Isolation And Identification Of An Antagonistic Endophyte From Mulberry And Preliminary Exploration Its Biocontrol Mechanism Against Boeremia Exigua

Boeremia exigua is a major pathogenic fungus that damages the shoots and leaves of mulberry(Morus L.).The disease is widespread and serious.After its onset,it leads to a large quantity of yield reduction of mulberry leaves,which cause great economic loss to mulberry farmers.Physical and chemical controls are the main control methods of B.exigua.However,physical control cost is high and the effect is limited.The degradation cycle of chemical control pesticide is long,which pollutes the environment.However,there are few reports on biological control.This study from stems of the resistant variety-Nongsang NO.8,isolating and screening of mulberry endophytes antagonistic pathogenic fungi with biocontrol potential against B.exigua.The antagonistic bacterium was identified through morphological features,cultural,physiological and biochemical characteristics.The biocontrol potential of antagonistic bacteria was evaluated by safety test,colonization test and biological control experiment of potted culture.Further,the whole genome and interaction transcriptome analysis of antagonistic strains were conducted to dig and clone the functional genes that exert biocontrol effect.Combined with the isolation and identification results of the main antibacterial active substances,the biocontrol mechanism of endophytic antagonists was preliminarily revealed.The main results of this study are as follows:1.Isolation,screening and identification of antagonistic endophytes from mulberryA total of 33 endophytes isolateed were obtained from the healthy stem of the mulberry cultivar Nongsang NO.8 by tissue culture method.B.exigua GXH1 was the indicator strain,an endophytic strain NPJ13 with the highest antibacterial rate was screened by the method of bacteriostatic zone and confrontation culture.The morphological,physiological and biochemical characteristics of NPJ13 strain were consistent with those of Bacillus.Based on the phylogenetic analysis of 16 S rDNA,gyrA and gyrB genes and single copy core genes of the whole genome,the strain NPJ13 was identified as Bacillus velezensis and named B.velezensis NPJ13 because it was the closest relative to Bacillus velezensis and was located in the smallest branch of the phylogenetic tree.The entire genome of B.velezensis NPJ13 consists of a single ring chromosome with a size of 4,021,183 bp.The results of prediction of secondary metabolites and gene functional annotation showed that they contained gene clusters related to the synthesis of bacteriostatic substances such as surfactants and bacteriocins.2.Biocontrol potential assessment of antagonistic bacteriaIt is necessary to ensure its safety to use the target strain for biological control.B.Velezensis NPJ13 suspension treated mulberry leaves did not cause adverse reactions to silkworms.There was no significant difference in the growth and development of silkworm,the weight of cocoon layer and other economic indexes between the treatment group and the control group.These results showed that B.velezensis NPJ13 had high biosafety.The mulberry seedlings inoculated with NPJ13/gfp strain were observed by microscopic observation.It was found that the labeled strains appeared successively in root,stem,leaf of mulberry.The fluctuation of NPJ13/gfp in various tissues and organs of mulberry seedlings was quantitatively analyzed by dilution coating method.These results showed that the colonization rules of labeled strains in roots,stems and leaves were consistent,that is,the first rise,then decline and finally stabilized.Under greenhouse conditions,the biological control effect of NPJ13 against B.exigua in pot was detected.These results showed that the antagonistic bacteria had no obvious negative effect on the growth of mulberry seedlings,and the control effect of the antagonistic bacteria on mulberry leaf rot was more than 50%.3.Biological control mechanism of antagonistic bacteriaTo explore the biocontrol mechanism of B.velezensis NPJ13,transcriptome sequencing technology was used.The eukaryotic transcriptome analysis was performed on the leaves treated with antagonistic bacteria and pathogenic fungi respectively.Moreover,prokaryotic and eukaryotic transcriptome analyses were conducted for biocontrol bacteria and pathogenic fungi during co-culture.The transcriptome data of mulberry leaves showed that antagonistic bacteria could induce up-regulation of genes related to the synthesis of diterpenoids and flavonoids in mulberry leaves,and enhance resistance to pathogen invasion.The transcriptome data of antagonistic bacteria showed that the pathogen would stimulate the significantly up-regulated expression of genes related to nitrogen metabolism of antagonistic bacteria to inhibit the growth of the pathogen.Based on the analysis of genomic information of strain NPJ13,eleven key biocontrol genes were screened and cloned from the genome of NPJ13 strain.Among them,there were three genes related to bacilysin synthesis(bacB,bacD,bacE),two genes related to surfactant synthesis(srfAD,sfp),three genes related to nitrate reductase(narX,narH,narG)and two genes related to siderophore synthesis and transport(dhbA,feuC).In combination with the biocontrol gene analysis and the thermal stability of the fermentation broth with antibacterial activity of B.velezensis NPJ13,a more pure small molecule was isolated by means of extraction and evaporation silica gel column chromatography and HPLC.Surfactant and iturin were detected by LC-MS from the metabolized crude extracts of B.velezensis NPJ13.In summary,a potential biocontrol strain of B.velezensis NPJ13 was obtained from healthy mulberry.The strain has good biocontrol effect,strong colonization ability and high safety.It can inhibit the growth of B.exigua by regulating nitrogen metabolism,producing surfactant,iturin and other small molecular substances.B.velezensis NPJ13 could induce the systemic resistance of mulberry host by inducing the production of sesquiterpenoids and flavonoids in mulberry leaves,and indirectly reduce the occurrence of mulberry diseases.This study provided a candidate strain for the biological control of B.exigua,laid a theoretical and experimental foundation for the use of this biocontrol strain,and also provided an important reference for the biological control of other plant diseases.