Colonization Of Endophytes In Casuarina Equisetifolia And The Transcriptomic And Metabolomic Analyses Based On Allelopathy

Casuarina equisetifolia is an excellent tree species for coastal windbreak and solidification.It was widely planted in the coastal zone around Hainan island in the 1980s and 1990s.With the growth of the forest age,the problem of difficulty in self-renewal appears in the C.equisetifolia forest.The main reason is the accumulation of allelochemicals.Our previous studies have shown that the soil microorganisms,roots and litter endophytes of C.equisetifolia may cause allelopathy of C.equisetifolia.Therefore,we selected four endophytes with strong allelopathic potential to infect the sterile seedlings of C.equisetifolia,analyzed the colonization and infection pathways of the interaction between the endophytes and seedlings by GFP fluorescence labeling and laser confocal microscopy;explored changes in gene expression related to allelochemicals synthesis in C.equisetifolia after endophyte infection by transcriptome technology;analyzed metabolome of changes in metabolites related to synthesis of allelopathic substances;and discussed the molecular basis of the interaction between C.equisetifolia and endophytes by transcriptome and metabonomic association analysis.The main research results were as follows:1.The colonization of endophytes was observed by using GFP labeling and laser confocal.When two GFP-labeled bacteria and fungi infect the seedlings of C.equisetifolia,the bacteria mainly colonized the branchlet stoma guard cells,and the fungus colonized a large number of root hairs,the overall colonization of bacterial strains was stronger than that of fungi.2.The transcriptome technology was used to study the gene expression profile changes between endophytes infecting C.equisetifolia seedlings and sterile seedlings.The results showed that after endophytes infection,there were 1016 significantly differentially expressed genes in C.equisetifolia seedlings at the transcriptional level,including 303 up-regulated genes and 713 down-regulated genes;analysis by GO and KEGG showd that there were 8up-regulated genes and 8 down-regulated genes related to the synthesis of allelochemicals.The down-regulated gene allene oxide synthase gene AOS was related to the process of linolenic acid metabolism,and was involved in the synthesis of methyl stearate and methyl palmitate;up-regulated gene 3,9-dihydroxypteranodon 6a-monooxy the gene-like CYP17 A and the down-regulated gene β-vanillin 28-monooxygenase gene CYP716 A were involved in the biosynthesis of phenylpropanoid,and affect the synthesis of 2,4 di-tert-butylphenol,an allelochemical substance through the shikimic acid pathway.In addition,there were differential genes related to photosynthesis,porphyrin and chlorophyll and glutathione metabolic pathways.3.Metabonomics technology was used to study the metabolic spectrum changes between endophytic bacteria infecting C.equisetifolia seedlings and sterile seedlings.The results showed that after endophyte infection,the seedlings of C.equisetifolia haved 29 differential metabolites at the metabolic level,of which 13 were up-regulated differential metabolites and16 were down-regulated differential metabolites;KEGG analysis showed that there were 2up-regulated metabolites and 3 down-regulated metabolites related to the synthesis of allelochemicals.Up-regulate the metabolites ionone and pipecolic acid involved in the biosynthesis of secondary metabolites,down-regulate the metabolites xanthine,thymol,ferulic acid and phenylpropanoid biosynthesis,participated in the terpenoid and polyketone metabolism process.In addition,most of the differential metabolites were mainly involved in the metabolism of other secondary metabolites,other amino acid metabolism and other metabolic pathways.4.Transcriptome differential genes and metabolome differential metabolites jointly participate in 26 pathways,mainly through the shikimic acid pathway in phenylpropanoid biosynthesis,which affects the allelopathy 2,4 di-tert-butylphenol biosynthesis,and when endophytes interact with C.equisetifolia,endophytes participate in plant physiological and biochemical activities to varying degrees.In summary,the research results show that the differential genes and differential metabolites related to the synthesis of allelochemicals under the infection of a variety of endophytes have significant changes,such as the pentose phosphate pathway,phenylpropanoid biosynthesis,shikimic acid pathway,porphyrin and chlorophyll metabolism are significantly enriched,it was suggested that the endophytes were involved in the synthesis of allelopathic substances through the above metabolic pathway,and the seedling’s photosynthesis might be affected by the differential expression of porphyrin and chlorophyll synthesis.This study provides a theoretical basis for the involvement of endophytes of C.equisetifolia in allelopathy,it also provides theoretical guidance for the subsequent use of engineered strains or microbial preparations to help renew and transform the C.equisetifolia forest.Innovation: For the first time,the synthetic community method was used to simulate the natural state,and to study the interaction between the C.equisetifolia and a variety of endophytes.