| Soft rot caused by Rhizopus stolonifer is one of the main causes of postharvest loss of sweet cherry(Cerasus avium(L.)Moench.).Due to the disadvantages of fungicide residues,environmental pollution and pathogen resistance of traditional chemical control,the research on biocontrol methods become hot topic in postharvest of fruit and vegetable.In this study,the change characteristics of the microbial community on the surface of sweet cherry under different storage conditions were identified.The decay-causing fungi from sweet cherry fruits were isolated and identified and their pathogenicity was evaluated.The bacillus velezensis(Q-84)was screened and selected by in vitro and in vivo,which has obvious inhibitory effect on the Rhizopus stolonifer.The effects of bacillus velezensis on the morphology of soft rot and the resistance of sweet cherry fruits were investigated.Through the whole genome analysis and transcriptome analysis of Bacillus velezensis and Rhizopus stolonifer,the biocontrol mechanism of Bacillus velezensis was studied.The main results are as follows.1.Succession regulations of microbial communities on the surface of sweet cherries under different storage conditions.We investigated the microbiota of bacteria and fungi on cherry surfaces under room-temperature storage conditions(25℃)and low-temperature storage conditions(0℃)using high throughput sequencing.A total of 9,652 bacterial OTU and 863 fungal OTU were detected on the surface of cherry.Erwinia,and Botrytis etc.were significantly enriched in the rotten samples,while Bacillus and Cryptococcus etc.were enriched in the unrotten samples,the fungi was more sensitive to postharvest rot,which may play a more important role in the process of the postharvest rotten;the microbial community composition on the surface of cherry was significantly influenced by temperature conditions.The microbiota on the cherry surfaces differed significantly between room-temperature storage and low-temperature storage.The postharvest rotten significantly reduced theα-diversity of the fungi,but the α-diversity of the bacteria was not vary significantly,and the fungi community had different evolution directions in the rot process during normal temperature and low temperature storage;The more complex microbial network under room-temperature storage conditions suggests that more microorganisms are involved in fruit rot at this temperature;significant correlations between potential pathogens and beneficial microorganisms indicate that microbiota,as well as pathogens,influence fruit rot.2.Isolation,identification and pathogenicity evaluation of the main decay-causing fungi in sweet cherry.The fungi on the samples of sweet cherry rotten fruits from 9producing areas were collected and 88 fungal isolates identified.The main decay-causing fungi in sweet cherry fruit in Shandong province belonged to Rhizopus,Aspergillus,Fusarium,Schizophyllum,Penicillium,Colletotrichum,Monilinia,Aureobasidium and Talaromyces.The results of the inoculation showed that,the pathogenicity of the three pathogenic fungi on sweet cherries was followed by Rhizopus stolonifer,Monilinia fructicola and Colletotrichum aenigma.3.Mechanism of endogenic antagonism of sweet cherry against soft rot(Rhizopus stolonifer).3.1 Effects of endogenous antagonism of sweet cherry on mycelial morphology and ultrastructure of Rhizopus stolonifer.Rhizopus stolonifer was selected as a target pathogen,the endogenous antagonistic bacterium Q-84 was obtained from healthy sweet cherry by pair culture method,and was identified as Bacillus velezensis by morphological,physiological and biochemical characteristics and 16 S r DNA sequence.The inhibition rate of Rhizopus stolonifer in vitro reached 87.12%,and the control effect in vivo reached 79.0%.It was observed by scanning electron microscope and transmission electron microscope that after co-culture with Bacillus velezensis,the morphology of mycelia and sporeswere distorted,and the substances in mycelia cells were leaking.Bacillus velezensis treatment had an obvious teratogenic effect on Rhizopus stolonifer,which might be related to the antibacterial substances produced by B.velezensis.3.2 Molecular Mechanism of Interaction between Bacillus velezensis and Rhizopus stolonifer.Illumina & Nanopore and Illumina & Pac Bio sequencing technologies were used to sequence the whole genome of Bacillus velezensis and Rhizopus stolonifer.Genomic analysis showed that there are 13 secondary metabolic synthesis gene clusters in the genome of B.velezensis strain to synthesize related secondary metabolites.There are 10 gene clusters that guide the synthesis of biologically active peptides and polyketides through non-ribosomal peptide synthetase(NRPS)and polyketide synthase(PKS).Among them,5 large gene clusters involved in the synthesis of surfactin,iturin,fengycin,bacillibactin and bacilysin,and 3 gene clusters direct the synthesis of antibacterial polyketones,namely bacillaene,difficidin and macrolactin.In addition,it was found that there are genes encoding molybdenum cofactor and butirosin in the strain.The similarity between the two strains is very low among different strains,especially molybdenum cofactor,which has not been reported in B.velezensis.After co-cultivation of B.velezensis and R.stolonifer,the prokaryotic chain-specific transcriptome and eukaryotic transcriptome were determined and analyzed.The differential expression analysis of all genes showed that a total of 616 differentially expressed genes werefound,of which 243 genes were up-regulated and 313 genes were down-regulated.According to KEGG aggregation analysis,the expression of pks IJ gene of bacillaene synthesis and tua AGE gene in teichuronic acid synthetic gene clusters were up-regulated.After the enrichment analysis of the GO,it was found that genes with significant expression differences were mainly enriched in a series of processes related to ribosome activity such as ribonucleoprotein complex assembly and subunit composition and ribosome assembly in biological processes.It could be speculated that in the early stage of interaction with pathogenic fungi,B.Velezensis mainly inhibit the proliferation of pathogenic fungi and invasive plant hosts through space and nutrient competition. |
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