Study On The Physiological And Biochemical Regulation Of The Endophytic Fungi On Casuarina Equisetifolia L. Under NaCl Stress

Casuarina equisitifolia L.,is a major species for protection forest on the southeast coast of China.However,in recent years,the salinization of coastal soils has gradually increased,which results in serious salt problems in the operation of protective forests.For understanding the relationship between the growth of C.equisetifolia and salt,and finding ways to alleviate the damage caused by salt stress effectively.We isolated and identified the endophyte fungus from C.equisetifolia,and chosen the fungi with certain growth-promoting and salt-tolerant strains.And the C.equisetifolia was infected by endophytic fungi.Then we studied the effects of NaCl stress(0,5%,10%,15%)on active oxygen scavenging system,osmotic adjustment,endogenous hormone,growth,biomass,and nutrient of infected C.equisetifolia(EI:EI-1 and EI-2)and C.equisetiolia with uninfected(EF).Meanwhile,transcriptomes of EF and EI under different salt stress concentrations were sequenced and differentially expressed genes were screened,and GO function and KEGG metabolic pathways of differentially expressed genes were analyzed.1.Isolation,purification,molecular identificatin and growth promotion of endophytic fungi from Casuarina equisetifolia LA total of 39 strains of endophytic fungi from C.equisetifolia were isolated and purified.DNA molecular identification showed that 27 strains were from 9 different genera,and Diaporthales were the most(6 strains);among the other 12 strains,2 strains were identified into the class(Dothideomycetes);lstrain identified as Ascomycota;4strains identified as Botryosphaericeae,and 5 strains were not identified.Salt tolerance test showed that 23 strains had better salt tolerance.The above 23 strains were tested biologically by the seedling growth.The biomass,growth and nutrient content of the whole plant after 30 days were determined.The results showed that 23 strains had different effects on seedling growth.Six strains(Z1,Y6,Z7,J10,J12,and J15)with better growth-promoting ability were selected according to biomass and growth.The nutrient content studies showed that the C content of J15 and G3 was significantly greater than Z1,and there was no significant difference in C content among other treatments;the N content of Y16,Y10,and J11 was significantly greater than Y5 and Y7,and there was no significant difference among the other treatments;J13 The content of P was significantly higher than all other treatments;there was no significant difference in K content between different strains;the content of Na in Y1 was significantly higher than al of other strains,while the content of Na in J10 was significantly lower than that of Y5,Y6,Y7,Y9,Y14,Y16,and Z1.The Ca content of Z9,Z7,J6,and G1;the Ca content of Y9 was the largest and significantly greater than that of Y1,Y7,Z1,Z7,J10,and G1;the Mg content of Y10,Z7,J13,and G3 was higher and significantly larger than Y6,Y7,and G1.In summary,endophytic fungi can affect host growth and nutrient content.From the point of plant growth and salt tolerance,Z1 and Y6 strain with good salt tolerance,for follow-up experiments.2.Effects of endophytic fungi on antioxidant system and membrane lipid peroxidation of Casuarina equisetifolia L.under NaCl StressWith the increase ofNaCl stress concentration,the content of hydro gen peroxide(H2O2)in EF decreased significantly,and the content of malondialdehyde(MDA)increased significantly.Superoxide dismutase(SOD),catalase(CAT)and peroxide on the activity of POD increased first and then decreased,while the contents of H2O2 and MDA in EI(EI-1 and EI-2)increased significantly.The activity of SOD,CAT,and POD increased first and then decreased.With the prolongation of stress time,H2O2 of EF and EI both increased first and then decreased significantly,and MDA content increased significantly.The activities of SOD,CAT,and POD increased first and then decreased.When EF and El were comprehensively compared,the H2O2 of EI was significantly lower than that of EF under mild stress(5%)and short-term(0 and 20 d)stress,and EI-1 in EI was significantly lower than that of EI-2.SOD,CAT,POD activity was significantly higher than that of EF,and EI-1 was significantly higher than EI-2;this indicated that under mild and short-term stress,the protective enzyme system of EF and EI was effective for the clearance of reactive oxygen species and the invasion of endophytic fungi.Staining may increase the activity of protective enzymes and remove more reactive oxygen species,leading to lower ROS of EI than EF,and the effects of the two endophytic fungi are significantly different;medium height(10%and 15%)and long-term(40d and 60d)Under stress,H2O2 of EI was significantly higher than that of EF,and EI-2 was significantly higher than EI-l,but the protective enzyme activity of EI was also significantly higher than that of EF,and the activity of POD and CAT of EI-1 was significantly higher than that of EI.2.The SOD was significantly lower than that of EI-2,indicating that although the ROS of El increased under medium altitude stress,the protective enzyme activity was also significantly greater than that of EF.It was speculated that endophytic fungi could affect the protective enzymes of Casuarina equisetifolia under NaCl stress.The system improves its resistance,and different endophytic fungi have different effects on the active oxygen metabolism and the protective enzyme system of the host.3.Effects of Endophytic Fungi on Osmotic Adjustment and Endogenous Hormone Content of Casuarina equisetifolia L.Seedlings Under NaC l StressWith the increase of NaCl stress concentration,the pro line(PRO)content of EF increased gradually,while EI increased first and then decreased.The conductivities of EF and EI increased first and then decreased.With the prolongation of stress time,the PRO content of EF and EI increased.Conductivity of EF and EI increase first and then decrease.Comparing the PRO and the conductivity of EF and EI,the PRO content of EI was significantly greater than that of EF at 0 stress concentration,and EI-2 was significantly greater than EI-1 in EI,whereas the conductivity of EI was significantly smaller than that of EF,and There was no significant difference between-1 and EI-2;the PRO content of EI was significantly less than EF at the other stress concentrations(5%,10%,15%),and EI-2 was significantly greater than EI-1,whereas the conductance of EI was not significant.The rate is significantly greater than that of EF,and EI-1 is significantly greater than EI-2,Whichindicating that NaCl stress may increase the permeability of the host protoplasmic membrane resulting in increased PRO content and conductivity,but PRO of El after endophytic infection,the low content indicates that endophytic fungi may protect the cell membrane to a certain extent and reduce the accumulation of pro line.With the increase of stress concentration,the auxin(IAA),zeaxanthin(ZR)and gibberellin 3(GA3)all showed a downward trend,while EI showed a trend of first increase and then decrease;EF and EI of abscisic acid(ABA)showed a trend of increasing volatility.With the increase of stress time,IAA,ZR,GA3 of EF decreased significantly,but ABA increased significantly;IAA of EI increased first and then decreased,ZR and GA3 decreased significantly,and ABA content increased significantly.Compared with EF and EI,the IAA of EI was significantly greater than EF at four different stress concentrations,and when the concentration of stress was 10%and 15%,EI-1 was significantly smaller than EI-2;when light stress,the ZR and GA3 content of EI was smaller than that of EF,but the difference was not significant.At medium and high stress,the ZR and GA3 of El were greater than EF but the difference was not significant.At each concentration of stress,the ABA of EI was Significantly less than EF,and under mild and moderate(5%and 10%)stress,ABA of EI-2 was significantly greater than EI-1,while EI-2 was significantly lower than EI at high stress.1.It can be seen that under NaCl stress,IAA,ZR,and GA3 of Casuarina equisetifolia decreased while ABA increased,and plant growth was inhibited.However,EI plants exhibited increased resistance to IAA and reduced ABA,which may be related to the infestation of endophytic fungi4.Effects of Endophytic Fungi on Growth,Biomass and Nutrient Content of Casuarina equisetifolia L.Seedlings under NaCl StressThere were significant differences in the growth of EF and EI under NaCl stress.Among them,under mild stress,the seedling height of EI-1 was significantly greater than that of EF and EI-2,and the caliber of EI was significantly greater than that of EF,but there was no significant difference between EI-1 and EI-2;Under the stress of and height,there was no significant difference in seedling height and ground diameter between EI and EF.Under light stress,there was no significant difference in fresh weight of dry weight between aboveground and underground parts of EI and EF.Under moderate and high stress,the aboveground weight of EI was significantly greater than that of EF.However,there is no significant difference between EI-1 and EI-2.The root-shoot ratio of EI was lower than that of EF under four stress concentrations.Under mild stress,the ratio of EI-1 root to shoot greater than that of EI-2,while that of medium and high altitude was less than EI-2,indicating that endophytic fungi infested.After the host was protected from NaCl stress and maintained normal growth in the aerial parts,and different endophytic fungi had different effects.Nutrient studies showed that under light stress,the C,N,and Mg contents of EI were significantly greater than those of EF,and the C content of EI-1 was significantly smaller than that of EI-2,while the Mg content of EI-1 was significantly greater than that of EI-2.There was no significant difference in N content;P,K and Na contents of EI were lower than EF but not significant.Under high stress,the contents of C,N,K,Ca,and Mg in EI were all higher than those in EF,but not significant,while the P content in EI was significantly lower than that in EF.In the underground part,the N of EI was significantly lower than that of EF under mild stress,but there was no significant difference between EI-1 and EI-2.The content of P,K,Ca and Mg of EI was lower than that of EF,but only Mg was significant;under concentration stress,the contents of C,N,P,and K in EI were lower than EF but not significant.The content ofNa in EI was significantly higher than that of EF,but the content of Ca and Mg in EI was significantly lower than that of EF.In conclusion,endophytic fungi can reduce the absorption of Na by roots and increase the content of C,N,and P under mild stress.However,under high stress,the absorption of Na by roots increases,which in turn reduces the content of Ca and Mg and ultimately affects the plant.The growth shows that the endophytic fungi may only work within a certain NaCl stress range.5.Transcriptome analysis of Casuarina equisetifolia L.seedling under NaCl stressThe transcriptomes of EF and EI were sequenced,and there were 25,631 genes annotated by 7 major databases.The differentially expressed genes of EF and EI were different under different salt stress concentrations.The three different salt stress concentrations of EF were 545,1117,and 795 compared with CK;the EI-1 were 599,243,respectively.And EI-2 were 1490,were 334,492 and 622 respectively.Analysis of differentially expressed genes at the same concentration of EF and EI revealed that there was a large number of overlapping genes between EF and EI,which indicated that there was a common response mechanism for different treatment Casuarina equisetifolia L.against salt stress.The GO function enrichment analysis of the differentially expressed genes revealed that the EF and EI concentrations were significantly higher than those of CK,and the expression of differential genes also included up-regulation and down-regulation.The differentially expressed genes of KEGG are also significantly enriched at different concentrations.Under the three treatments,the expression pathways of differentially expressed genes in KEGG were different under different stress concentrations.EF is mainly reflected in the flavonoid biosynthesis pathway and the dialdehyde and dicarboxylic acid metabolic pathways.EI-1 mainly reflected in the plant circadian rhythm pathway and flavonoid synthesis.Casuarina EI-2 is mainly reflected in the tryptophan metabolism pathway,plant circadian rhythm pathway and starch sucrose metabolism pathway.Comprehensive analysis of the three differentially expressed genes revealed that the biosynthesis pathway of flavonoids and the circadian rhythm of plants were all present,which indicated the important role of flavonoid biosynthesis in response to salt stress in Casuarina equisetifolia L.