| Flue-cured tobacco,as the dominant crop with the largest planting area and highest yield in Liangshan Prefecture,faces problems such as excessive fertilization,resulting in a decrease in tobacco yield.Microbial agents are an effective way to increase crop yield,and their endophytic bacteria have great potential application value.Endophytes have the advantages of promoting growth and increasing yield,biological control,and improving soil microecology.However,currently,the excavation of endophytes in plants mainly focuses on bacteria and actinomycetes,and there are few research reports on endophytes in flue-cured tobacco.Therefore,this study conducted isolation,cultivation,initial screening,and re screening of flue-cured tobacco,and screened a fungus SICAU-Z1 with good IAA production,antibacterial activity,iron carrier,phosphorus solubility,and cellulose degradation effects.It was identified as Rhizopus dellemar.Pot experiments were conducted on the strain SICAU-Z1,and the optimal medium composition and fermentation conditions were obtained through single factor experiments and response surface methodology design.Transcriptome analysis was used to explore the molecular mechanism of Rhizopus delemar in response to p H stress through the differential expression of genes in the strain SICAU-Z1 under different p H stress.Enrich the microbial resource pool in tobacco areas,reduce fertilizer usage,in order to improve tobacco production and provide support for sustainable agricultural development.The main research results obtained are as follows:1.Isolation,cultivation,and preliminary screening of endophytic fungi from flue-cured tobacco.A total of 120 fungal strains were isolated from tobacco stem samples,and after multiple purification and cultivation,65 fast growing and pollution-free strains were preliminarily screened.2.Conduct research on the growth promoting characteristics of endophytic fungi.The results showed that 11 strains（accounting for 16.9%of the total isolated strains）had strong IAA production ability(IAA>30 mg·L-1),and 11 strains（4.62%）could resist two or more pathogenic bacteria;30 strains（46.2%）had phosphorus solubilization effect,and 7 strains had cellulose degradation zone（>2 cm）,accounting for 10.8%of the total isolated strains;There are three strains producing iron carriers,accounting for4.61%of the total isolated strains.Among all strains,strain SICAU-Z1 showed the best overall performance,with an IAA production of 53.0 mg·L-1 and the ability to resist the growth of four pathogenic bacteria.The phosphorus solubilizing transparent circle was2.20±0.36 cm,the phosphorus solubilizing ability was 34.2 mg/L,the cellulose degradation circle was 2.60±0.66 cm,the iron carrier orange halo circle was 2.05±0.17 cm,and the iron carrier production ability was 31.7%.3.Based on the study of the growth promoting characteristics of strain SICAU-Z1,a phylogenetic tree was constructed by ITS sequencing of endophytic fungi in flue-cured tobacco.The strain was preliminarily identified as Rhizopus delemar,and the sequence of strain SICAU-Z1 was submitted to NCBI for gene registration number ON584326.Understanding the effect of strain SICAU-Z1 on the growth and development of tobacco seedlings,the results showed that strain SICAU-Z1 could significantly improve various indicators of tobacco seedlings.Compared with the control group,the above ground increased by 67.0%,the leaf area increased by 56.6%,the number of roots increased by 58.0%,the underground length increased by 8.84%,the above ground fresh weight increased by 35.5%compared to CK,and the underground fresh weight increased by 75.8%,The above results indicate that strain SICAU-Z1 has a significant promoting effect on the growth and development of tobacco seedlings.4.An optimization experiment was conducted on the optimal medium composition and fermentation conditions of Rhizopus delemar SICAU-Z1 strain.The single factor test results showed that the biomass was 4.35 g at p H 7,4.23 g with glucose as carbon source,3.84 g with glucose concentration of 15 g/L,4.34 g with yeast powder as nitrogen source,4.19 g with yeast powder concentration of 10 g/L,and 4.11 g with potassium dihydrogen phosphate as phosphorus source When the concentration of potassium dihydrogen phosphate is 7 g/L,the biomass is 4.12 g,and when the medium capacity is 150 m L,the biomass is 3.87 g.The optimal medium group for strain SICAU-Z1 is p H 7.27,glucose concentration 15.53 g/L,yeast powder concentration 10.81 g/L,potassium dihydrogen phosphate concentration 6.78 g/L,and medium capacity 152.23m L.Under these conditions,the predicted biomass of the model can reach 4.0371 g.5.Sequence analysis of transcriptome of strain SICAU-Z1 under different p H stress.Using p H 7 as the control group,the results showed that a total of 1629differentially expressed genes were found in the four groups.In the p H 4 treatment,the most differentially expressed genes were found in the p H 7 control group,with 567genes upregulated and 343 genes downregulated.The main response pathway under different p H stresses is pal,and Pac C is a key regulatory factor in this pathway.After comparing these differentially expressed genes with the carbohydrate enzymes（CAZymes）database,a total of 1721 CAZymes differentially expressed genes were obtained.In GO enrichment,glutamate synthase metabolism and ribosome generation process enrichment are most significant.In KEGG enrichment analysis,ko03008（eukaryotic ribosome biosynthesis）and ko00040（pentose and glucuronate metabolism）were the most significant.In summary,this study aims to investigate the molecular mechanism of Rhizopus delemar response to p H stress by screening and identifying endophytic fungi in flue-cured tobacco,optimizing cultivation conditions,and promoting the growth and development of tobacco seedlings.This provides a foundation for microbial inoculum and a scientific basis for their application in agriculture. |
