Background: To explore the optimum fermentation conditions for tobacco leaves and also screen the microbiota and metabolites that are beneficial for fermentation. Methods: Tobacco leaves were fermented at 25 °C, 35 °C, and 45 °C for 2, 4, and 6 weeks, respectively. For identification of the best fermentation temperature, physicochemical properties and sensory quality of fermented tobacco were investigated. Subsequently, based on the appropriate temperature, 16 s rRNA sequencing and metabolomics analysis of tobacco were performed to monitor the change of microbes and metabolites during fermentation process (from 2 to 6 weeks). Results: Sensory quality analysis indicated that fermentation at 45 °C for 6 weeks represented the optimum condition. Metabolomics analysis showed that a total of 415 metabolites were annotated. The increase of fermentation period led to significant changes of metabolites. Results revealed an increase in concentration of L-phenylalanine and sphingosine as well as decreased concentration of betaine and phytosphingosine with the prolongation of fermentation period (2 to 6 weeks). Distinct changes in the microbiota were also observed with prolongation of the fermentation time. Results revealed that Pseudomonas, Pantoea, and Burkholderia were dominant bacteria in fermentation at 45 °C for 6 weeks. With the extension of the fermentation time, the abundance of Pseudomonas increased, while that of Sphingomonas and Methylobacterium decreased. Furthermore, microbiota profiles were tightly relevant to the altered metabolites, especially compounds involved in the sphingolipid metabolism. Conclusion: Suitable fermentation conditions were 45 °C for 6 weeks; phytosphingosine and sphingosine might affect tobacco fermentation via the sphingolipid metabolism pathway. This study provides a theoretical basis for guiding tobacco fermentation and gives insights into reducing harmful substances during tobacco fermentation.

In order to explore the effects of biochar on root system and growth characteristics of flue-tobacco, three years of field experiments were conducted to study the influence of different biochar application levels [600 (T₁), 1200 (T₂), 1800(T₃), 2400 (T₄), 3000 (T₅) kg/ha] and no fertilizer (CK) on the root physiological indexes and growth index of tobacco. Compared with local conventional fertilization, the application rate of N fertilizer in each treatment (except for control) was reduced by 40% to analyze the effects of different amount of biochar on the physiological indexes of tobacco roots and leaf photosynthesis during flourishing. The results showed that tobacco plants' root development status in the flourishing period was consistent with the photosynthetic physiological indexes, chlorophyll content, and leaf-area coefficient. Compared with the control, the application of biochar could increase the root vigor by 177.8%. Biochar improved the roots, increasing the total root area by 91.35% and the number of root tips by 100.9%. Meanwhile, biochar increased the net photosynthetic rate of tobacco leaves by 77.3% and the total tobacco biomass by 72.5%. Studies have shown that biochar can promote the development of tobacco roots, and then enhance the photosynthesis of leaves, so that tobacco plants can grow healthily, which is conducive to the tobacco production and the cultivation of soil.

In this study, four different biochar application rates and a control were set up using indoor potted tobacco, to study the effects of biochar on the microbial diversity and metabolism of tobacco-growing soil. The five treatments were as follows: control—0% biochar (w/w) + 26 g fertilizer/pot; biochar treatments—1% biochar (w/w) + 26 g fertilizer/pot, 2% biochar (w/w) + 26 g fertilizer/pot, 3% biochar (w/w) + 26 g fertilizer/pot, and 4% biochar (w/w) + 26 g fertilizer/pot. We found that biochar increases the microbial diversity of soils and simultaneously changes the microbial community structure. Under the influence of biochar, soil urease activity increased by 18%, invertase activity increased by 23.40%, polyphenol oxidase activity increased by 59.50%, and catalase activity increased by 30.92%. Biochar also significantly increased the microbial biomass carbon and nitrogen content of the soil. Soil microbial biomass nitrogen had a positive correlation on bacterial diversity, with the highest coefficient, while soil microbial biomass carbon had a positive correlation on fungal diversity, with the highest coefficient. The microbial diversity and metabolic capacity of soil are improved under the influence of biochar, and soil enzyme activity and microbial biomass carbon and nitrogen have positive impacts on soil microbial diversity.

This study aimed to investigate drought resistance of the LY1306 tobacco strain. Seedlings of tobacco strains LY1306, ZhongYan 100 (ZY100) and Hong Hua Da Jin Yuan (HHDJY) were treated with polyethylene glycol (PEG)-6000 to induce osmotic stress. As validation, water-deficit-induced drought treatments, including mild drought (MD; watering 1.5 L/week) and severe drought (SD, without watering) were carried out. Changes in cell morphology, leaf water potential, antioxidant enzyme activity, as well as contents of malondialdehyde (MDA) and proline were determined for each treatment. Transcriptome sequencing was performed for the seedlings treated with 15% PEG-6000. No obvious changes were observed in morphology of LY1306 and ZY100 under osmotic or drought stress; whereas, visible wilting was observed in HHDJY. Superoxide dismutase and peroxidase activities of LY1036 and ZY100 under osmotic stress were significantly higher than those of HHDJY. Under SD, the MDA content of LY1306 was significantly lower and the proline content of LY1306 was significantly higher than that of HHDJY. Differential genes between LY1306, ZY100 and HHDJY were enriched in functions about alpha-linolenic acid, and arginine and proline metabolisms. LY1306 could increase its antioxidant enzyme activities and proline accumulation in response to drought stress, probably by regulating drought resistance-related pathways and genes.