| Endophytes are fungi or bacteria that living in healthy plant tissues and organs at certain stage or at all stages.Endophytes hve been commonly found in higher plants,which are able to survive and reproduce independently in plants,but are not susceptible to outside environments.Endophytes exist in plants and undergo long-term co-evolution with host plants.Endophytes have the biological functions enhancing the resistance of plants,promoting growth of host plants,fixing nitrogen in non-leguminous plants and being applied as foreign gene carrier and in biological remediation.Recently,a variety of endophytes have been used as potential microbial pesticides and Fertilizers.Endophytes in bryophyte have been sporadically studied these years,but within few groups and fields in comparison with so many bryophyte species and their rich habitats.Brachythecium plumosum,Trachycystis microphylla,Taxiphyllum taxirameum and Fissidnes bryoides are four widely-distributed moss species in Eastern China,with high horticultural values,especially for Brachythecium plumosum.The experiments hot to select best method of surface sterilization for separating and determining endophytes were conducted by using Brachythecium plumosum.Functional bacteria within Brachythecium plumosum,which are able to promote the growth of host plants or control pathogenic bacteria were screened.High-throughput sequencing method was applied to analyze the composition and diversity of endophytes in three populations of Brachythecium plumosum from different habitats,and in three moss species from the same environment,for understanding the influences of environments on moss endophytes,and the differences of endophytes in different moss species,to clarify whether endophyte selectively grow within host mosses.The main results are as follows:(1)The sterilization effects of different methods(ultraviolet irradiation,the concentration of 75% ethanol solution,hydrogen peroxide solution,mercuric chloride solution,sodium hypochlorite solution and two different combinations)on moss plants for endophyte separation and determination were compared.The best method is a combination of 30 s of ethanol + 9 min H2O2 hydrogen peroxide(30%).With this method,moss materials could be sterilized thoroughly and kept alive.(2)The grinded gametophytes of Brachythecium plumosum were coated on medium plate.Abundant endophytes were obtained.These endophytes were further purified.The liquid with endophytes were made by shaking the endophytes into cultural liquid.A series functional experiments were conducted to screen special endophytes which are able to promote the growth of host mosses or inhibit the spread of germs.Bacillus thuringiensis serovar konkukian STR.97-27 was found to promote the growth of the gametophytes of Brachythecium plumosum,while Bacillus pumilus was able to control the spread of some pathogenic germs among the gametophytes of the host species.(3)By using the high-throughput sequencing method,we compared the differences of the endophytes within the gametophytes of Brachythecium plumosum in three different habitats(urban environment,the filed environment,and cultivated environment).We found no obvious differences in the OUT of endophytes within the gametophytes from three different environments.The main endophytes of Brachythecium plumosum included Proteobacteria,Actinobacteria,Pseudonocardia,Comamonadaceae,Micromonospra,Oxalobacteraceae.Based on the abundance of endophytes,the samples of Brachythecium plumosum could be divided into three groups,but no correspondence with their original environmental sourves.Thefore,the endophytes within the gametophytes of B.plumosum are not significantly influenced by their environments.We also found that the samples from wild environments have the endophytes with strong functional signs,while those from urban environments with weak functional signs.(4)The composition and diversity of endophytes in three moss species(Trachycystis microphylla,Taxiphyllum taxirameum and Fissidnes bryoides)sampled from samle environment were determined by using high-throughput sequencing method.We found that the main endophytes of these three moss species included Proteobacteria,Actinobacteria,Bacteroidetes,Flarobacterium,Comamonadaceae.Based on the abundance of their endophytes,by using Cluster and Ordination analyes,we found a distinct difference in endophyte composition among these three moss species.For Taxiphyllum taxirameum,the abundance of Mycobacterium,Micromonospora,Actinoplanes,Bacillus and Hyphomicrobium was higher,Within the gametophytes of Trachycystis microphylla,the abundance of Pedomicrobium,Pirellula,and Comamonas,were higher;within the gametophytes of Fissidnes bryoides,the endophytes such as Aureimonas and Spirosoma have relatively high abundance.The main metabolisms detected in the endophytes of these three moss species were exogenous material degradation and metabolism,nucleotide metabolism,terpenoids and polyketides metabolism,amino acid metabolism,vitamin metabolism,lipid metabolism,polysaccharide biosynthesis metabolism,enzyme family,energy metabolism,carbohydrate metabolism and other secondary metabolites biosynthesis and metabolism of amino acid products.For Taxiphyllum taxirameum,its endophtyes were of strong exogenous material degradation and metabolism,energy metabolism,carbohydrate metabolism function,and other secondary metabolites biosynthesis;For Fissidnes bryoides and Trachycystis microphylla,their endophytes were of high energy metabolism function.Therefore,there was significant differences in the diversity and function of endophytes among these three moss species,and the endophytes within the gametophytes of mosses have certain selectivity to their host plants.Our study also found some important functions of moss endophytes.For example,in the endophytic bacteria of the above four species of moss,we found a high metabolic activity associated with ATP binding protein in the antibacterial transport system,To some extent,it is reasonable that most mosses are antibacterial. |
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