Introduction

The cigar is renowned for its profound cultural heritage and exceptional taste1. Its flavor is characterized by a strong, mellow, and varied when compared with flue-cured tobacco, which exhibits a range of aromas including fruity, floral, nutty, chocolate, coffee, and milk2. A cigar is a hand-rolled tobacco product comprising filler, binder, and wrapper. Its sensory characteristics are closely related to the aroma profile of tobacco leaves. Flavor is the fundamental attribute of cigar tobacco leaves (CTLs), determining the quality and style characteristics3. The chemical constituents of CTLs, in terms of their composition, content, and balance, serve as the basis for establishing the flavor profile. These chemical constituent profiles are influenced by a range of factors, including variety, planting environment, and production technology.

The elaboration of a cigar encompasses a series of processes, including cultivation, air-curing, fermentation, and aging. These processes, along with the accumulation, degradation, and transformation of macromolecules and the corresponding biosynthesis of aroma precursors and volatile flavor compounds, are influenced by the growth and metabolism of microorganisms4,5,6. Microorganisms can regulate metabolic levels through the production of specific signaling molecules, which in turn affect the expression of genes associated with metabolites in plants, alternatively, they may participate in synthetic signaling pathways7,8,9,10. The distinction in the structure and functionality of the microbial community gives rise to variations in flavor11. Consequently, a thorough comprehension of the microbial community and its role is of paramount importance in regulating cigar flavor12,13.

Right now, the roles of microorganisms in forming aroma profiles during air-curing and fermentation are well documented14. Bacteria are mainly involved in sugar metabolism, lipid metabolism, and amino acid metabolism, while fungi are mainly involved in saprophytic degradation of lignin, cellulose, and pectin15,16. For example, the microbial communities in the middle CTLs had higher abundances of metabolic pathways related to carbohydrates and amino acids than those in the upper CTLs, potentially leading to the formation of more flavor compounds during air-curing17. The predominant bacteria in cigars hold significant potential for applications in the degradation of aromatic compounds, hydrocarbons, aliphatic compounds, and lignin, while fungi primarily served a saprotrophic role after fermentation18. The starch in wrapper, binder, and filler CTLs has a significant positive effect on fungal microbes and thus indirectly on the formation of nitrogenous flavor compounds19. Besides, the influence of functional microorganisms, fermentation media, and origins on the microbial communities and corresponding aroma profile of CTLs are also analyzed. For example, inoculation with Staphylococcus nepalensis resulted in higher consumption rates of reducing sugars and total nitrogen contents during the CTL fermentation, and it was positively correlated with carotenoid degradation products, indicating its potential role in promoting flavor formation20. Bacillus siamensis inoculation during CTL fermentation resulted in a significant enrichment of Pseudomonas, which played an important role in sugar metabolism and pectin degradation21. The microbial functions of cigar filler leaves supplemented with the Tremella aurantialba SCT-F3 fermentation medium were enhanced, including nucleotide biosynthesis, amino acid biosynthesis, fatty acid and lipid biosynthesis, nicotine degradation, and nicotinate degradation22.

The tobacco aroma quality is mainly determined by the genetic factors of the variety and the environmental factors of the origin. The variety influences the nature and variety of aroma constituents, while the origin mainly affects the content and composition of aroma constituents. The breeding and selection of superior varieties is a fundamental prerequisite to producing tobacco leaves with an exceptional aroma profile23. The variety has a direct impact on the quality of the tobacco leaf, influencing gene expression regulation. The differences in genotype and genetic basis result in varying contents of aroma constituents among the various tobacco leaf varieties. It is challenging to rectify the deficiencies in the aroma profile of tobacco varieties through cultivation methods alone24. Biological factors exert a considerable influence on the concentration of plant metabolites. However, there is a paucity of literature examining the regulation of cigar flavor both from the perspective of varieties and microorganisms.

Flue-cured tobacco is very sensitive to the ecological environment, and the ecological conditions of the origin basically determine the flavor profiles and quality of the tobacco and form the basis of the style of different brands of cigarettes. Many tobacco origins have been declared as protected geographical indications to establish and maintain the characteristics and quality of the tobacco from which they originate. Comprehensively analyzing the composition and characteristics of aroma constituents in tobacco from different origins helps recognize the characteristics of tobacco, trace the origin of tobacco, maintain geographical indications, protect the rights and interests of tobacco farmers, and regulate the quality of tobacco. Besides, the microbial communities were changed among different origins, thereby affecting the aroma profile of CTLs.

The current study was carried out to investigate the effect of the filler and wrapper from three varieties and the filler from four origins on the aroma profile of CTLs. The key aroma constituents to these CTLs and the difference in the microbial community diversity and structure were explored. The relationship between both dominant microorganisms and aroma constituents was also revealed. It laid a foundation for the efficient regulation of the aroma quality of cigars.

Results

Effect of variety on aroma profiles and differential constituents of wrapper CTLs

A total of 60 aroma constituents were detected in three varieties of wrapper CTLs, which were divided into 7 classes according to their chemical structures, including esters (6), alcohols (6), acids (1), aldehydes (7), ketones (19), nitrogen heterocycles (13), and alkenes (8), and their total content was between 2.40 mg/kg and 3.08 mg/kg (Table S1). There were significant differences in the varieties and contents of aroma constituents, and the changes in aroma constituents exhibited obvious variety specificity. Among them, the contents of alcohols, nitrogen heterocycles, and total aroma constituents in AQ1 increased, the aldehydes, ketones, and alkenes in AQ2 enhanced, and the esters in AQ3 increased (Fig. 1A). Besides, there were significant differences in the proportions of the aroma profile between different varieties of wrapper CTLs, including the esters (5.53–8.50%), alcohols (2.81–5.20%), nitrogen heterocycles (19.45–43.82%), and alkenes (2.40–8.52%). Volatiles were one of the most important factors in terms of aroma and taste, especially in tobacco, where many volatile compounds could be transferred from the tobacco to the smoke by volatilization without any structural changes25. Therefore, cigar varieties could be determined by their volatiles.

Fig. 1
Fig. 1
Full size image

The aroma profiles of cigar tobacco leaves (n = 3). (A) Wrapper of different varieties. (B) Filler of different varieties. (C) Filler of different origins.

Esters provide sweet and fruity flavors to tobacco26. The total esters content of AQ3 increased by 20.01% compared with AQ1, due to isobutyl isobutyrate was newly detected (Fig. 2A), and dihydroactinidiolide and norambreinolid increased by 18.99% and 38.33%, respectively. Methyl 2-methylbutyrate and lavender lactone were newly detected in AQ1 and AQ2. Alcohols help to enhance the floral and fruity aromas of tobacco27. The total alcohols content of AQ1 increased significantly, as the content of newly detected hexa-hydro-farnesol reached 107.01 μg/kg. 2-Hexyldecanol was newly detected and the contents of 3,7,11,15-tetramethyl-2-hexadecen-1-ol and thunbergol were significantly increased in AQ2. Viridiflorol was newly found and the content of 3-methylpentanoic acid increased in AQ3. The total aldehydes content of AQ2 increased significantly, due to the newly detected trans-2-hexenal, and the contents of hexanal and decanal increased to 6.45 μg/kg and 16.14 μg/kg, respectively. Octanal was newly found and the contents of prenal and benzaldehyde increased in AQ1.

Fig. 2
Fig. 2
Full size image

The heatmap cluster of aroma constituents of cigar tobacco leaves. (A) Wrapper of different varieties. (B) Filler of different varieties. (C) Filler of different origins.

Ketones could form important aromatic molecules through carotenoid degradation28. The total ketones content of AQ2 reached 1.56 mg/kg, which was 23.87% and 59.07% higher than AQ1 and AQ3, respectively, due to the newly detected 5-ethyl-6-methyl-3(E)-hepten-2-one, 4,8-dimethylnona-3,7-dien-2-one, and hexahydropseudoionone, the content of hexahydropseudoionone reached 111.28 μg/kg. The contents of butenone, methyl heptenone, ketoisophorone, dihydro-β-ionone, pseudoionone, geranyl acetone, and megastigmatrienone also increased significantly, and geranyl acetone and megastigmatrienone reached 978.89 μg/kg and 181.27 μg/kg, respectively, the former increased by 21.90% and 40.22% compared with AQ1 and AQ3, and the latter increased by 1.63 and 5.10 times, respectively. Megastigmatrienone mainly imparted woody and floral flavors to tobacco29. Acetone, butanone, propione, solanone, nootkatone, and solavetivone were newly detected in AQ1.

The total nitrogen heterocycles content of AQ1 increased significantly, reaching 1.35 mg/kg, which was 1.61 and 0.64 times higher than AQ2 and AQ3, respectively, mainly due to the newly detected methylpyrazine, 2,5-dimethylpyrazine, methylpyrrolidone, cotinine, and pyridine, and the contents of 2-methylpyrrole, nicotinonitrile, 3-acetopyridine, ethylmethylmaleimide, and myosmine increased significantly. The contents of 3-acetopyridine and myosmine reached 588.41 μg/kg and 335.63 μg/kg, respectively, the former increased by 53.81% and 43.22% compared with AQ2 and AQ3, while the latter increased by 2.70 and 1.40 times, respectively. Compounds derived from N-heterocyclic carbenes such as pyridine play a key role in shaping the aroma of cigars30. The total alkenes content of AQ2 reached 227.14 μg/kg and increased by 208.22% and 18.57% compared with AQ1 and AQ3, respectively, as the newly detected β-ocimene and 1-nonadecene. The contents of 1-tetradecene and cembrene increased in AQ2 and AQ3.

As shown in Fig. 3A, the flavor constituents of the first six high OAVs in the three wrapper varieties of CTLs included methyl 2-methylbutyrate, nonanal, decanal, methyl heptenone, isophorone, and dihydro-β-ionone. Among them, the OAVs of nonanal, decanal, methyl heptenone, and dihydro-β-ionone were the highest in AQ2, which imparted strong citrus, orange peel, and floral aromas. The OAVs of methyl 2-methylbutyrate and isophorone were the highest in AQ1 and AQ3, giving apple and cedarwood aromas, respectively.

Fig. 3
Fig. 3
Full size image

OAV flavor profile analysis of cigar tobacco leaves. The OAV profile was expressed as the log of OAV from main volatiles. (A) Wrapper of different varieties. (B) Filler of different varieties. (C) Filler of different origins.

Effect of variety on aroma profiles and differential constituents of filler CTLs

There were 63 aroma constituents detected in filler CTLs of three varieties, including esters (8), alcohols (13), acids (1), aldehydes (10), ketones (16), phenols (1), nitrogen heterocycles (10), and alkenes (4), and their total contents were 2.72–4.27 mg/kg (Table S2). Each of these components has a different concentration, which gave cigar its unique olfactory properties and distinguished it from other types of tobacco31. There were also significant differences in the varieties and contents of aroma constituents. Among them, the contents of ketones, alkenes, and total aroma constituents of QDQX2 increased, the contents of esters, alcohols, and aldehydes of QDQX3 increased, and the contents of nitrogen heterocycles increased both in QDQX2 and QDQX3 (Fig. 1B). There were significant differences in the proportion of aroma profiles between different filler varieties for esters (2.36–7.46%), alcohols (3.46–11.62%), aldehydes (1.24–3.42%), ketones (22.34–47.56%), and alkenes (1.23–5.16%). The filler, which occupied a central position in a cigar, was prioritized as the main ingredient, and it usually accounted for 70% to 85% of the total weight of the cigar, which has a significant impact on the style and characteristics of the cigar32.

The total esters content of QDQX3 increased significantly, reaching 241.01 μg/kg, due to the increased contents of dihydroactinidiolide and methyl butanoate. Acetoxyethane was newly detected and the contents of lavender lactone and norambreinolid increased in QDQX1. The total alcohols content of QDQX3 reached 375.09 μg/kg, which was 56.59% and 153.98% higher than QDQX1 and QDQX2, respectively, as the newly detected 3-acetopropanol and methylundecylcarbinol (Fig. 2B), and the contents of 4,8-dimethylnonanol, phytol, hexa-hydro-farnesol, thunbergol, and geranylgeraniol were significantly increased. Dodecanol and isophytol were newly found in QDQX2. The total aldehydes content of QDQX3 increased by 83.93% and 109.38% compared with QDQX1 and QDQX2, respectively, due to the newly detected nonanal and the increase in octanal content. Neral was newly detected and the contents of methacrylaldehyde, valeraldehyde, prenal, hexanal, benzaldehyde, and 3-methylpentanoic acid increased significantly in QDQX1.

The total ketones content of QDQX2 reached 2.03 mg/kg, which was 1.50 and 1.81 times higher than that of QDQX1 and QDQX3, respectively, due to the newly detected geranyl acetone and cis-ψ-ionone, whose contents were 1.21 mg/kg and 83.19 μg/kg, respectively, and the contents of pseudoionone and farnesyl acetone increased significantly. Methyl tridecyl ketone was newly detected, and the contents of acetone, butenone, methyl heptenone, 5-ethyl-6-methyl-3(E)-hepten-2-one, and ketoisophorone were increased in QDQX1, while isophorone, solanone, and megastigmatrienone were newly detected in QDQX3. As tobacco was processed and aged, solanone underwent a further transformation to produce compounds such as somnirol, solanofuran, and norsolandione, and these derivatives have a considerable effect on the taste of tobacco products33. Besides, dihydrooxophorone and 4,8-dimethylnona-3,7-dien-2-one were detected, and the content of hexahydropseudoionone increased in QDQX1 and QDQX2.

The total nitrogen heterocycles content of QDQX3 was significantly higher than that of QDQX1, due to the newly detected nicotyrine in QDQX3, with the content reaching 637.07 μg/kg. Pyridine, methylpyrazine, and 3-methylpyrrole were newly detected, and the content of 2,5-dimethylpyrrole increased in QDQX2. The contents of 2-methylpyrrole and nicotinotrile in QDQX1 increased, and the contents of myosmine and isonicoteine in QDQX1 and QDQX2 increased significantly. The total alkenes content of QDQX2 increased significantly, reaching 219.98 μg/kg, as 1-nonadecene was detected, and its content was 193.75 μg/kg. Amorphadiene and cembrene were detected in QDQX1 and QDQX3, respectively, while the 1-acetylcyclohexene content of QDQX1 and QDQX2 increased significantly.

As shown in the Fig. 3B, the flavor constituent of the first six high OAVs in the three filler varieties of CTLs included nonanal, decanal, neral, methyl heptenone, geranyl acetone, and 3,5-dimethylphenol. Among them, the OAVs of neral, methyl heptenone, and 3,5-dimethylphenol were the highest in QDQX1, which imparted strong lemon, citrus, and balsamic aromas. The OAVs of decanal and geranyl acetone were the highest in QDQX2, and orange peel and fruity aromas were prominent.

Effect of origin on the aroma profiles and differential constituents of filler CTLs

A total of 64 aroma constituents were detected in four filler origins of CTLs, including esters (9), alcohols (10), acids (1), aldehydes (11), ketones (18), phenols (1), nitrogen heterocycles (9), and alkenes (5), and their total content was ranged from 1.74 mg/kg to 2.87 mg/kg (Table S3). There were significant differences in the varieties and contents of aroma constituents, which showed the origin specificity. Among them, the contents of esters, ketones, nitrogen heterocycles, alkenes, and total aroma constituents of FXQX1 increased, while the contents of alcohols and aldehydes of QDQX1 and QZQX1 increased (Fig. 1C). Besides, there was a significant difference in alcohol proportion (2.06–11.88%) between filler CTLs from different origins. Ketones and nitrogen heterocycles were dominant, with the proportions of 38.66–44.72% and 36.74–43.63%, respectively, the esters, aldehydes, and alkenes were 3.17–11.74%, 2.13–4.90%, and 0.39–2.80%, respectively. The aroma precursor varieties and contents of CTLs from different origins were significantly different, which showed the obvious origin specificity, while different origins in proximity had high similarity in their aroma profiles34.

The total esters content of FXQX1 and ZCQX1 increased significantly, due to the increase in dihydroactinidiolide content, which were 182.36 μg/kg and 181.47 μg/kg, respectively. Ethyl acetate and farnesyl acetate were newly detected (Fig. 2C), and the contents of lavender lactone and sclareolide were increased in QDQX1. Methyl carbonate, methyl propylate, and methyl 2-methylbutyrate were newly detected, and the content of methyl butanoate increased in QZQX1.

The total alcohols content of QDQX1 reached 239.54 μg/kg, which was 1.67–4.33 times higher than that of other origins, due to the newly detected nerolidol, hexa-hydro-farnesol, and 2-hexyldecanol, and the contents of 6,10,14-trimethylpentadecan-2-ol and thunbergol significantly increased. Tetradecanol, isophytol, and phytol were detected, and the content of 3,7,11,15-tetramethyl-2-hexadecen-1-ol increased in FXQX1. The total aldehydes content of QZQX1 increased by 32.76–75.68% compared with other origins, as the newly detected octanal and pentadecanal, and the contents of trans-2-hexenal, nonanal, and decanal increased significantly. Methacrylaldehyde and pentanal were newly detected, and the contents of prenal, hexanal, and benzaldehyde increased in QDQX1.

The total ketones content of FXQX1 increased significantly, reaching 1.24 mg/kg, which increased by 28.70–84.14% compared with other origins, due to the significant increase in the contents of geranyl acetone and megastigmatrienone, reaching 856.40 μg/kg and 124.01 μg/kg, respectively, and the newly detected isophorone and cis-ψ-ionone. Isophorone, a degradation by-product of carotenoids, belonged to the terpenoid family and was known for its powerful and long-lasting aroma, which significantly enhanced the flavor of different tobacco types35. Acetone and 5-ethyl-6-methyl-3(E)-hepten-2-one were newly detected, and the contents of butenone, methyl heptenone, ketoisophorone, dihydrooxophorone, 4,8-dimethylnona-3,7-dien-2-one, hexahydropseudoionone, methyl tridecyl ketone, farnesyl acetone, and 3,5-dimethylphenol increased in QDQX1, and hexahydropseudoionone and farnesyl acetone reached 260.63 μg/kg and 358.53 μg/kg, respectively. 5,6-Epoxy-β-ionone was newly found, and the contents of pseudoionone, solavetivone, and 3-methylpentanoic acid increased in ZCQX1.

The total nitrogen heterocycles content of FXQX1 reached 1.25 mg/kg, which increased by 43.82–96.45% compared with other origins, due to methylpyrrolidone being newly detected, and the nicotyrine content increased significantly, reaching 884.36 μg/kg. The contents of 2-methylpyrrole, myosmine, and isonicoteine in QDQX1 were also significantly increased, reaching 57.43 μg/kg, 233.41 μg/kg, and 402.42 μg/kg, respectively. Pyridine was newly detected, and the content of 1-methylpyrrole increased in QZQX1. The total alkenes content of FXQX1 increased significantly, as the newly detected bicylogermacrene and cembrene, and amorpha-4, 11-diene and β-ocimene were detected in QDQX1 and QZQX1, respectively.

The flavor constituents of the first six high OAVs included methyl 2-methylbutyrate, nonanal, decanal, neral, methyl heptenone, and 3,5-dimethylphenol (Fig. 3C). Among them, the OAVs of neral, methyl heptenone, and 3,5-dimethylphenol were the highest in QDQX1, giving it strong lemon, citrus, and balsamic aromas. The OAVs of methyl 2-methylbutyrate, nonanal, and decanal were the highest in QZQX1, with apple, citrus, and orange peel aromas.

Effect of variety on the microbial community diversity and structure of wrapper CTLs

The high-throughput sequencing technology was used to explore the microbial community characteristics of CTLs from different varieties and origins. The effective sequence range of bacteria and fungi in all samples were 62,338–83,465 and 19,537–98,236, respectively. As the sequencing depth increased, the dilution curve gradually became flat, indicating that the sequencing results were sufficient to reflect the diversity of samples.

As shown in Tables 1 and 2, for the bacterial community, Chao1 and Observed_OTUs indices were higher in AQ2, thereby the community richness was higher. The Shannon and Pielou_e indices and community diversity and evenness were high in AQ3, while the community richness, Shannon, and Pielou_e indices were lower in AQ1. For the fungal community, the community richness, diversity, and evenness were the highest in AQ1, while they were the lowest in AQ2.

Table 1 The bacterial community α-diversity indices of cigar tobacco leaves from different wrapper and filler varieties and filler origins.
Full size table
Table 2 The fungal community α-diversity indices of cigar tobacco leaves from different wrapper and filler varieties and filler origins.
Full size table

NMDS analysis showed that there were obvious differences in the community structure of different varieties of wrapper CTLs (Fig. 4A,B). For the bacterial community, Corynebacterium and Staphylococcus were dominant in the three varieties, but their abundances were different. For the fungal community, the abundances of Alternaria, Nigrospora, and Cladosporium were higher in AQ1. Alternaria was dominant, and Aspergillus abundance was higher in AQ2, while Aspergillus was dominant in AQ3.

Fig. 4
Fig. 4
Full size image

Non-metric multidimensional scaling (NMDS) analysis of the microbial community of cigar tobacco leaves at the genus level (OTU > 1%). (A) Bacterial community of different wrapper varieties. (B) Fungal community of different wrapper varieties. (C) Bacterial community of different filler varieties. (D) Fungal community of different filler varieties. (E) Bacterial community of different filler origins. (F) Fungal community of different filler origins.

The bacterial communities of different varieties of wrapper CTLs included 23 phyla, 43 classes, 104 orders, 156 families, and 915 genera. The dominant bacterial genus composition and abundance of the three varieties of CTLs were slightly different at the genus level (Fig. 5A). The dominant bacteria were Corynebacterium and Staphylococcus, which was consistent with other finding36, with abundances of 45.78–55.34% and 15.16–34.60%, respectively, and the total abundances of the two and Aerococcus were 79.50–90.89%. The abundances of Brachybacterium and Enteractinococcus in AQ2 increased slightly. When cigars undergo stack fermentation, Staphylococcus and Aerococcus could utilize sugars and acids in the CTLs, leading to an increase in stack temperature and pH, which in turn promoted the growth of Corynebacterium37. These three strains exhibited stronger resistance to alkali and salt, giving them a competitive advantage during the stacking fermentation process37. Staphylococcus played a crucial role in carbohydrate catabolism, amino acid conversion, protein hydrolysis, and lipolysis. It mediated the breakdown of branched-chain amino acids into methyl-branched-chain alcohols, aldehydes, carboxylic acids, and their corresponding esters, establishing itself as a key genus in flavor development38. It could quickly metabolize malic acid and citric acids in tobacco, and these organic acids influenced the smoking quality of the tobacco37. Corynebacterium produced aldehydes and ketones, which were crucial in enhancing the flavor profile of cigars39.

Fig. 5
Fig. 5
Full size image

The microbial community composition of cigar tobacco leaves at the genus level (OTU > 1%). (A) Bacterial community of different wrapper varieties. (B) Fungal community of different wrapper varieties. (C) Bacterial community of different filler varieties. (D) Fungal community of different filler varieties. (E) Bacterial community of different filler origins. (F) Fungal community of different filler origins.

The fungal communities included 8 phyla, 27 classes, 66 orders, 135 families, and 194 genera. The composition and abundance of the fungal communities were significantly different (Fig. 5B). Among them, the abundances of Alternaria, Nigrospora, and Cladosporium in AQ1 were higher, with abundances of 27.95%, 10.09%, and 9.76%, respectively. Alternaria was dominant and Aspergillus was higher in AQ2, with abundances of 63.24% and 23.01%, respectively. Aspergillus was dominant with an abundance of 51.90%, while the abundances of Filobasidium, Dolichousnea, and Alternaria increased to 9.98%, 9.23%, and 7.85% in AQ3, respectively. The role of fungi in modulating CTLs may be associated with the degradation of lignin, cellulose, and pectin40. Alternaria was capable of degrading cellulose in CTLs, which contributed to the biosynthesis of sugar derivatives41. Aspergillus promoted the degradation of sugars and proteins as well as the synthesis of aroma compounds in tobacco, which capable of producing various enzymes such as proteases and amylases, to enhance the production of aromatic compounds, to convert carbohydrates into organic acids, alcohols, and esters, thus improving the quality of cigars. Cladosporium was a key filamentous fungus that produced amylase, protease, and cellulase enzymes, which were responsible for breaking down macromolecules and producing characteristic flavor components42.

Effect of variety on the microbial community diversity and structure of filler CTLs

Based on the α-diversity indices analysis, the richness, diversity, and evenness were the highest in QDQX1 both for the bacterial and fungal communities, while they were the lowest in QDQX3. According to the NMDS analysis (Fig. 4C,D), there were obvious differences in the bacterial and fungal community structures of different filler varieties of CTLs. Among them, Corynebacterium and Staphylococcus were dominant in the bacterial communities, but there were significant differences in their abundances. For fungal communities, the abundances of Wallemia, Aspergillus, and Alternaria were higher in QDQX1, and the abundances of Alternaria and Stemphylium were higher in QDQX2, while Aspergillus was dominant in QDQX3.

The bacterial communities of different filler varieties of CTLs included 25 phyla, 40 classes, 99 orders, 148 families, and 228 genera. The compositions of dominant bacterial genera were similar, but there were obvious differences in abundance at the genus level (Fig. 5C). The dominant bacteria were Corynebacterium and Staphylococcus, and their total abundances were 76.67–94.47%, but the abundances of Corynebacterium and Staphylococcus in QDQX1 were 29.97% and 46.70%, they were 60.90% and 22.12% in QDQX2, while they were 54.04% and 40.43% in QDQX3, respectively.

The fungal communities included 10 phyla, 28 classes, 61 orders, 121 families, and 179 genera. There were significant differences in the composition and abundance of the fungal community (Fig. 5D). For example, the abundances of Wallemia, Aspergillus, and Alternaria were higher in QDQX1, with abundances of 23.20%, 18.84%, and 11.50%, respectively. The abundances of Alternaria, Stemphylium, Aspergillus, and Acremonium were higher in QDQX2, with abundances of 28.17%, 12.47%, 8.74%, and 7.89%, respectively. Aspergillus was dominant in QDQX3, with an abundance of 77.17%. The dominant microorganisms possess the ability to degrade, transform, metabolize, and utilize the matrix components of CTLs, thereby playing a vital role in shaping their quality characteristics43. The secretion of α-amylase and amylase by Staphylococcus and Aspergillus was the primary cause of the changes in starch and total sugar content44.

Varieties have little effect on the bacterial community structures of wrapper and filler varieties of CTLs, and there were only differences in their abundances, but there were obvious differences in fungal community composition and abundance. For example, in the fermented wrapper, binder, and filler CTLs, the bacterial community structures were more similar compared to the fungal microbiota, and seven fungal genera were identified as functional microorganisms that may play a crucial role in maintaining the sustainability and stability of the phyllosphere ecosystem19. The wrapper CTLs contained a greater diversity of characteristic fungal genera compared to the filler and underwent fermentation more rapidly, which suggested that the specifics of the fermentation process should be appropriately adjusted to ensure consistent quality when dealing with plant leaves of different genotypes45. The dominant microbes during the cigar stacking fermentation of eight varieties of CTLs primarily influenced the microbial community structure and characteristic microorganisms through microbial interactions, thereby affecting the transformation of volatile flavor compounds6. However, the dominant bacterial and fungal genera of different CTL varieties during fermentation were similar6, which indicated that the fungal communities were more sensitive to environmental conditions than bacteria after fermentation.

The distinctive flavor of CTLs was largely attributed to the metabolic activities of the microbial community composition, which included the degradation of carbohydrates and chlorogenic acid, the breakdown of proteins, the biosynthesis of fatty acids and lipids, and the biosynthesis of amino acids and aromatic compounds37,46. The main pathways used by bacteria to break down macromolecules during CTL fermentation included carbohydrate metabolism, lipid metabolism, and amino acid metabolism16. Intermediates from carbohydrate metabolic pathways could act as raw materials for synthesizing a variety of substances. Amino acids could participate in the Maillard reaction with reducing sugars, a crucial reaction that contributed to the development of aroma in tobacco47. The microbial-mediated hydrolysis of peptides and/or proteins, along with the advancement of the Maillard reaction, played a significant role in modifying the distribution of free amino acids, thereby shaping the distinct flavor profile of CTLs48. The methylerythritol phosphate pathway and the carotenoid synthesis pathway were key contributors to flavor development in CTL from different origins36.

Effect of origin on the microbial community diversity and structure of filler CTLs

The richness, diversity, and evenness of FXQX1 were the highest in the bacterial community, while the richness of QZQX1 and the diversity and evenness of ZCQX1 were the lowest. For the fungal community, the richness of QZQX1 and the diversity and evenness of QDQX1 were the highest, while the richness of FXQX1 and the diversity and evenness of QZQX1 were the lowest. Based on the NMDS analysis (Fig. 4E,F), the bacterial community structures of QZQX1 and ZCQX1 were similar, and there were obvious differences between the two and FXQX1 or QDQX1. Among them, Corynebacterium was dominant and Staphylococcus abundance was higher in the communities of QZQX1 and ZCQX1, while Pseudomonas was dominant in FXQX1, Staphylococcus was dominant and Corynebacterium abundance was higher in QDQX1. Besides, the fungal community structures of ZCQX1 and QDQX1 were similar, and there were obvious differences between the two and QZQX1 or FXQX1. The abundances of Aspergillus, Wallemia, and Alternaria were higher in communities of ZCQX1 and QDQX1, while the abundances of Alternaria and Wallemia were higher in QZQX1, and the abundances of Aspergillus and Wallemia were higher in FXQX1.

The diversity and dynamics of microbial communities were closely related to environmental factors such as climate, soil composition, and agricultural practices, and varied across tobacco-growing regions. The bacterial communities of filler CTLs from different origins included 23 phyla, 37 classes, 73 orders, 166 families, and 275 genera. There were significant differences in the composition and abundance of bacterial communities at the genus level (Fig. 5E). The Corynebacterium abundance was higher in QZQX1 and ZCQX1, the former was 48.42% and the latter was 83.25%, the abundances of Salinicoccus, Brachybacterium, Enteractinococcus, and Brevibacterium in QZQX1 were also higher, with abundances of 12.28%, 11.01%, 6.72%, and 6.10%, respectively. Staphylococcus was dominant, and Corynebacterium abundance was higher in QDQX1, with abundances of 46.70% and 29.97%, respectively. The abundances of Pseudomonas, Blautia, and Corynebacterium were higher in FXQX1, with abundances of 32.48%, 7.06%, and 5.02%, respectively. Pseudomonas promoted the enzymatic degradation of proteins, enriching the pool of amino acids, which significantly contributed to flavor development. Some strains were also capable of degrading nicotine in tobacco, helping to adjust tobacco strength and improve the quality of upper tobacco leaves49,50.

The fungal communities included 12 phyla, 36 classes, 278 orders, 143 families, and 226 genera. The dominant fungal genus composition was similar, but the abundance was different (Fig. 5F). Alternaria, Aspergillus, Wallemia, and Cladosporium were dominant in filler CTLs from different origins, and their total abundances were 58.43–78.41%. Among them, the abundances of Alternaria and Wallemia were higher in QZQX1, which were 46.73% and 24.84%, respectively. The abundances of Aspergillus, Alternaria, Wallemia, and Cladosporium in ZCQX1 were higher, with abundances of 26.63%, 16.68%, 11.80%, and 10.89%, respectively. The abundances of Aspergillus, Wallemia, and Cladosporium in FXQX1 were higher, with abundances of 32.24%, 23.07%, and 9.54%, respectively. The abundances of Wallemia, Aspergillus, and Alternaria in QDQX1 were higher, with abundances of 23.20%, 18.84%, and 11.50%, respectively. Pseudomonas spp., Aspergillus fumigatus, and Rhodococcus spp. degraded nicotine in tobacco into niacin, ethanesulfonic acid, oxidized nicotine, nicotinamide, N-methylnicotinamide, and adamantane51.

Microbial communities from different origins differed significantly from each other, with bacterial microorganisms being more diverse and abundant than fungi, which was in agreement with other study36. It was the bacterial community, rather than the fungal community, that played a key role in differentiating cigars from the Americas, Southeast Asia, and East Asia, which in turn influenced microbial metabolism52. For CTLs from Dominica, Brazil, Indonesia, and China, the relationships between the fungal community and volatile flavor compounds were weaker compared to those with the bacterial community, and most of these relationships were negative, indicating that the bacterial community had a greater impact on CTL flavor12. However, for CTL sourced from six regions of Yunnan Province in China, the influence of fungal microflora on metabolites was more pronounced. In this case, fungi exhibited a stronger regional impact on microbial communities than bacteria, likely due to the shorter geographical distances between the CTLs compared to other studies.

The microbial community on CTLs primarily originated from the soil and surrounding environment during field growth, as well as from the air during the drying process. Geographic factors were the dominant influence shaping the composition of the tobacco microbial community, and differences in geospatial locations led to variations in microbial community structure53. Through intricate interactions, microorganisms from various origins collaborate to influence the structure and function of the entire microbial community, thereby impacting the flavor quality of CTLs. Changes in community structure and composition were largely associated with shifts in filler tobacco leaves, which stemmed from alterations in their metabolic activities54. Besides, geographical location primarily influenced the structure of phyllosphere microbial communities through the following mechanisms: (1) Climate factors, climatic variations, soil properties, and phyllosphere functional traits generally varied with geographical location. (2) The spatial distribution patterns of contemporary natural vegetation were linked to geographical location. (3) Historical changes in elevation have reshaped the spatial distribution of climate variables and vegetation55,56. Plant species or varieties influenced the structure of the phyllosphere microbial community primarily through the following mechanisms: (1) Differences in the assembly mechanisms of the phyllosphere microbial community, ecological strategies, leaf morphological structures, and physicochemical properties among various plant species or varieties57. (2) Parental “vertical transmission” served as a significant source of phyllosphere microorganisms, and the capacity for this vertical transmission may vary among different plant species or varieties. (3) In the case of phyllosphere microbial communities associated with trees or perennial species, the migration of microorganisms from the soil to the phyllosphere may be less frequent due to greater physical distance and the extended time required for host adaptation58.

Microbial recruitment in the phyllosphere appeared to be evolutionarily complex and was thought to occur based on the functional significance of different microbial communities to plant traits and the ecological strategies of the host plant. Temporal or seasonal variations bring about significant changes in temperature, humidity, and solar radiation levels, which in turn induce corresponding shifts in the structure and composition of the foliar microflora59. The seasonality and dynamics of bacterial and fungal communities in the phyllosphere have been effectively predicted60. It was now widely recognized that short-term variations in the microbial communities of the phyllosphere were driven by stochastic weather events, such as strong winds and heavy rainfall, which directly influenced the selective growth and colonization of microbes on leaf surfaces61. The long-term seasonal changes in these communities were attributed to variations in seasonal climate, microbial succession, and the age of the plant host62. The predicted biogeography was a key factor distinguishing foliar microbial communities, even among individuals of the same plant species. This distance-decay relationship suggested that plants growing closer together have more similar phyllosphere microbial communities, with this similarity decreasing as geographic distance increased63.

The correlation between aroma constituents and dominant microorganisms

As shown by correlation analysis (Fig. 6A), Romboutsia in the bacterial community of different wrapper varieties of CTLs was strongly correlated (P < 0.001) with the aroma constituents of isobutyl isobutyrate, 2,6-dimethylpyrazine, viridiflorol, and bicylogermacrene. Lactobacillus was associated (P < 0.05) with decanal and farnesyl acetone. Fungal communities of Nigrospora, Dirkmeia, Moesziomyces, Phaeosphaeria, Archaeorhizomyces, Zeloasperisporium, Russula, Spencerozyma, Neobulgaria, and Symmetrospora were correlated (P < 0.01) with methyl 2-methylbutyrate, hexa-hydro-farnesol, octanal, acetone, butanone, propione, solanone, nootkatone, solavetivone, methylpyrazine, 2,5-dimethylpyrazine, methylpyrrolidone, and cotinine (Fig. 6B).

Fig. 6
Fig. 6
Full size image

Correlation analysis of aroma constituents and dominant microbes (OTU > 1%, P < 0.05, and Spearman’s |RHO|> 0.6). (A) Bacterial community of different wrapper varieties. (B) Fungal community of different wrapper varieties. (C) Bacterial community of different filler varieties. (D) Fungal community of different filler varieties. (E) Bacterial community of different filler origins. (F) Fungal community of different filler origins.

Rothia and Pseudocercospora in the microbial communities of different filler varieties of CTLs were closely related (P < 0.001) to the aroma constituents of δ-decenolactone, dodecyl alcohol, isophytol, crotonaldehyde, geranyl acetone, cis-ψ-ionone, pyridine, methylpyrazine, 3-methylpyrrole, and 1-nonadecene (Fig. 6C,D), and Brachybacterium (P < 0.01) and Iodophanus (P < 0.001) were associated with acetoxyethane, 6,10,14-trimethylpentadecan-2-ol, cis-citral, methyl tridecyl ketone, and amorphadiene. Corynebacterium in the bacterial community was associated (P < 0.05) with isobutyl isobutyrate, pseudoionone, and 2,5-dimethylpyrrole. Filobasidium in the fungal community was correlated (P < 0.05) with lavender lactone, 5-ethyl-6-methyl-3(E)-hepten-2-one, nicotinonitrile, prenal, and ketoisophorone.

Alternaria (P < 0.05), Salinicoccus, Gracilibacillus, Virgibacillus, and Brevibacterium in the microbial communities from different filler origins of CTLs were closely related (P < 0.001) to the aroma constituents of tetradecanol, isophytol, isophorone, cis-ψ-ionone, methylpyrrolidone, bicylogermacrene, and cembrene (Fig. 6E,F). Bacterial communities of Pseudomonas, Ruminococcus_torques_group, Faecalitalea, Prevotella, Atopostipes, Facklamia, and Dorea were closely associated (P < 0.001) with methyl carbonate, methyl 2-methylbutyrate, 4,8-dimethylnonanol, octanal, pentadecanal, butanone, pyridine, β-ocimene. Fungal communities of Iodophanus and Uwebraunia were closely associated (P < 0.001) with 5,6-epoxy-β-ionone and 2,5-dimethylpyrrole. Bulleromyces and Coprinellus were closely associated (P < 0.01) with methyl carbonate, methyl 2-methylbutyrate, 4,8-dimethylnonanol, octanal, pentadecanal, butanone, pyridine, and β-ocimene, whereas Phaeosphaeria was associated (P < 0.05) with prenal, hexanal, myosmine, butenone, farnesyl acetone, and 1-acetylcyclohexene.

Correlation analysis has facilitated the screening of functional microorganisms and the development and application of microbial agents aimed at enhancing flavor quality and producing characteristic aromatic cigars. For example, in vitro isolation and bioaugmentation inoculation with Candida parapsilosis and Candida metapsilosis have been shown to significantly decreased alkaloid content while increasing the concentration of flavor components in tobacco leaves64. The addition of Bacillus altitudinis enhanced the transformation of macromolecules in CTLs and effectively promoted aroma production, the total aroma production increased by 43% compared to natural fermentation65. The contents of solanone, 6-methyl-5-hepten-2-one, benzeneacetic acid ethyl ester, cyclohexanone, octanal, acetophenone, and 3,5,5-trimethyl-2-cyclohexen-1-one in CTLs were significantly elevated following the inoculation with Acinetobacter sp. 1H866. The changes in microbial communities were primarily associated with their varied functions during fermentation. This suggested that when the fermentation effect of the original microbial community in CTLs was not optimal, we could optimize or design the microbial community based on the specific fermentation functions needed67. Besides, the correlation analyses have shown that microorganisms affect tobacco aroma through synergistic effects, therefore, when searching for functional microorganisms to improve the aroma quality of tobacco, it was important to consider the synergistic effects between microorganisms68.

Conclusion

Aroma constituents of the ketones and nitrogen heterocycles were dominant in wrapper and filler CTLs from different varieties and filler CTLs from different origins. The proportions of the two in different CTL varieties varied greatly, while the differences between various origins were not obvious. There were significant differences in the varieties and contents of aroma constituents in CTLs of different varieties and origins, which exhibited obvious variety and origin specificity. For example, the contents of alcohols, nitrogen heterocycles, and total aroma constituents in the wrapper variety of AQ1 increased significantly, while the contents of aldehydes, ketones, and alkenes of AQ2 increased. The contents of ketones, alkenes, and total aroma constituents in the filler variety of QDQX2 increased significantly, while the contents of esters, alcohols, and aldehydes of QDQX3 increased. The contents of ketones, nitrogen heterocycles, alkenes, and total aroma constituents in the origin of FXQX1 increased significantly, while the alcohols and aldehydes in QDQX1 and QZQX1 increased, respectively. Besides, the microbial community diversity and structure of CTLs were significantly affected by variety and origin. There were obvious differences in the α- and β-diversity of microbial communities in different varieties and origins. Varieties have little effect on the bacterial communities of wrapper and filler varieties of CTLs, Corynebacterium and Staphylococcus were dominant and there were only differences in their abundances, but there were obvious differences in fungal community composition and abundance. Different from the influence of variety, the composition and abundance of the bacterial community were significantly changed by the origin. Alternaria, Aspergillus, Wallemia, and Cladosporium were dominant in the fungal communities, with only differences in their abundances. The correlation analysis showed that the formation of aroma profiles of CTLs from different wrapper and filler varieties and filler origins was closely related to the microbial communities, with the synergistic effect of different bacterial and fungal communities, and the microbial communities that promoted the formation of the aroma profiles were obviously different among different wrapper and filler varieties and filler origins.

Materials and methods

Materials and reagents

The chemical reagents utilized in the experiments were procured from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). The phenethyl acetate standard was obtained from Anpel Laboratory Technologies Inc. (Shanghai, China). The CTLs were harvested in 2021 and obtained from the production regions in Shandong, China (Table 3).

Table 3 The cigar tobacco leaves used in this study.
Full size table

Analysis of aroma constituents

The aroma constituents of CTLs were analyzed by HS–SPME–GC–MS, in accordance with the method of the reference29. In summary, 0.5 g of CTL powder was weighed and placed in a 20 mL headspace vial. Subsequently, 2 μL of 105 mg/L phenethyl acetate was added, the vial was rapidly sealed and shaken, and the mixture was heated at 70 °C for 30 min. Then the mixture was extracted using a 50/30 μm DVB/CAR/PDMS fiber (Supelco Inc., Bellefonte, PA, USA) for 30 min.

The analysis was conducted using a GC–MS (7890A/5977C, Agilent Technologies Inc., Santa Clara, CA, USA) equipped with an elastomeric quartz capillary column (DB-5MS, 30 m × 0.25 mm × 0.25 μm, Agilent Technologies Inc.). The heating program was set to an initial temperature of 40 °C and maintained for 2 min. Thereafter, the temperature was increased to 220 °C at a rate of 6 °C/min and maintained for 0 min. Subsequently, the temperature was elevated to 280 °C at a rate of 20 °C/min and maintained for 10 min. The temperature of the inlet was 240 °C, and the carrier gas was helium (99.999%). The conditions of mass spectrometry were as follows: electron impact (EI) with an ionization voltage of 70 eV, the temperatures of the ion source and transmission line were 230 °C and 290 °C, respectively, and a full scanning mode with a mass scanning range of 33–325 amu. The aroma constituents were qualified by comparative analysis of the mass spectrometry data according to the NIST 17 standard library, and semi-quantification was performed by comparing the area of the total ion chromatogram with that of the internal standard.

Analysis of microbial community

The DNA of the microorganisms present in the samples was extracted using either the CTAB or SDS methods. Following this, the purity of the extracted DNA was detected using agarose gel electrophoresis. The samples were then diluted to a concentration of 1 ng/μL using sterile water. The 16SV34 and ITS1 regions were selected for sequencing using the diluted DNA as a template. Primers 341F and 806R, and the primers ITS1-F and ITS2 were employed for the purpose of PCR amplification. The PCR products were subjected to electrophoresis on an agarose gel at a concentration of 2%. The library was constructed using a library construction kit. Following quantification using the Qubit and qPCR methods, the constructed libraries were quantified and then sequenced using the HiSeq2500 PE250 platform.

The effective tags were obtained by splicing the reads of each sample using FLASH, followed by strict filtering and tags quality control according to the specifications outlined in the Quantitative Insights Into Microbial Ecology (QIIME) protocol. The complete set of effective tags from all samples was clustered using Uparse software, with sequences clustered into operational taxonomic units (OTUs) by default at a 97% similarity. The species annotation analysis was conducted using the Mothur method and the SSU rRNA database. The complexity of the samples was analyzed using the α-diversity indices, which were calculated with the QIIME software.

Statistical analysis

All analyses were conducted in triplicate, and the resulting data were presented as mean ± standard deviation. One-way analysis of variance (ANOVA) and Duncan’s multiple comparison test were employed by IBM SPSS Statistics 25 (IBM Corp., Armonk, NY, USA) to assess the discrepancies between samples69. The odor activity value (OAV) was the ratio of the volatiles concentration to the volatiles odor threshold. Non-metric multidimensional scaling (NMDS) was conducted using the OmicShare tool (https://www.omicshare.com/tools). Pearson correlation analysis was conducted using the R to establish a correlation between the aroma constituents and microorganisms.