Characterization Of Bacteria At Different Growth And Development Stages Of Hypsizygus Marmoreus And Functional Study Of Isolation Bacteria

Hypsizygus marmoreus is a popular edible fungus widely cultivated in China for its rich nutritious ingredients and great flavour.The factory production development of H.marmoreus is sharply limited because of long growth cycle,high cost,and low productivity.In this study,the high-throughput sequencing technology?HST?was used to analyze the community structure,dynamics and succession of bacteria at different growth and development stages of H.marmoreus.The dominant bacteria were isolated from cultivation bags and the function of these bacteria was investigated to verify their growth-promoting effects on H.marmoreus,which will lay a solid foundation to shorten production cycles and increase production efficiency in factory cultivation of H.marmoreus.The main results are as follows:1.Analysis of bacteria community structure and diversity at different growth and development stages of H.marmoreusThe HST results showed that the total 827880 effective tags were obtained from five groups of hyphae samples in plate and cultivation bags at different stages?blank bag,half-bag period,full-bag period,matured period?,and clustered into 690 Operational Taxonomic Units?OTUs?,which were assigned to 34 phyla,79 classes,127 orders,228 families,and 417 genera.The dominant phyla were Firmicutes and Proteobacteria and the dominant genera were Rummeliibacillus,Paenibacillus and Lysinibacillus.At the phylum level,Firmicutes bacteria decreased gradually in abundance from half-bag period to matured period.By contrast,Proteobacteria underwent an increase and reached a peak?54.72%?in the matured period of H.marmoreus.At the genus level,Rummeliibacillus,Lysinibacillus and Bacillus in abundance increased at the full bag period and decreased at matured period.The abundance of Paenibacillus had a totally different trend,decreased at the full bag period and increased at matured period.Serratia genera increased exponentially in cultivation bags in the matured period,and the relative abundance reached 55.74%compared to the abundance?<1%?during the period from half bag to the full bag,suggesting that Serratia played a critical role in the decomposition of substrates,and facilitated the formation of the primordium after the completion of maturation.In addition,RDA analysis showed that Rummeliibacillus,Paenibacillus,and Serratia genus was in good accord with the growth cycle of H.marmoreus.By contrast,a negative correlation was observed for the growth cycle of H.marmoreus versus the abundance of Lysinibacillus,Lactobacillus,Escherichia-Shigella,Acinetobacter,Bacillus,and Sporolactobacillus.2.Isolation,identification and location of dominant bacteriaOn the basis of analyzing the bacteria composition structure at different growth stages of H.marmoreus,the dominant bacteria were isolated from the samples taken from cultivation bags at the matured period.The obtained strains were identified as Bacillus altitudinis,Bacillus stratosphericus,Lysinibacillus massiliensis,Lactobacillus amylovorus,Paenibacillus illinoissensis and Serratia odorifera genus.In these bacteria,the most eye-catching strain was Serratia odorifera HZSO-1 and its quantity changed considerably during the matured period of H.marmoreus.Therefore,subsequent work focused on this strain to explore its symbiosis effects on the growth and development of H.marmoreus.A plasmid?p UC19-bud-kan-gfp?expressing the green fluorescent protein?GFP?was constructed and electroporated into the Serratia odorifera HZSO-1 cells.Under the fluorescence microscope,the intense green fluorescence was observed inside and surfaces the hyphae of H.marmoreus co-cultured with the GFP-labelled gfp-HZSO-1.The existence of S.odorifera HZSO-1 in hyphae of H.marmoreus was verified by PCR amplification targeting the variable region 4?V4?of 16S r DNA gene of S.odorifera HZSO-1.3.Effects of HZSO-1 fermentation broth on the growth and development of H.marmoreus and composition analysis of metabolitesThe experimental results showed that the addition of sterilized fermentation broth of HZSO-1 in cultivation substrates could significantly promote the growth of H.marmoreus fruiting body,increase the pileus diameter and the stipe length,improve the weight of single bundle,shorten the fruiting cycle,and make the harvest time earlier for3-4 days.The ultra-high liquid phase-mass spectrometry?UHPLC-QTOF-MS?was used to analyze the metabolites composition of HZSO-1 flitered steriled fermentation broth?FSFB?.The results showed that peptides,organic acids,antibiotics,hormones and other antagonistic growth promoting substances were involved in HZSO-1 FSFB.Besides,four types of acyl-homoserine lactones?AHLs?signal molecules mediating quorum sensing in S.odorifera HZSO-1 were discovered in the structure analysis of first-level mass spectrometry.The four molecules included N-?3-Oxohexanoyl?-homoserine lactone?3-oxo-C₆-HSL?,N-Isovaleryl-L-homoserine lactone?C₅-HSL?,N-Heptanoyl-homoserine lactone?C₇-HSL?,and N-3-Hydroxyoctanoyl-L-homoserine lactone?3-OH-C₈-HSL?.The addition of the exogenous 3-oxo-C₆-HSL as signal molecule into the culture medium can significantly promote the mycelial growth of H.marmoreus,indicating AHLs signal molecules secreted by S.odorifera HZSO-1 may play a key role in stimulating the growth and development of H.marmoreus.