Microbial Community Characteristics Of Moldy Tobacco Leaves And Their Mold-inducing Mechanisms

In the storage ecosystem,tobacco leaves are the most important raw material for cigarette production,but they are easily infected by various fungi and then develop mildew or rot.Tobacco mildew not only cause serious economic problems but also serious health and social problems due to the secretion of mycotoxins and the spread of mold spores.The occurrence of tobacco mildew resulted from the interaction of the ecological relationship of tobacco leaves,tobacco microbiome,molds,and environment.Although a lot of researches have been carried out,the characteristics of microbial community related to mildew and the mildew mechanism are still not clear.In this study,we used high-throughput sequencing to analyze the diversity,composition,and network characteristics of microbial community for storage mildew tobacco leaves,and analyzed the distribution pattern and influencing factors of mold community combing with climatic and physical and chemical factors.The source of tobacco mold was studied by analyzing the mold community structure during tobacco processing,and the mildew condition and mildew ability were discussed by studying the growth characteristics and mildew characteristics of the dominant strains.Then,we simulated the mildew process of tobacco leaves in laboratory,and integrated the data of amplicon sequencing,metagenomic sequencing and physicochemical factors to analyze the succession and construction mechanism of mold community during the mildew process.Finally,LC-MS was used to characterize the metabolic profiles of tobacco leaves of natural mildew and artificial mildew,and microbiome data were combined to further reveal the mildew mechanism of tobacco leaves.The following are the main results of the research.1)Microbial community had low diversity and richness and unstable network structure in mildew tobacco leaves,which was dominated by mesophilic xerophilic fungi.Tobacco microorganisms were mainly composed of bacteria,fungi,archaea and viruses,and bacteria was the predominant microorganisms in healthy tobacco leaves(abundance > 90%),fungi was dominant in mildewed tobacco leaves(abundance >83%).Compared with healthy tobacco leaves,the total microbial and fungal community diversity in mildewed tobacco leaves decreased significantly,while the bacterial community diversity did not change significantly.Fungal communities of mildewed tobacco leaves were regulated by abundant taxon,among which saprophytic fungi such as Aspergillus,Xeromyces and Wallemia,were dominant microflora.Aspergillus and Xeromyces had strong mildew ability.Among them,X.bisporus,A.proliferans,A.pseudoglaucus,A.ruber,A.niger,A.flavus were the main mildew-causing fungi.The optimum growth temperature was 20-35 °C and the optimum water activity was 0.656-0.975.The distribution of fungal communities in tobacco leaves had significant environmental heterogeneity,temperature and humidity were the driving forces of these differences.The ecological network analysis showed that fungal network of healthy tobacco leaves was dominated by rare sub-community,while the fungal network of mildew tobacco leaves was dominated by abundant and intermediate sub-community,which were significantly negatively correlated with rare groups(p < 0.05).The stability,connectivity and complexity of the fungal network in mildew tobacco leaves were significantly reduced due to the lack of core functional microflora.2)Post-harvest pollution was the main source of molds in stored tobacco leaves.About 50% of the microflora in stored tobacco leaves were "field fungi" associated with green tobacco leaves,while the rest were mostly arid or semi-arid "storage fungi" accumulated in the post-harvest processing environment.The dominant molds,such as Aspergillus,Xeromyces and Wallemia,were significantly enriched in tobacco leaf samples collected in the period between the completion of flue-curing and the start of re-curing,indicating that this period was the main window period for mildew fungal infection.3)Environmental selection and nutritional competition were the important driving forces for the succession and assembly of tobacco fungal communities.In the process of tobacco mildew,Proteobacteria and Mucoromycota were gradually reduced,while Ascomycota and Firmicutes were gradually increased.In the fungal community,the dominant microbiota changed from Dothideomycetes,Sordariomycetes,Tremellomycetes and Cystobasidiomycetes to Microbotryomycetes and Mucoromycetes,and then to Eurotiomycetes.Under dry conditions,the assembly of mold community was a random process,but with the increasing of temperature and humidity,the influence of deterministic process on mold community assembly gradually increased.p H,water content,TOC,temperature and humidity were important factors affecting mold community assembly.The interactions among microorganisms in tobacco leaves were intimate,mutual benefit were dominant within-domain,and competition were dominant between-domain.The niche width of microorganisms in mildewed tobacco leaves was significantly larger than that in healthy tobacco leaves,suggesting that nutritional competition was one of the important driving force of microbial community recombination in moldy tobacco leaves.Metagenomic analysis showed that the functional genes involved in organic matter and carbon degradation(GH31,GH51 and amy A)were significantly enriched in mildewed tobacco leaves,while the functional genes involved in nitrogen and phosphorus cycling were inhibited in midew tobacco leaves.These indicated that moldy microorganisms gained competitive advantages by degrading carbon-containing compounds.4)Microbial metabolism was one of the important drivers of tobacco mildew.Microbial action was related to the degradation and formation of metabolites.Mildew microorganisms promoted the formation of phenols,indole,benzene and derivatives,carbonyl compounds,and the degradation of flavonoid amino acids,fatty acids,carbohydrates,glycerol phospholipids.The contents of 21 metabolites,such as dulcitol,indolelactic acid,arbutin,gallic acid,were significantly enriched in mildewed tobacco leaves.However,the contents of 15 metabolites,such as sucrose,L-phenylalanine,lactosamine,were significantly decreased in mildewed tobacco leaves.These results indicated that these metabolites might be effective indicators for identifying mildew in tobacco leaves.Metagenomic and metabonomics conjoint analysis showed that carbohydrate metabolism and amino acid metabolism were the main metabolic functions in the tobacco ecosystem.Molds played dominant roles in the process of tobacco mildew mainly through tryptophan metabolism,tyrosine metabolism,aminobenzoate degradation,toluene degradation,among which tryptophan metabolism was the most significant.In conclusion,tobacco mildew was probably a natural phenomenon of ecological unbalance which was caused by the rapid growth and multiplication of one or a few environmentally adapted dominant fungi under suitable temperature and humidity conditions.In the process,dominant fungi could compete for ecological niches and resources of rare taxa in the interaction,and degrade and transfer tobacco compounds through relevant metabolic pathways.Thus,the physicochemical and microecological environment of tobacco leaves were changed,and finally the tobacco ecosystem was out of balance.