| Walnut(Juglans regia L.)is the most widely distributed and the largest economic value in the Juglans plants.The nutrition of walnut is extremely beneficial to human health and the walnut was considered as "super food in twenty-first Century".Walnut is also Chinese important "woody grain and oil" strategy tree and had good economic value and social benefit.Studied on the endophyte bacteria species diversity,community composition and the seasonal dynamic variation of dominant population in the walnut root cortex,to understand the potential biocontrol bacteria etc.beneficial microorganisms in walnut vivo.Walnut root rot pathogens bacteria Fusarium solani was as the indicator bacteria to screening antagonistic endophytic bacteria against it and made a study on how the antagonistic bacteria play the role of disease prevention.Then,lay the foundation for the future development of biological pesticides to has a good control effect on the root rot disease.Through the observation of colony morphology,physiological and biochemical tests and molecular biology identification,to research the district analysis and seasons dynamic changes of endophytic bacteria,and preliminarily study on the resistance mechanism of the antagonistic strain inhibited root rot disease.The main research results were following:(1)There were 61 strains isolate from walnut root cortex after 12 times of separation and purification,belonging to 11 genera,29 species.The 16SrDNA sequence and ITS sequence analysis,physiological and biochemical identification results showed that endophytic bacteria strains belonging to bacteria,fungi,actinomycetes.The bacteria were the dominant species,the separation ratio was 95.27%.It included Bacillus,Brevundimonas,Pantoea,Enterobacter,Pseudomonas.the Bacillus and Pseudomonas was the dominant bacteria,isolation rates were 61.31%,25.34%,two of the total proportion was more than 80%;the segregation ratio of fungi was 3.27%,including Psathyrella,Basidiomycota,Aspergillus,Trametes;The segregation ratio was 1.01%and mainly was Streptomyces.It illustrated the richness and diversity of the endophytic microorganisms in walnut root cortex.(2)The endophytic strains quantity analysis in different seasons showed that the endophytic strains quantity exist obvious difference changes in different seasons.The data played that the quantity of endophytic strains gradually decreased and increased with seasonal variation,showing the spring>winter>summer>autumn,endophytic strains number was the highest in spring and winter;The quantity of endophytic bacteria gradually decreased and increased with seasonal variation,showing the spring>winter>summer>autumn,endophytic bacteria number was the highest in spring and winter;The quantity of endophytic actinomycetes gradually decreased with seasonal changes,showing the spring>summer>autumn>winter;the quantity of endophytic fungi was not significantly change with the seasons variation.Overall,the number of endophytic bacteria in spring and winter was more than in summer and autumn.(3)The analysis of the types and isolated proportion of strains in different seasons showed that isolated species in summer and autumn is greater than in winter and spring.In spring,the dominant bacteria was Pseudomonas,isolated rate was 89.51%;In summer,the dominant population were Bacillus and Pseudomonas,the isolated rate were 57.14%,31.75%which total isolation ratio greater than 85%;In autumn,the dominant bacteria was Pseudomonas,the isolated rate was 48.72%,second was Bacillus,the isolated rate was 43.59%;In winter,the dominant species were Pseudomonas,Bacillus and Pantoea,the isolated rate was 47.54%,29.51%and 22.13%respectively.Each season of dominant bacteria were showing consistency changes,the dominant bacteria was Pseudomonas and Bacillus in summer,autumn and winter,indicated that the suitability and colonization ability of Pseudomonas and Bacillusis stronger than other endophytic bacteria in walnut root cortex,and occupy the stability dominant position for a long time.(4)Taking the walnut root rot fungus Fusarium solani as the tested bacteria to determine the antagonistic effect of 61 strains endophytic bacteria.The advantages antagonistic bacteria strain 1A was identified as Bacillus amyloliquefaciens The GenBank accession number was KJ865856.The inhibition rate reach to 72.56%.In addition,strain 1A had a significant inhibitory effect on several forest pathogenic bacteria such as Cryphonectria.The result of antibiosis mechanism indicated strain 1A can produce protease and cellulase to degradate the protein and cellulose composition of fungal cytoderm,damage the Fusarium solani mycelium to inhibite the growth of Fusarium solani.The strain 1A could strongly inhibit the Fusarium solani fungus hypha growth and germination of spores,the spore germination inhibition rate was 74.89%.The mycelium was distortion,fracture,branch increased and winding,deepen color.The antagonistic bacterium strain 1A performed good control potential on Walnut root rot in the semi-field experiment and the pre-treatment of the antagonistic strain 1A had a better biocontrol effect. |
