lolC gene out of 11 LOL cluster genes was present in the three isolates.Chemotype analyses revealed infected H.brevisubulatum contained only peramine,and not ergine,ergonovine,ergovaline or lolitrem B.Biological and physiological characteristics showed that the optimal temperature of WBE1 were 22²5?.The optimal pH ranges for mycelial growth were 5.09⁶.1.The most preferred of WBE1 for carbon source was maltose,D-xylose inhibited the mycelial growth.The most preferred of WBE1 for nitrogen source was casein tryptone,ammonium molybdate also inhibited the mycelial growth.WBE1 grew significantly better on potato dextrose agar?PDA?,oat meal agar?OMA?,corn meal agar?CMA?and potato sugar agar?PSA?than on Czapek medium,and most slowly on water agar?WA?and wild-barley agar?WBA?.3.The results of paraffin section showed that endophyte infection influenced some root,stem and leaf structure parameters.In terms to root structures,endophyte infection alleviated the decrease of cross-sectional area of meta-xylem and thickness of cortex under salt stress.Endophyte infection strengthened the increase of the thickness of the endodermis,while having no effects on the cross-sectional area of stele and thickness of the epidermis under salt stress.In terms to stem structures,endophyte infection alleviated the decrease of down-epidermis,vascular bundle and xylem vessels under salt stress,while having no effects on the cross-sectional area of pith cavity.In terms to leaf structures,endophyte infection alleviated the decrease of leaf thickness,cross-sectional area of xylem and phloem under salt stress.4.The results of qPCR and HPLC showed that salt stress significantly increased the endophyte concentration and peramine concentration in basal stem of H.brevisubulatum,each of them increased gradually with increasing salt stress concentration and stress period.There was a significant positive correlation between fungal concentration and peramine concentration,the correlation coefficient was 0.633.These results showed that the endophyte significantly affected the peramine concentration.5.The presence of endophyte strengthened the increase of tyrosine and proline,alleviated the decrease of threonine,glutamic acid,phenylalanine,histidine,arginine,while having no significant effects on the serine,glycine,alanine,valine,methionine,isoleucine,leucine and lysine.Endophyte infection significantly increased aspartic acid and total amino acid content,but significantly lowered cystine content.Endophyte infection increased the total phenols and total soluble sugar contents in shoots under salt stress,but lowered flavonoid contents both in control and under salt stress,while having no effects on the free fatty acid contents and total soluble sugar contents in roots.6.The presence of endophyte enhanced conversion of putrescine to spermidine and spermine,as well as improved shift ability from free forms and soluble conjugated forms of polyamines to insoluble bound forms of polyamines under salt stress in H.brevisubulatum.7.Exogenous spermidine enlarged the growth parameter difference between E+plants and E-plants under salt stress,promoting or inhibiting the function of endophytic fungi on effects of amino acid content.Exogenous spermidine improved potassium and calcium absorbing ability but lowered sodium absorbing ability in E+and E-H.brevisubulatum.Spermidine enlarged the role of endophyte in the absorption of sodium-potassium-calcium ions under salt stress,strengthened the decrease of Na⁺/K⁺ratio under salt stress with the decreasing trend of E+plants was higher than that of E-plants.Exogenous spermidine enlarged the role of endophyte in increasing carbon,nitrogen and shoot phosphorus absorption,furthermore,modified the nutrient stoichiometry of H.brevisubulatum under salt stress.8.The presence of Epichlo???endophyte infection on H.brevisubulatum under both salt stress?molar ratio of NaCl:Na₂SO₄=1:1?and alkali stress?molar ratio of NaHCO₃:Na₂CO₃=1:1?led to significant amelioration of both stresses.Epichlo???infection significantly increased photosynthesis,chlorophyll content,total antioxidant capacity and glycine betaine content,whilst lowering leaf malondialdehyde content.Furthermore,Epichlo???infection reduced Na⁺content,the Na⁺/K⁺ratio and shoot Ca²⁺content but increased K⁺content and the root Ca²⁺content.Epichlo???infected plants also had higher C,N and P contents but lower ratios of C:N,C:P and N:P than uninfected plants.The possible mechanisms by which the presence of Epichlo???endophyte enhances growth of plants exposed to those two stresses include improved photosynthetic ability,increased antioxidant potential,increased nutrient absorption,and osmotic and ionic adjustment.The study also found that alkali stress is more harmful to wild barley than salt stress.