| Mulberry fruit sclerotiniosis,also known as white fruit disease,is a major fungal disease of mulberry fruit.The disease is divided into three types according to the phytopathogens and the symptoms that appear on the infected fruits,mulberry sorosus hypertrophic sclerote disease,mulberry sorosus parvulling sclerote disease,and mulberry sorosus diminuting sclerote disease.The infection cycle of sclerotiniosis,which caused by different pathogens,is basically similar,and it has occurred in many mulberry fields in Chongqing,Sichuan,Zhejiang.Once the fruits are infected,they will lose the commercial value and edibleness,which bring great economic losses to the production of mulberry fruits.It is difficult to control the sclerotiniosis disease because of its fast transmission speed,wide spread range,and high degree of harm.At present,controlling this disease mainly relies on chemical fungicides,long-term overuse of agrochemicals,however,not only causes environmental pollution,but also threatens food safety.Biological control of mulberry fruit sclerotiniosis through the application with plant endophytes is conducive to the healthy development of the mulberry fruit-related industry,and it also provides a new strategy for the prevention of other mulberry diseases.Our team screened out an endophytic bacteria Bacillus sp.7PJ-16（previously identified as Bacillus tequilensis 7PJ-16）,which isolated from healthy mulberry trees and exhibited good biocontrol potential against mulberry fruit sclerotiniosis.To further explore the biocontrol effects and mechanisms of this strain,we first evaluated the biocontrol potential of Bacillus sp.7PJ-16 through animal safety experiments and infection colonization experiments.On the basis of optimizing the fermentation conditions of the antifungal substance produced by the 7PJ-16 strain,a large number of bacterial suspensions and cell-free supernatant were prepared to detect their effects on the growth of pathogens and soil microecology in mulberry fields,as well as the effects of 7PJ-16 field disease prevention experiment and greenhouse growth promotion assay.Further,based on the whole genomics and interaction transcriptomics of Bacillus sp.7PJ-16,the functional genes that play a biocontrol role were excavated and verified,in combination with the isolation and identification of antifungal substances and the detection of related growth-promoting components,the biocontrol mechanism of Bacillus sp.7PJ-16 were systematically revealed.The main results of this study are as follows:1.Evaluation of the biocontrol potential for endophytic Bacillus sp.7PJ-16Safety is the basic requirement for the biological control of mulberry fruit sclerotiniosis through the application with target strain.In the mouse experiments,all the mice grew well after treated with Bacillus sp.7PJ-16 bacterial cells and cell-free supernatant by two methods of intragastric administration and intramuscular injection.There were no significant differences in body weight changes,blood routine indexes,and organ indexes compared with the control groups.In the silkworm experiments,the mulberry leaves treated with Bacillus sp.7PJ-16 cell suspension and cell-free supernatant did not cause the death of silkworms.The growth of the silkworm in each treatment group and economic indicators of silkworm cocoon,such as total cocoon volume,cocoon layer volume,and cocoon layer rate,were roughly equivalent to those of the control group.The results of safety experiments showed that Bacillus sp.7PJ-16 cell suspension and cell-free supernatant did not affect the normal growth of mice and silkworms,and strain’s safety was high.Effective colonization in plants will be beneficial for biocontrol bacteria to better play the role of disease prevention and growth promotion.Bacillus sp.7PJ-16 has good abilities of biofilm formation and motility,which makes it have extraordinary colonization potential in plants.Bacillus sp.7PJ-16/gfp with no significant difference in biological characteristics from the wild-type 7PJ-16 strain and carrying green fluorescent protein and chloramphenicol resistance markers,was successfully obtained by electroporation plasmid pGFP22 to 7PJ-16.Microscopic observation of mulberry seedlings inoculated with 7PJ-16/gfp showed that after1 day of inoculation,the labeled strain adsorbed and aggregated at the root tip,lateral root,and root hair,appearing in the epidermal region of root infection sites.After 2-7 days of inoculation,the bacterial cells gradually colonized into the cortex and vascular tissues,and appeared dispersed or aggregated in the cells.After 14-16 days of inoculation,the strain that completed colonization of the roots migrated to the stem tissues through the xylem vessel,and successfully colonized the leaf intercellular space and vein after 20 days of inoculation.Furthermore,the population fluctuation of 7PJ-16/gfp in the tissues of mulberry seedlings were quantitatively analyzed on LB medium containing chloramphenicol by dilution coating plate method.Results showed that the population of marked strain had the same dynamic change in all tissues of mulberry seedling,that is,it rose first and then fell and finally became stable.Microscopic observation and re-separation experiments showed that Bacillus sp.7PJ-16 could successfully colonize mulberry root,stem,leaf,and flower（fruit）.In addition,a total of 608 endophytic bacteria were isolated from the stems of winter,spring,and autumn（six seasons）and four fruit mulberry varieties（‘Changguo Sang’,‘Chuan Sang No.7637’,‘Xin Lunjiao’,and‘Hong Guo No.2’）in 2015 and 2016 using the fragmentation technique.The distribution of seasonal bacterial community showed that Bacillus spp.accounted for more than 20%of the endophytic bacteria in each season of the two consecutive years,especially the members of isolates in the winter of 2016（37.21%）.Meanwhile,the population distribution of Bacillus spp.seemed no obvious preference for the host cultivars.The genus Bacillus also belonged to the predominant flora among endophytic bacterial community isolated from different mulberry cultivar.The distribution of endophytic bacteria in healthy mulberry trees shows that Bacillus spp.can stably exist in different mulberry cultivars for a long time with high abundance.2.A study on the disease prevention and growth promotion effects of Bacillus sp.7PJ-16To provide sufficient experimental materials for subsequent indoor antifungi and outdoor field biocontrol,single factor experiment and orthogonal experiment were used to optimize the fermentation conditions of the antifungal substance produced by the 7PJ-16 strain.Results indicated that the optimal fermentation conditions were peptone（0.5%）,ammonium sulfate（0.75%）,maltose（3%）,and Na2HPO4（0.3%）,temperature 30°C,initial pH 7.5,inoculation volume 4%,the bottling volume 30%,and fermentation time 120 h.The optimized fermentation medium and culture conditions were conducive to the production of bioactive substances.Bacillus sp.7PJ-16 was fermented in an optimized medium,the cell suspension and cell-free supernatant were harvested,and their effects on the growth of phytopathogens and soil microecology were examined.Results showed that 7PJ-16 cell suspension and cell-free supernatant can significantly inhibit sclerotia germination,mycelial growth,fruiting body growth and development,and spore viability.Compared with water control,the proportion of Actinobacteria in the mulberry field soil after inoculated with biocontrol bacteria was significantly increased,and the abundance of Bacillus in the phylum Firmicutes was also increased,and the results ofβdiversity analysis showed that the soil flora structure after inoculated with biocontrol bacteria was more stable.In field studies of two consecutive years,different concentrations of bacterial suspensions and different dilutions of cell-free supernatant can control the mulberry fruit sclerotiniosis at different levels,and applying7PJ-16 cell suspension of 1.0×109 CFU/mL three times not only resulted in the highest biocontrol efficacy（90.84%）,performing better than that of the fungicide（90.52%）,but also accelerated the ripening of mulberry fruits.In greenhouse experiments,different concentrations of bacterial suspensions and different dilutions of cell-free supernatant had a great effect on the germination of mulberry seeds and the growth of mulberry seedling,and the cell suspension（1.0×106 CFU/mL）and the cell-free supernatant（100-fold dilution）significantly stimulated mulberry seed germination and promoted mulberry seedling growth.3.Molecular mechanism of the disease prevention and growth promotion effects of Bacillus sp.7PJ-16The results of Bacillus sp.7PJ-16 whole genome sequencing analysis showed that7PJ-16 genome was comprised of two circular plasmids and a 4.2M circular chromosome.The biocontrol strain was reconfirmed as Bacillus subtilis（previously identified as B.tequilensis）by analyzing the phylogenetic tree of related species.Gene function annotation and prediction of secondary metabolites showed that 7PJ-16 genome contains 6 gene clusters related to the biosynthesis of antifungal substances and 19 genes related to the plant growth-promoting substances.These genes are related to the biosynthesis of biocontrol-related metabolites such as surfactin,fengycin,siderophores,IAA,and cellulases.Moreover,21 genes related to the infestation and colonization process（biofilm formation,motility）were also found in the genome of the strain.To further dig biocontrol-related genes and clarify the interaction process between B.subtilis 7PJ-16 strain and sclerotinia pathogens（S.sclerotiorum PZ-2,Scleromitrula shiraiana SXSG-5）,this study used transcriptome sequencing technology to conduct prokaryotic transcriptome for the antagonistic bacteria and eukaryotic transcriptome for the pathogenic fungi,respectively.Through analysis of prokaryotic transcriptome data,it was found that the growth of pathogenic fungi led to the down-regulation of genes related to flagella assembly and chemotaxis of the antagonistic bacteria,but it induced the up-regulation of genes related to the synthesis of various biocontrol metabolites such as fengycin,siderophores,and bacilysin.In the eukaryotic transcriptomes,after antagonizing bacterial biological stress,most of the genes related to antioxidant enzymes of pathogenic fungi were up-regulated,and the genes related to pathogenicity,such as the synthesis of cell wall components（e.g.,chitin）,metabolism of plant cell wall degrading enzymes（e.g.,pectinase and cellulase）,and melanin synthesis,were down-regulated expression.Based on the information of biocontrol-related genes detected from 7PJ-16 genome and transcriptome,this study successfully screened and cloned 13 key biocontrol-related genes from the strain genome,including 5 genes related with surfactin synthesis（srfAA、srfAB、srfAC、srfAD、sfp）,7 genes related with bacilysin synthesis（bacA、bacB、bacC、bacD、bacE、bacF、bacG）,and 1gene related with fengycin synthesis（fenE）.Two biocontrol-related genes,fenE and bacG,were successfully knocked out based on the homologous recombination technique,stable knockout strains?FenE and?BacG were obtained,and their abilities of biofilm formation and antifungal activity were reduced to varying degrees.4.Study on biocontrol metabolites of endophytic Bacillus sp.7PJ-16Combining the analysis and functional verification of biocontrol related genes,to further reveal the possible biocontrol metabolites produced by Bacillus sp.7PJ-16,the physical and chemical properties of bioactive cell-free supernatant was firstly tested.Results showed that the supernatant had better ability to resist high temperature and pH,and it was not sensitive toα-chymotrypsin and trypsin,but sensitive to pepsin and proteinase K.Based on the physicochemical properties of active metabolites,purification strategy was designed,and six small molecule substances,including dipeptide cycle（Pro-Phe）,were isolated from 7PJ-16fermentation broth with antifungal activity through many substance separation techniques（such as extraction,silica gel column chromatography,thin-layer chromatography,and high-performance liquid chromatography）,in combination with antifungal activity tracking approach.Furthermore,by a combination of acid precipitation and methanol extraction,a crude lipopeptide with strong antifungal activity was successfully obtained from the 7PJ-16cell-free supernatant.After semi-preparative separation by HPLC and mass spectrometry identification,it was found to contain two kinds of lipopeptide antibiotics,surfactin and fengycin,which was consistent with the results of the functional gene analysis.In addition,7PJ-16 strain can also produce siderophores and plant exogenous hormones such as indole acetic acid and gibberellin.Collectively,this study clarified that the antagonism and growth promotion are the main biocontrol mechanisms of B.subtilis 7PJ-16 against mulberry fruit sclerotiniosis from the aspects of genome,transcriptome,gene function verification,and metabolites.The target strain has high safety performance and extraordinary colonization ability.It not only inhibited the phytopathogens growth and further successfully prevented the occurrence of mulberry fruit sclerotiniosis in the field by producing a variety of antibiotics such as surfactin,fengycin,and dipeptide small molecules（eg.,cyclo-dipeptides and bacilysin）,but also promoted the germination of mulberry and the growth of mulberry seedlings by producing siderophore,indole acetic acid,and gibberellin,thereby enhancing the resistance of the host system and indirectly reducing the occurrence of disease.Our work will lay a theoretical basis and experimental basis for the biological control of mulberry fruit sclerotiniosis,and also provide an important reference for the biological control of other plant diseases. |
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