Air-curing is the initial step in the processing of cigar tobacco leaves. However, the dynamics of microbial community and metabolic functions in different parts of tobacco leaves during this process remain largely unclear. In this study, amplicon-based high-throughput sequencing revealed that Pseudomonas (9.0 to 29.9%) and Sphingomonas (0.5 to 13.8%) were the dominant bacterial genera in the early stages of air-curing, while Pantoea (1.7-90.4%) became predominant after air-curing. Microbial community diversity analysis indicated that species richness and diversity were significantly higher during the fresh leaf and withering periods. Functional prediction based on PICRUSt2 suggested that the microbial communities in the middle leaves exhibited higher abundances of metabolic pathways related to carbohydrates and amino acids than those in the upper leaves, potentially leading to the formation of more flavor compounds. The volatile flavor compounds were detected during the air-curing process by HS-SPME-GC-MS, with alkaloids and esters being the most prominent, although their accumulation periods differed across leaf parts. Furthermore, based on PLS-DA, 17 and 38 significantly changed flavor components were identified in the upper and middle leaves, respectively. Finally, the potential relationships between characteristic microbes and flavor components were explored based on Spearman correlation coefficient. It was found that multiple bacteria such as Rhodanobacter, Gemmatimonas, and Ramlibacter present in the middle leaves exhibited significant positive correlations with multiple flavor compounds such as 3,3-dimethylacrylic acid, phenylacetone, 2,3-butanedione, and geranylacetone, potentially promoting the flavor formation of cigar tobacco leaves during air-curing process. This study provides scientific insights into the role of microorganisms during the air-curing process of cigar tobacco leaves and offers a scientific basis for screening of specific functional microorganisms to improve and stabilize cigar tobacco flavor in the future.

Nitrosamines are considered carcinogens that threaten human health and environment. Especially, high contents of Tobacco-specific nitrosamines (TSNAs) are generated during the fermentation process of cigar tobacco. To control the accumulation of TSNAs, one novel strain WD-32 was isolated by comprehensively evaluating the reduction characteristics of nitrate, nitrite, and TSNAs, and this strain was identified as Bacillus siamensis by 16S rRNA gene analysis and MALDI-TOF MS evaluation. Subsequently, whole genome sequencing of B. siamensis WD-32 was carried out to excavate important genes and enzymes involved, and the possible reduction mechanism of TSNAs was explored. More importantly, the reduction of TSNAs by B. siamensis was significantly promoted by knockout of narG gene. During the practical agricultural fermentation process of the cigar tobacco leaves, the treatment by the WD-32∆narG cells resulted in a 60% reduction of the total TSNAs content compared with the control, and the concentrations of the NNN and NNK were decreased by 69% and 59%, respectively. In summary, this study offers efficient strains for reduction of the TSNAs in cigar tobacco, and provides new insights into the reduction mechanism of TSNAs, which will promote the application of microbial methods in control of TSNAs and nitrite.

Cigar variety CX-010 tobacco leaves produce localized green spots during the air-curing period, and spraying exogenous sucrose effectively alleviates the occurrence of the green spots. To investigate the alleviation effect of exogenous sucrose spraying, the total water content and the number and size of green spots on tobacco leaves were investigated during the air-curing period under four treatments; CK (pure water), T1 (0.1 M sucrose), T2 (0.2 M sucrose) and T3 (0.4 M sucrose). The results showed that the total water content of tobacco leaves showed a trend of T3 < CK < T2 < T1 in the early air-curing stage, and the number and size of green spots showed a trend of T3 < T2 < T1 < CK. All sucrose treatments alleviated the green spot phenomenon, and T3 had the fewest green spots. Thus, the tobacco leaves of the T3 and CK treatments at two air-curing stages were used to perform metabolomics analysis with nontargeted liquid chromatography‒mass spectrometry to determine the physiological mechanism. A total of 259 and 178 differentially abundant metabolites (DAMs) between T3- and CK-treated tobacco leaves were identified in the early air-curing and the end of air-curing stages, respectively. These DAMs mainly included lipid and lipid-like molecules, carbohydrates, and organic acids and their derivatives. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the T3 treatment significantly altered carbohydrate metabolism (pentose phosphate pathway, sucrose and starch metabolism and galactose metabolism) and amino acid metabolism (tyrosine metabolism and tryptophan metabolism) in air-curing tobacco leaves. Sucrose treatment alleviated green spots by altering DAMs that affected chlorophyll degradation, such as tyrosine and citric acid, to promote the normal degradation of chlorophyll.

The harvest time is a key factor for cigar leaves with high quality, which varies greatly depending on the environment. In this study, we performed a growth characterization of the cigar tobacco CX-26 ( Nicotiana tabacum L.) from the cluster stage to the vigorous stage and mature stage. Further, soil plant analysis development measurement of three middle leaves with 67 (T1), 71 (T2), and 75 days (T3) of growth after transplant indicates that the maturity might change from 71 to 75 days of growth. A genome-scale transcriptome was conducted to explore the gene expression dynamics of this maturity change. A total of 80,502 genes were detected in the CX-26 leaves, of which 64,611 genes were annotated on the reference genome. T1 and T2 leaves had fewer differential expressing genes (DEGs), while T3 leaves had 26,456 DEGs from T2 leaves, supporting the distinct growth of T3 leaves. Analyses of Gene ontology, Kyoto encyclopedia of genes and genomes identified that DEGs are involved inkey pathways including photosynthesis-antenna proteins, plant hormone/mitogen-activated protein kinase signaling pathway, and plant-pathogen interaction. Abscisic acid and jasmonate acids positively regulate leaf senescence and chlorophyll degradation and were higher in the T3 leaves compared with T1 and T2 leaves. In contrast, the total sugar was reduced in T3 leaves compared with T1 and T2 leaves, indicating the overripe state of the T3 leaves. Furthermore, several photosynthesis-related enzymes and a transcription factor were highlighted in the gene regulatory network, which might regulate the dynamics of carbohydrate metabolism, lipid metabolism, and energy metabolism. In summary, our study provides insight into the growth state of CX-26 cigar leaves.

The harvest time is a key factor for cigar leaves with high quality, which varies greatly depending on the environment. Here, we performed a genome-scale mRNA transcriptomic analysis on the cigar cultivar CX-26 (Nicotiana tabacum L.) to evaluate the relationship between gene expression and growth state. The leaves were harvested with 67 (T1), 71(T2) and 75 (T3)-day growth. A total of 80,502 genes were detected in the CX-26 leaves, of which 64,611 genes were annotated on the reference genome. Principal component analysis showed that T1 and T2 leaves had a high overlapping pattern, while T3 leaves were distinct. Indeed, T1 and T2 leaves had fewer differential expressing genes (DEGs), while T3 leaves had 26,456 DGEs from T2 leaves, supporting the distinct growth of T3 leaves. GO annotations mainly enriched the photosynthesis-related metabolic process, catalytic activity and binding biological processes. KEEG analysis identified the key pathways including photosynthesisantenna proteins, plant hormone/MAPK signaling pathway and plant-pathogen interaction. The maturity regulation and defense response-associated hormones abscisic acid and jasmonate acid were higher in the T3 leaves than that in T1 and T2 leaves confirming the KEEG analysis. Furthermore, several photosynthesis-related enzymes and a transcription factor were highlighted in the gene regulatory network, which might regulate the dynamics of carbohydrate metabolism, lipid metabolism and energy metabolism. In summary, our study provides insight into the growth state of CX-26 cigar leaves.

Nitrogen (N) is an essential nutrient element in plants that participates in physiological and biochemical regulation. However, the effects of different N applications on cigar tobacco ( Nicotiana tabacum L.) growth are not well known. In this study, the differences in agronomical characteristics and nutritional quality of cigar tobacco leaves at the mature and curing stages under different exogenous N applications were explored. The dry matter accumulation of cigar tobacco leaves increased with increasing N application rates. In the two stages, the nutritional quality of cigar tobacco leaves was similar, but the concentrations of N and Cl were increased. To understand the effect of exogenous N on cigar tobacco metabolism, untargeted metabolomics was applied. Six significantly differential metabolites, including phenylalanine, phosphoserine, glutamate, oxoproline, succinylhomoserine, and homoserine, were identified, and the main metabolic pathways around the tricarboxylic acid cycle were identified. These results provide a better understanding of the effects of exogenous N application on the physiological, biochemical, and metabolic processes of cigar tobacco and provide a reference for fertilization control in cigar tobacco production.

Abstract Shading is a conventional cultivation method in crop production. However, the effects of shading on photosynthesis and metabolism of cigar leaves remain unclear. Here, the effects of shading on tobacco photosynthesis characteristics and metabolome were studied by setting light transmittance of 100%, 90%, 80%, 70% and 60%. The soluble sugar concentration and soluble protein concentration of cigar leaves at 70% and 60% shading treatments increased by 149.18% and 117.98%, and 174.48% and 195.91%, respectively, compared with that at 100% light transmittance at mature stage. A total of 702 differential metabolites were detected in cigar leaves among the different shading treatments. The metabolites mainly contain flavonoids, such as lignin, sesquiterpenoids, Metabolic pathways are enriched in amino acid metabolism, secondary metabolites biosynthesis and lipid metabolism. Six key metabolites are detected: Chlorogenic Acid, D-Glucosaminide, D-Urobilin, Farnesylcysteine, Hydroxyatrazine and Sucrose. Token together, our study showed that shading could significantly affect the photosynthesis and metabolism of cigar leaves, resulting in the accumulation of primary metabolites, soluble sugar and soluble protein in leaves, which was beneficial to cigar plant growth and quality formation.

Studying the relationship between rhizosphere microorganisms and root exudates is of great significance for the interaction between rhizosphere microorganisms and plants, and the prevention and control of soil-borne diseases. This article analyzed the effects of different microorganisms on tobacco root exudates and rhizosphere microorganisms. It was found that the bacterial wilt pathogen can greatly increase acids and amines, while the probiotic B. amyloliquefaciens ZM9 can eliminate some acids and amines. The results of the study show that the root exudates of pathogenic bacteria may contain a variety of allelochemicals that cause soil-borne diseases.

The role of hydrogen sulfide (H 2 S) in regulating the pathogenic bacteria has been well documented. However, whether exogenous H 2 S addition inhibits the pathogens in soil is not understood, and whether H 2 S can suppress the plant disease caused by pathogen R. Solanacearum is not clear. In the present study, different concentrations of H 2 S donor NaHS were applied to the tobacco field to explore the interrelation among NaHS, tobacco baterial wilt, soil physicochemical properties and microbial community. In order to decipher the disease suppression mechanism from the perspective of soil microecology. Application of NaHS significantly reduced the disease incidence and disease index of TBW, increased soil pH, alkali-hydrolyzed nitrogen (AN), available phosphorus (AP), available phosphorus (AP) and organic matter (OM). NaHS addition also changed soil microbial community composition and structure. Furthermore, NaHS addition significantly reduced the abundance of Ralstonia and Fusarium , and increas pathogenic ed beneficial microorganisms S olirubrobacter , Rhodococcus , Rhizobium , Pseudomonas , Paenibacillus , Microvirga , Lysobacter , Haliangium , Granulicella , Flavobacterium , Bacillus , Trichoderma and Aspergillus at the genus level. Our findings suggested that exogenous application of NaHS significantly suppressed TBW caused by R. Solanacearum through regulated soil microecology. This study revealed the potential of NaHS in control of bacterial wilt.

To explore the effect of lignin composition on cellulase adsorption, dehydrogenation polymers (DHPs) were prepared from p-glucocoumaryl alcohol/coniferin/syringin, giving rise to H-DHP, G-DHP, and S-DHP, respectively. The structures of DHPs were thoroughly characterized and compared by GPC and NMR techniques, and the Langmuir isotherm protocol was applied to determine the cellulase adsorption behaviors of these different types of DHPs. The adsorption study indicated that the binding strength between the DHPs and cellulase varied in the following order: G-DHP > H-DHP > S-DHP. The inhibition of different types of DHPs on enzymatic hydrolysis of cellulose was in the same order as the cellulase adsorption, indicating that non-productive adsorption was the main way to influence cellulase. The correlation analysis results showed a positive association between the phenolic hydroxyl group content in DHPs and their maximum adsorption capacity toward enzymes. A negative correlation between the PDI and binding strength was also observed. It was also found that the adsorbed cellulase could be desorbed and retained normal enzyme activity, and so it was presumed that DHPs and cellulase were mainly linked by physisorption such as hydrogen bonding. This study clearly showed that the composition of lignin had a great impact on cellulase, and that G-type lignin exhibited the most detrimental effect. The results could provide useful information on the mechanism of cellulase adsorption onto lignin using DHPs as lignin model compounds.

To understand the influence of lignin characteristics on their antioxidant activities, lignins were isolated from Broussonetia papyrifera by organosolv pretreatment at different temperatures.

为有效防治烟草青枯病,采用细胞培养和毛细管法研究了烟草化感自毒物质邻苯二甲酸二丁酯对青枯菌的影响及趋化诱导效应,同时采用梯度训化法筛选邻苯二甲酸二丁酯降解菌,并通过盆栽试验测定了邻苯二甲酸二丁酯降解菌处理后的烟草青枯病发病率.结果表明,邻苯二甲酸二丁酯对青枯菌具有趋化诱导作用,并且在低浓度条件下能促进青枯菌生长;筛选的邻苯二甲酸二丁酯降解菌(Acinetobacter sp.Ed2)在土壤中对邻苯二甲酸二丁酯降解效率为51.17％;土壤中添加邻苯二甲酸二丁酯的处理(T1),与对照(CK)相比青枯菌数量显著提高59.89％;经邻苯二甲酸二丁酯降解菌Ed2处理(T2)后,与对照和T1处理相比,土壤中青枯菌数量分别降低84.63％和90.38％,青枯病发病率分别降低52.23％和136.42％.因此,利用邻苯二甲酸二丁酯降解菌消耗土壤中的邻苯二甲酸二丁酯,既减少青枯菌的营养供给,又截断邻苯二甲酸二丁酯对青枯菌的趋化诱导作用,从而显著降低了青枯病发病率.

In order to improve the degradation efficiency of lignin and cellulose in tobacco straw and shorten the decomposing time of tobacco straw, the strains with the ability to degrade lignin and cellulose were screened out by plate screening and enzyme activity determination experiments. The effects of different strain combinations on tobacco straw degradation were discussed. The results showed that the obtained Lysinibacillus fusiformis (N019a), Trametes hirsuta (MA), and Rhizopus oryzae (imp) were effective in degrading lignin, hemicellulose, and cellulose in tobacco straw. The composite flora I composed of N019a, MA and imp was used for solid fermentation of tobacco stalk powder for 25 d. The degradation rates of hemicellulose, cellulose and lignin were 67.23±0.73%, 61.17±0.45% and 60.12±0.48%, respectively. B. tequilensis (B4), B. subtilis (B26) and A. niger (M90) composed a complex flora II with degradation rates of hemicellulose, cellulose and lignin being 72.15±0.35%, 65.68±0.55%, and 38.15±0.76%. The degradation rates of hemicellulose, cellulose and lignin by compound flora III composed of 6 strains was were 83.32±0.45%, 75.21±0.71% and 66.13±0.53%. Different degrading bacteria degrade different components in wood fiber with certain specificity, and the combination of multifunctional strains can significantly improve the biomass degradation effect of tobacco straw. The research results provide a basis for the development of a highly effective compound bacterial agent for tobacco straw degradation.

In order to improve the spore yield of compound Bacillus spp. (B. amyloliquefaciens, B. laterosporus and B. megaterium), the effects of nutrient conditions including carbon source, nitrogen source, mineral salt and fermentation conditions including the inoculum age, inoculation amount, loading volume of liquid and initial pH on the spore yield were studied. The results indicated that the optimized medium was glucoses 20 g/L, soybean meal 30.0 g/L, K2HPO4 1.0 g/L; fermentation temperature is 37℃, the inoculum age 12 h, initial pH 7.0, 2% inoculation amount, loading volume of liquid 20 mL/250 mL. Under the optimized conditions of culture medium and fermentation for compound Bacillus spp., spore yield was 10.24 times more than the initial medium, and the spore formation rate reached more than 90%.

The research on relationship between rhizosphere microbes and root exudates has a great significance on discussion of interaction between rhizosphere microbes and plants, as well as control of soil-borne diseases and insect pest. GC-MS was used to analyze changes of tobacco root exudates under the antagonistic action of tobacco bacterial wilt and black shank. It turned out that when pathogens of tobacco bacterial wilt and black shank in tobacco root microorganisms increase, tobacco root exudates augmented rapidly among of which organic acids have the biggest growth, followed by amines. When the pathogens of tobacco bacterial wilt and black shank are inhibited by the active substance of antagonistic antibacterial, 20 - 23 kinds of root exudates are added; besides, the content of 7 substances was reduced to 0. Another inter-esting finding was that the fluctuations of phthalic acid, isophthalic acid and benzoic acid, which have caused continuous cropping obstacles, were very dis-tinct. The results of this study have provided novel clues for the exploration of continuous tobacco cropping obstacles and soil-borne diseases.

O-phthalic acid is a kind of important pollutant, which accumulates in the environment with the extensive use of plastics and other products. Meanwhile, phthalic acid is one of the high content of allelopathic autotoxic substances secreted by tobacco. The accumulation of phthalic acid in soil is an important cause of tobacco continuous cropping effect. In order to degrade phthalic acid accumulated in environment, the barrier effect of tobacco continuous cropping caused by phthalic acid accumulation in soil can be removed. A strain capable of degrading phthalic acid was isolated from sludge of sewage treatment plant and compared with 16 s DNA. The homology between this strain and Enterobacter sp. is 99%. The optimum growth conditions are as follows: pH7 at 30°C, 500 mg/L of o-phthalic acid, inoculation concentration ≥ 1.2% and its highest degradation rate of o-phthalic acid is 74%. The results of pot experiment showed that the degradation efficiency of o-phthalic acid in soil was about 40%, which alleviated the inhibitory effect of o-phthalic acid accumulation on tobacco growth.

Through the analysis of ecological colonization and antimicrobial substances of Bacillus amyloliquefaciens YH-22, the resistance mechanism of YH-22 against bacterial wilt which was caused by Ralstonia solanacearum was preliminarily investigated. The tolerance dosage of YH-22 resistance strain against rifampicin reached 400μg/mL by resistance induction with rifampicin. The results of pot experiments showed that YH-22 resistance strain firstly colonized on the rhizosphere soil. Then, it intruded into the root and stem successively. The antimicrobial substances in broth could be dissolved by methanol, but not by chloroform and ethyl acetate. They could maintain high stability under the conditions of heat treatment, protease hydrolysis and Ultraviolet. Moreover, they had the character of oil displacement and droplet collapse. In conclusion, strain YH-22 has colonization ability in rhizosphere soil as well as tobacco tissue. The antibacterial components of strain YH-22 may consist of proteins and lipopeptides.