| Shiraia bambusicola P.Hennings is a pathogenic fungus that parasitizes on the branches of bamboos(Brachystachyum).The infestation will lead to the decline of bamboo forest by the brown spot disease.The Shiraia fruiting body has been a traditional Chinese herbal medicine(Zhu Huang).It can treat rheumatoid arthritis,tracheitis,toothache,sciatica,lumbar muscle strain and muscle soreness.Hypocrellins are main active substances of S.bambusicola,which have anti-inflammatory,anti-virus,anti-tumor and anti-bacterial effects due to their photosensitive properties.Hypocrellins have been widely used in clinic for photodynamic therapy.The main source of hypocrellins is still the fruiting body of S.bambusicola.There are a large number of bacterial communities in the fruiting bodies with unknown structure and function.Among them,Bacillus is an important microbial resource with important biological activity.In this study,we isolated and purified the bacteria associated with Shiraia fruiting body by means of pure culture,analyzed the diversity of the associated Bacillus bacteria by 16S rDNA sequence alignment,and screened a strain of B.cereus No.1 with a capacity to influence on hypocrellin content.The whole genome of this bacterium was analyzed.Through co-culture,the effects of strain No.1 on the growth and hypocrellin biosynthesis of Shirai sp.S9 and its mechanism were further explored,and the application of the active strain in biological control was preliminarily practiced.The main results are as follows:1.Firstly,31 strains of associated bacteria were isolated and purified from the fruiting body of Shirai sp.S9 by pure culture.By 16S rDNA sequence alignment analysis,15 strains of associated bacteria belonged to Bacillus spp.Further analysis on the diversity of associated Bacillus spp.showed that 10 strains belonged to B cereus,4 strains belonged to B.subtilis and 1 strain belonged to B.safensis.The antagonistic experiment of bacteria fungi co-culture on the plate showed that 15 strains of Bacillus had significant inhibitory effect on hypocrellins accumulation.B.cereus No.1 showed very significant inhibitory activity.Therefore,we further explored the relationship between this associated bacterium and the synthesis of hypocrellins.2.We used the second-generation Illumina miseq platform and the third-generation Promethion platform sequencing technology to sequence the whole genome of B.cereus No.1 strain from the fruiting body.Based on the genome of strain No.1,the genome function was annotated by using COG database,GO database,KEGG database and CAZy database.Six secondary metabolite gene clusters of strain No.1 were predicted by using antismash 5.0 online website,which encoded bacteriocin,siderophores,non-ribosomal peptides(NRPs)and betalactone respectively.These results indicate that the bacteria may compete with the outside world to obtain iron ions through iron carriers or exert their biological activity through bacteriocin mediation.3.Then,we studied on the effect of strain No.1 on the growth and active metabolites of host Shirai.Firstly,strain No.1 inhibited the fungal growth and the hypocrellin production.The treatment of living bacterium changed the mycelium to be thick and loose.After the treatment the conidia shrunk and the number of spores increased.No.1 strain had the most significant inhibitory effect on the production of hypocrellin within 24 h,while the yield of hypocrellin was suppreseed to 0.02±0.01 mg/g.In addition,No.1 bacteria promoted the laccase production.The laccase production reached 6475±214 U/L on the 8th day,about 1.44 times of the control group.No.1 strain significantly inhibited the yield of exopolysaccharide,and the yield of polysaccharide in treatment group was 13.3±1.14 mg/L only.4.In order to explore the mechanism of No.1 strain on hypocrellin biosynthesis,we compared the inhibitory effects of the living bacteria,bacterial polysaccharide(BPS),ethyl acetate extract(EAE)of bacterial fermentation broth and non-contact with living bacteria.It was found that the inbitory effect of living bacteria was the most obvious,followed by BPS and EAE,and the non-contacts.Furthermore,7 genes related to HA synthesis and transport were all down-regulated by strain No.1.The expression of multicopper oxidase(MCO)was down-regulaed by 6.6 times.In addition,the expreesion of polyketide synthase(PKS),O-methyltransferase(Omef),monooxygenase(Mono),FAD/FMN-containing dehydrogenase(FAD),Zinc finger transcription factor(Zftf)and ATP-binding cassette(ABC)genes were down-regulated by 2.0,3.7,2.2,1.5,5.3 and 3.7 fold,respectively.In addition,it was found that strain No.1 could reduce the carbonyl group of HA to hydroxyl group and bioconvert it into HAH2 by LC-MS/MS analysis.The results showed that strain No.1 inhibited the synthesis of hypocrellin by living bacterium contact,down-regulated the genes related to the synthesis of hypocrellin and degradated extracellular hypocrellins.5.The antagonistic effect of strain No.1 to pathogenic fungi and its biocontrol effect on bamboo leaf infection were investigated preliminarily,and the production of extracellular enzymes of strain No.1 was detected.The results showed that strain No.1 had obvious inhibitory on the pathogenic fungi,and the inhibition zones against Shirai sp.S9 and Alternaria sp.were 9.27 mm and 16.40 mm,respectively.We found that strain No.1 could produce protease and cellulase,and the diameters of their hydrolysis circles were 13.53 ± 0.59 mm and 9.30 ± 0.53 mm,respectively.The infection experiment of bamboo leaves showed that strain No.1 had inhibitory on the infection of bamboo leaves.In this study,a strain of B.cereus No.1 was screened from the Shirai fruiting body by pure culture to inhibit the biosynthesis of hypocrellin.The diversity of the genus Bacillus associated with host Shirai was analyzed and the whole genome sequence of strain No.1 was analyzed.The functional annotation of the coding gene was elucidated.The secondary metabolites of strain No.1 were predicted by antismash 5.0 online website.The effect of No.1 on the growth and hypocrellin synthesis of host Shirai and the mechanism was further studied in the co-culture.The application in biological control was preliminarily practiced.Our study is helpful to for understanding the relationship between fruit-body associated bacteria and host fungi,and for the exploration on the regulation of biosynthetic pathway of hypocrellin.Also,we wish our study could provid the basis for the biological control of pathogen Shirai of bamboos and other crop plants. |
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