Dynamic changes and potential contributions of arbuscular mycorrhizal fungi in cigar tobacco fermentation

Dynamic changes and potential contributions of arbuscular mycorrhizal fungi in cigar tobacco fermentation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Dynamic changes and potential contributions of arbuscular mycorrhizal fungi in cigar tobacco fermentation Hui Zhang, Xueru Song, Qi Zhou, Yuming Yin, Ying Yang, Jilai Zhang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7416527/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Arbuscular Mycorrhizal Fungi (AMF) are key species in plant-microbe interactions, and this study is the first to discover their dynamic survival in the fermentation system of cigar tobacco. To explore the functional significance of AMF in cigar tobacco fermentation, this study focused on the Yunxue variety of cigar tobacco. We combined multi-time point sampling over a 35-day fermentation process and used Internal Transcribed Spacer (ITS) gene high-throughput sequencing to analyze the AMF community structure. Diversity indices, species correlation networks, and Mantel tests were employed to explore the relationship between AMF and chemical components. The study revealed a significant dynamic succession within the arbuscular mycorrhizal fungi (AMF) community throughout the fermentation process, identifying 22 species (comprising 524 operational taxonomic units [OTUs]), with Paraglomus being the predominant species. Core functional flora included OTU217 and OTU88, whose abundance variations aligned with the generation of volatile flavor compounds. AMF diversity peaked during the mid-fermentation stage and exhibited a negative correlation with total nitrogen (TN), total sulfur (TS), and reducing sugars (RS), indicating that sugar and nitrogen metabolism were driving factors in the reorganization of the AMF community. Notably, Glomus-group-B-Glomus-lamellosu-VTX00193 demonstrated a marked increase in abundance towards the end of fermentation, suggesting its crucial role in the degradation of complex organic compounds. Analysis specific to different tobacco varieties revealed a significant increase in the number of OTUs unique to Yunxue 6, with fluctuations in total acidity (TA) content significantly associated with changes in AMF abundance. The findings highlight the regulatory role of AMF in modulating the chemical composition of tobacco leaves through carbon and nitrogen metabolism, with Paraglomu s and Glomus identified as core functional flora. These results offer a foundational framework for targeted manipulation of AMF communities and the design of innovative fermentation processes. Cigar Tobacco Fermentation Arbuscular Mycorrhizal Fungi Physicochemical Components Microbial Community Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Key Points The dynamic survival of arbuscular mycorrhizal fungi (AMF) during cigar tobacco fermentation and their functional roles are revealed for the first time. Paraglomus is identified as the dominant AMF genus, influencing tobacco chemical composition through modulation of carbon and nitrogen metabolism. AMF community diversity varies significantly across tobacco cultivars, with Yunxue No. 6 exhibiting the highest richness of rare species, while Glomus-lamellosu-VTX00193 increases markedly in late fermentation stages, potentially contributing to lignin degradation and flavor enhancement. Introduction The fermentation of cigar tobacco is a critical step in enhancing tobacco leaf quality. Its primary objectives are to improve aroma, optimize the smoking experience, and ensure product consistency and safety (Liu et al., 2021 ). Fermentation of tobacco leaves via microbial metabolism serves to degrade impurities and irritants, resulting in a purer aroma and a smoother taste. This process also facilitates the conversion of carbohydrates and amino acids, leading to the production of various aromatic compounds like ketones, alcohols, and terpenes. These transformations contribute to the distinctive aroma and multifaceted flavor profile characteristic of cigars (Zhang et al., 2024 ). Additionally, fermentation improves the physical properties of the tobacco leaves, enhancing their flexibility and combustion performance. More importantly, the fermentation process effectively reduces the content of harmful substances such as nicotine and nitrosamines, thus improving the health quality of the cigars. In conclusion, fermentation not only determines the aroma and taste of cigars but also ensures their high quality and market competitiveness (Zhang et al., 2024 ). The study of microbial community changes during cigar tobacco fermentation has become a key focus of current research. It not only helps to reveal the mechanisms behind the generation of aromatic compounds but also facilitates the improvement of cigar quality, consistency, and safety through the regulation of fermentation conditions. This makes it a crucial aspect of enhancing cigar production and quality control (Su et al., 2024 ). During the fermentation of cigar tobacco leaves, the microbial community exhibits significant dynamic changes, which can be divided into three main stages. In the early stage, the diversity of bacteria predominates, with the major phyla being Firmicutes and Proteobacteria . Studies have shown that Corynebacterium , Pseudomonas , and Staphylococcus genera exhibit high abundance in this stage. These microorganisms are involved in the breakdown of carbohydrates in the tobacco leaves, such as starch, pectin, and cellulose. In the mid-fermentation stage, after rehydration, microbial communities begin to undergo significant shifts as humidity and total acidity increase. Aerococcus dominates during this stage, utilizing reducing sugars and organic acids (such as malic acid and citric acid) in the tobacco leaves. This leads to increased temperature and pH in the fermentation pile, promoting the growth of other microorganisms. At the same time, the structure of the fungal community remains relatively stable, with Aspergillus , Alternaria , and Cladosporium continuing to dominate. In the final fermentation stage, the drying phase, microbial diversity decreases. Firmicutes continues to dominate, while Aspergillus remains the primary fungal genus. At this stage, the dynamic changes in the microbial community gradually stabilize, accompanied by an increase in volatile compounds (such as ketones, aldehydes, and terpenes), which intensifies the aroma (Guo et al., 2024 ; Wu et al., 2024 ; Si et al., 2023 ; Xue et al., 2023 ). It can be observed that throughout the fermentation process, microorganisms such as Aerococcus , Pseudomonas , Staphylococcus , and Aspergillus play key roles. They are not only responsible for the transformation of chemical components in the tobacco leaves but are also closely associated with the generation of various volatile organic compounds (VOCs). In particular, Pseudomonas is positively correlated with several ketones and aldehydes (such as isophorone and acetophenone), which significantly influence the aroma characteristics of cigars, imparting them with a rich, sweet, and roasted fragrance (Liu et al., 2021 ; Zhang et al., 2023 ). Additionally, Aspergillus and other fungi in the community are related to the generation of terpenes and aromatic compounds (such as indole), which contribute to the unique floral and fruity characteristics of cigars (Zhang et al., 2024 ; Wu et al., 2023 ). Arbuscular Mycorrhizal Fungi (AMF), belonging to the phylum Glomeromycota , are obligate symbiotic fungi (Willis et al., 2013 ). They form mutualistic symbiotic relationships with over 80% of terrestrial plants by penetrating plant roots to form arbuscules, which serve as the core structures for nutrient exchange between the host plant and the fungi. This symbiotic relationship significantly enhances the plant's ability to absorb essential mineral elements such as phosphorus, nitrogen, and zinc. In particular, in poor or arid soils, the fungal mycelial network of AMF can greatly extend the root system’s ability to absorb nutrients (Gosling et al., 2006 ). Furthermore, AMF secretes glomalin (a sticky glycoprotein) to improve soil aggregation, enhancing water retention and organic matter content. Its ecological functions have been widely applied in the fields of sustainable agriculture and soil remediation (Singh et al., 2013 ; Purin et al., 2007). However, traditional views hold that AMF is highly dependent on living plant roots, and its survival is strictly limited by the active carbon sources and cellular signals provided by the host. Consequently, AMF is generally considered unable to survive once the plant tissue dies or during processing, leading to its potential role in food and tobacco fermentation being largely overlooked, with relevant research being nearly nonexistent. It is noteworthy that recent studies have found that AMF may maintain metabolic activity through spore dormancy or by utilizing residual carbon sources in plant remains, providing new insights into understanding the ecological adaptability of AMF in non-symbiotic environments (Pepe et al., 2018 ; Wei et al., 2019 ; Gavito et al., 2003). However, whether these phenomena have functional significance remains unknown. In the current cigar tobacco fermentation process, the known and widely used strains mainly include pectinase-producing bacteria, protease-producing bacteria, lactic acid bacteria, yeasts, acetic acid bacteria, cellulose-degrading bacteria, and antioxidant bacteria. These strains improve tobacco leaf quality by degrading macromolecular organic compounds, generating flavor precursors, or regulating redox states (Su et al., 2024 ; Ren et al., 2023 ; Song et al., 2024 ; Zheng et al., 2022 ). While the utilization of current strains is well-established, their functional constraints pose a barrier to the advancement of fermentation strain innovation. This research represents the initial investigation into the dynamic shifts of the arbuscular mycorrhizal fungi (AMF) community within a cigar fermentation system. The findings validate that AMF can sustain metabolic activity via spore dormancy or residual carbon sources in plant remnants. The alterations in microbial communities during cigar fermentation are pivotal for the development of cigar aroma and excellence. Hence, delving into the functional importance and potential impact of AMF in the cigar fermentation process not only elucidates the microbial mechanisms underlying cigar aroma formation from a novel perspective but also introduces fresh concepts and a theoretical framework for enhancing cigar production techniques and elevating cigar quality. This study aimed to elucidate the role of arbuscular mycorrhizal fungi (AMF) in tobacco degradation, flavor development, and stress response. Additionally, it sought to assess the suitability of AMF as a novel fermentation agent and propose a novel approach for targeted modulation of the cigar fermentation microbiome. Correlation analysis was conducted to investigate the relationship between physicochemical constituents and fungal populations during tobacco leaf fermentation. By elucidating the intricate interplay between fungi and the physicochemical characteristics of tobacco leaves, the study unveiled the potential impact of fungi on cigar fermentation. Materials and methods Cigar tobacco fermentation management and sample collection The fermentation site was located at the Ede Cigar Drying and Fermentation Management Workshop in Mosha Town, Xinping County, Yunnan Province, Yuxi City, China, at an altitude of 493 meters. The selected cigar varieties included Yunxue No. 1, Yunxue No. 2, and Yunxue No. 6. The fermentation facility measured 10m x 8m x 5m, employing the fermentation method with each fermentation pile weighing 500kg. Each of the three cigar varieties was processed in a separate fermentation plant, totaling three plants. The fermentation process maintained a temperature of 35°C and a humidity level of 75%. Fermentation lasted for 35 days, with sampling conducted weekly, removing 500g of tobacco leaves from each fermentation pile using a 5-point sampling technique. The tobacco samples were promptly stored at -80°C in an ultra-low temperature refrigerator. Determination of physicochemical components The collected samples were ground into powder using a mortar in liquid nitrogen and stored in a − 80°C ultra-low temperature freezer. Subsequently, the total nitrogen, total sugar, reducing sugar, total alkaloids, and other physicochemical components of the tobacco leaves were measured. The standards for the determination methods are shown in Table 1 (Fu et al., 2018; Jia et al., 2023 ; Ren et al., 2023 ). Table 1 Methods and equipment for testing physicochemical composition of cigar Physicochemical composition Detection method follows the standard Total nitrogen (TN) Tobacco and tobacco products-Determination of total nitrogenContinuous flow method (YC/T 161–2002) Total alkaloid (TA) Tobacco and tobacco products-Determination of total alkaloids-Continuous flow (potassium thiocyanate) method (YC/T 468–2013) Total sugar (TS) Determination of water-soluble sugars in tobacco and tobacco products by continuous flow method (YC/T 159–2019) Reducing sugar (RS) Determination of reducing sugar in sugar beet root (NY/T 1751–2009) Tobacco leaf DNA extraction and PCR amplification Genomic DNA was extracted and detected using 1% agarose gel electrophoresis. Based on the specified sequencing regions, specific primers with barcodes were synthesized. Low cycle number amplification was used to ensure consistent amplification cycle numbers across all samples. A pre-experiment randomly selected representative samples to ensure that, under low cycle numbers, most samples generated products of appropriate concentration. PCR amplification was performed using TransGen AP221-02 TransStart Fastpfu DNA Polymerase, with an ABI GeneAmp® 9700 PCR instrument. The amplification primers used were AML1F-AML2R and AMV4-5NF-AMDGR (Ma et al., 2024 ). In the formal experiment, each sample was run in triplicate, and the mixed products were detected using 2% agarose gel electrophoresis. The PCR products were recovered from the gel using the AxyPrep DNA Gel Extraction Kit, eluted with Tris-HCl, and rechecked using 2% agarose gel electrophoresis. Based on the preliminary quantification results from electrophoresis, PCR products were quantified using the QuantiFluor™-ST blue fluorescence quantification system from Promega. The samples were then mixed in proportions according to sequencing requirements. Library construction and sequencing In Illumina sequencing, linker sequences were initially incorporated at both ends of the target region using PCR technology. Subsequently, the PCR products were fragmented and isolated utilizing gel extraction kits, eluted with Tris-HCl buffer, and assessed for recovery efficiency via 2% agarose gel electrophoresis. The resulting product was then denatured with sodium hydroxide to yield single-stranded DNA fragments, which were preserved using the TruSeq™ DNA Sample Prep Kit. Upon progression to the sequencing phase, the linker sequences at the fragment ends could hybridize with complementary bases immobilized on the chip surface, thereby anchoring the fragment to the chip. Utilizing the immobilized DNA fragment as a template, the chip-bound sequence served as a primer for bridge PCR amplification, facilitating the synthesis of a new complementary strand. Subsequent denaturation and annealing allowed the other end of the newly synthesized strand to randomly hybridize with an adjacent primer, forming a "bridge" structure. Numerous DNA clusters were generated through iterative bridge amplification and subsequently linearized into single strands. During sequencing, a specialized DNA polymerase and dNTPs containing fluorescent labels and termination groups were utilized, with the introduction of only one base per reaction cycle. A laser scanner identified the fluorescently labeled base incorporated into each DNA template during synthesis, followed by the removal of the fluorescent and termination groups to reactivate the 3' end for the subsequent cycle of base addition. Ultimately, the precise nucleotide sequence of the desired DNA fragment was determined through statistical analysis of the fluorescence signals from each sequencing cycle. Statistical analysis Statistical analysis of tobacco leaf physicochemical components and sequencing data was performed using Excel 2019 and SPSS 26.0 software. One-way ANOVA was used to evaluate the differences in the changes of physicochemical components and AMF microbial diversity during cigar tobacco fermentation. Pearson correlation analysis was conducted to explore the potential correlation between the AMF microbial community and the physicochemical components of tobacco leaves. Alpha diversity, based on Sobs, Shannon, and Chao indices, was used to study the dynamic changes in species diversity during fermentation. The relevant calculations and visualizations for AMF community analysis were performed in R software (version 4.1.1, https://www.r-project.org ) and its “vegan,” “phyloseq,” “DESeq2,” and “picante” packages(Aci, et al., 2025 ). All data are presented as "mean ± standard error," and graphs were generated using Excel 2019, GraphPad Prism 8.0, and MATLAB 2019 software. Results Dynamic succession patterns and variety-specificity of the AMF community OTUs The species annotation results of the AMF community in this cigar tobacco fermentation study encompass species quantities across different taxonomic levels. A total of one phylum and one class were identified, further subdivided into five orders, five families, five genera, and 22 species, resulting in the detection of 524 Operational Taxonomic Units (OTUs). Through OTU analysis (Fig. 1 ), it is clear to observe and record the dominance of specific AMF communities at particular stages during the fermentation of cigar tobacco and how these communities change over time. As shown in Fig. 1 , both shared and unique OTUs between varieties were evident before and after fermentation, demonstrating the diversity and similarity of microbial communities. During fermentation, the number of unique OTUs in Yunxue No. 6 and the shared OTUs among all three varieties increased significantly, while the shared OTUs between Yunxue No. 1 and Yunxue No. 6, as well as between Yunxue No. 2 and Yunxue No. 6, showed a downward trend. This suggests that the AMF communities of different cigar tobacco varieties exhibit significant differences in OTU numbers as fermentation progresses. The distribution of overlapping and unique OTUs highlights the impact of different fermentation conditions on the diversity of the AMF community. The number of OTUs in all three varieties showed different trends during fermentation, particularly from the early to mid-fermentation stages. Yunxue No. 2 and Yunxue No. 6 displayed a more stable OTU number, showing a trend of decrease followed by an increase, while Yunxue No. 1 exhibited significant fluctuations during the mid-fermentation stage. These fluctuations may reflect the competition and interactions among different AMF species, that is, the successional dynamics of AMF across different habitats and time scales, while also revealing the differences in physicochemical properties among the three cigar tobacco varieties (Hart et al., 2001 ). Due to the differences in the content of macromolecules (such as proteins, starch, pectin, etc.) between tobacco leaf varieties, these physicochemical properties may significantly impact the composition and metabolic activity of the AMF community during fermentation, leading to significant fluctuations in the community structure of AMF throughout the fermentation process. This variability also suggests that the variety-specific chemical composition plays an important regulatory role in the adaptability and ecological succession of microorganisms. However, for all three varieties, the number of OTUs showed a clear increase from the early stages of fermentation to the end of fermentation (Wang et al., 2024 ; Wu et al., 2024 ). During the 35-day fermentation process, the AMF communities of Yunxue No. 1, Yunxue No. 2, and Yunxue No. 6 exhibited dynamic changes. Table 2 represents taxonomic statistics of species in different OTU sets. OTU217 consistently dominated across all varieties, and its significant presence indicates the core role of AMF fungi in the cigar tobacco fermentation process. During fermentation, certain saprophytic fungi may play an important role in the chemical composition changes of tobacco leaves. The sustained high abundance of OTU217 may suggest its potential positive impact on tobacco leaf quality through processes such as organic matter degradation and nutrient cycling during fermentation (Zhao et al., 2024 ; Yang et al., 2024 ). At the same time, the abundance changes of OTU217 and OTU88 were prominent across the three varieties, which aligns with the role of AMF as "key mutualists" in soil ecosystems, indicating that this phenomenon also exists in cigar tobacco fermentation (Yang et al., 2014 ). In the later stages of fermentation, the presence of these OTUs will be related to the flavor and texture of the cigar tobacco, as studies have shown that any high-abundance specific fungi can alter the chemical composition in tobacco leaves during fermentation, thereby affecting the sensory properties of the final product (Zhang et al., 2023 ). Table 2 OTU taxonomic statistics Order Family Genus Species OTU Total Percent Prevalence o__Paraglomerales f__Paraglomeraceae g__Paraglomus s__unclassified_g__Paraglomus OTU217 176698 0.592819 96.15% o__Paraglomerales f__Paraglomeraceae g__Paraglomus s__unclassified_g__Paraglomus OTU88 33198 0.111379 92.31% o__unclassified_c__Glomeromycetes f__unclassified_c__Glomeromycetes g__unclassified_c__Glomeromycetes s__unclassified_c__Glomeromycetes OTU216 11678 0.03918 96.15% o__Paraglomerales f__Paraglomeraceae g__Paraglomus s__Paraglomus-Glom-1B.13-VTX00308 OTU82 4239 0.014222 26.92% o__Paraglomerales f__Paraglomeraceae g__Paraglomus s__unclassified_g__Paraglomus OTU76 3458 0.011602 26.92% o__unclassified_c__Glomeromycetes f__unclassified_c__Glomeromycetes g__unclassified_c__Glomeromycetes s__unclassified_c__Glomeromycetes OTU219 2530 0.008488 61.54% o__unclassified_c__Glomeromycetes f__unclassified_c__Glomeromycetes g__unclassified_c__Glomeromycetes s__unclassified_c__Glomeromycetes OTU24 1594 0.005348 3.85% o__unclassified_c__Glomeromycetes f__unclassified_c__Glomeromycetes g__unclassified_c__Glomeromycetes s__unclassified_c__Glomeromycetes OTU265 1657 0.005559 61.54% o__unclassified_c__Glomeromycetes f__unclassified_c__Glomeromycetes g__unclassified_c__Glomeromycetes s__unclassified_c__Glomeromycetes OTU470 3047 0.010223 69.23% o__unclassified_c__Glomeromycetes f__unclassified_c__Glomeromycetes g__unclassified_c__Glomeromycetes s__unclassified_c__Glomeromycetes OTU260 1614 0.005415 50% AMF microbial diversity index analysis The Sobs index represents the number of observed species and reflects the trend in community richness (Fig. 2 A). Before fermentation, the richness of the three cigar tobacco varieties was relatively low, showing a relatively small number of OTUs. By the second week, the Sobs index of Yunxue No. 1 and Yunxue No. 6 increased rapidly, with a significant rise in richness, peaking in the third week. Afterward, it showed a declining trend, reaching its lowest point in the fifth week, indicating that these two varieties may share a similar AMF community succession pattern. In contrast, Yunxue No. 2 showed a decrease in the second week and did not exhibit a continuous upward trend. This suggests that the chemical components in Yunxue No. 2 may be more difficult for AMF to utilize directly in the early fermentation stages, or that the available substrates for AMF are limited. For example, if certain macromolecules in Yunxue No. 2 are difficult to degrade, the growth of AMF may be inhibited. This is consistent with the relationship between AMF and substrate availability observed in the study by Gryndler et al. (2022). During fermentation, microbial communities must continuously adapt to the changing chemical environment. The chemical composition of Yunxue No. 2 may lead to fluctuations in its AMF community's adaptability, resulting in a decrease during the second week. The succession pattern of AMF in the fermentation process of all three cigar tobacco varieties followed a "rapid growth—peak—gradual decline" trend, indicating that the AMF succession pattern is similar to that of typical microbial community succession. Despite differences in the physicochemical properties of these varieties, the succession of the AMF community during fermentation is driven by similar external factors, such as nutrient depletion and environmental changes. This phenomenon suggests that the microbial dynamics during fermentation in different cigar tobacco varieties may follow a common pattern, which is of significant importance for the standardization and optimization of fermentation processes (Michele et al., 2007 ; Si et al., 2023 ). The Shannon index (Fig. 2 B) shows the microbial community diversity of the three cigar tobacco varieties, with significant differences in the trends observed during fermentation. Yunxue No. 1 displayed a wavy trend, with the highest diversity occurring in the third week. Yunxue No. 2 and Yunxue No. 6 followed a "decrease followed by increase" trend, with Yunxue No. 6 reaching the highest microbial diversity in the first week. Yunxue No. 1 may have introduced more competitive microorganisms during fermentation, which dynamically competed with AMF and other microorganisms at different fermentation stages (Atta et al., 2022 ). For example, in the early fermentation stage, certain microbial communities may grow rapidly, causing the Shannon index to decline; later, other microorganisms reoccupy ecological niches, leading to an increase in diversity. This frequent competitive replacement process results in the wavy fluctuations of the Shannon index. For Yunxue No. 2 and Yunxue No. 6, the competitive effect may be weaker, mainly due to the gradual depletion of nutrients by the early dominant microorganisms, followed by the recovery of secondary microorganisms, showing a single "decrease then increase" pattern. The Chao index (Fig. 2 C) also shows significant fluctuations during fermentation for all three varieties, with clear differences in the patterns observed. Notably, the highest and lowest points for each variety's index do not align. For Yunxue No. 1, the richness of rare species peaked in the second week and was lowest in the fifth week; for Yunxue No. 2, it was highest in the fifth week and lowest in the third week; and for Yunxue No. 6, it was highest in the fourth week and lowest in the first week. The Chao index for the three cigar tobacco varieties exhibited different fluctuation patterns during fermentation, particularly the differences between the highest and lowest points. This may be due to differences in the physicochemical components and nutritional substrates of each variety, which cause rare species in the microbial community to respond differently to specific nutrients at different fermentation stages (Li et al., 2024 ; Little et al., 2008 ). Specifically, the composition of Yunxue No. 1 promotes the rapid growth of rare species in the early fermentation stage, resulting in the highest richness in the second week. In contrast, the composition of Yunxue No. 2 and Yunxue No. 6 favors the continued colonization and growth of rare species in the later stages of fermentation, leading to peak richness in the fourth and fifth weeks, respectively. These differences in substrate composition may affect the adaptability and colonization dynamics of rare species, causing them to reach their peak richness at different stages of fermentation. Dynamic differences of AMF at the genus level As shown in Fig. 3 , during fermentation, the AMF communities of the three varieties exhibited significant differences at the genus level. Paraglomus was the absolute dominant genus, with the highest relative abundance across all varieties and fermentation stages, indicating its strong adaptability and stability in the fermentation environment. This may be closely related to the high metabolic activity and competitive ability of Paraglomus in the high-temperature, low-oxygen, and nutrient-rich fermentation environment, allowing it to dominate and become the core functional group in the fermentation process. Furthermore, the dominance of Paraglomus may play an important role in key metabolic functions, such as carbon-nitrogen cycling and organic matter degradation (Gosling et al., 2014 ; Mello et al., 2013 ). Unclassified_c__Glomeromycetes ranked second to Paraglomus and showed a gradual increase in relative abundance towards the end of fermentation, reflecting its adaptation to the environmental changes in the later fermentation stages. This phenomenon is related to its specific metabolic functions, such as the degradation of residual materials or participation in nutrient cycling, thereby reflecting the dynamic balance and functional division within the microbial ecosystem (Oehl et al., 2011 ). In the later stages of fermentation, the environmental changes caused by nutrient depletion and the accumulation of metabolic products likely provided a new ecological niche for this genus, promoting its increased abundance. Additionally, an interesting observation was made that the AMF community of Yunxue No. 2 exhibited relatively high diversity in the first week of fermentation, with a significant presence of Glomus_f__Glomeraceae and a small amount of unclassified_o__Archaeosporales . However, as fermentation progressed, the community structure gradually stabilized and became more consistent with the other two varieties. This initial high diversity reflects the more complex microbial ecosystem of Yunxue No. 2 in the early stages of fermentation, which may be related to the unique metabolic demands or greater microbial adaptability under specific conditions of this variety. The fluctuations in the microbial community observed in the early fermentation stages could be linked to the initial phase of material degradation and the rapid response of the community to environmental changes (Zhang et al., 2023 ). Overall, these findings suggest that environmental selection pressures during fermentation drive the concentration of dominant AMF populations, while also revealing the functional division of different microbial communities at various stages of fermentation. These results provide important insights into the potential role of AMF in tobacco fermentation and suggest that future efforts could optimize community functions by regulating fermentation conditions, thereby improving fermentation quality. Figure 4 illustrates the changes in the relative abundance of the two most abundant species, Paraglomus and unclassified_c__Glomeromycetes , across the three varieties. Paraglomus consistently dominated throughout the fermentation process but exhibited significant fluctuations in abundance across the different varieties. In Yunxue No. 1, the relative abundance of Paraglomus followed a "decrease, increase, then decrease" trend, with the highest abundance occurring in the mid-fermentation phase. This may suggest that the Paraglomus community has slightly weaker adaptability to the early fermentation environment, but its metabolic activity increases as fermentation progresses. However, by the end of fermentation, its abundance likely decreased again due to nutrient depletion or the inhibitory effects of secondary metabolites. For Yunxue No. 2, the fluctuations of Paraglomus were more pronounced, with a significant decrease in abundance during the third week, and the highest abundance observed in the pre-fermentation stage. This reflects that the initial microbial activity in this variety was high, but during the mid-fermentation stage, the community faced greater environmental pressures, possibly influenced by pH fluctuations or nutrient competition (Wu et al., 2024 ). In contrast, the fluctuations of Paraglomus in Yunxue No. 6 were smaller, with abundance gradually increasing after fermentation began, stabilizing in the mid-fermentation phase, and decreasing as fermentation neared its end. This suggests that the AMF community in Yunxue No. 6 was more stable, with its AMF population possibly better adapted to the changes in the fermentation environment. Overall, the relative abundance of Paraglomus at the end of fermentation was generally lower than in the pre-fermentation stage, indicating the presence of limiting factors that restricted its growth during fermentation. Meanwhile, the abundance trend of unclassified_c__Glomeromycetes was the opposite of Paraglomus , with the two genera showing reciprocal fluctuations in abundance during fermentation. In the early stages of fermentation, Paraglomus was dominant, leading the community diversity, while the abundance of unclassified_c__Glomeromycetes was relatively low. This may be related to the nutrient breakdown demands and the competitive ability of Paraglomus in the early fermentation phase (Wang et al., 2022 ; Li et al., 2022 ; Song et al., 2024 ). However, in the later stages of fermentation, the abundance of unclassified_c__Glomeromycetes increased significantly, suggesting that it may play specific metabolic roles in the later stages, such as breaking down residual fermentation materials or utilizing metabolic products generated in mid-fermentation. At the same time, its abundance rebound reflects the dynamic balance of the microbial ecosystem, especially in the later stages of fermentation, where non-dominant microbial groups may play compensatory roles in nutrient cycling or environmental regulation (Yang et al., 2024 ). Community succession of AMF at the species level and functional potential analysis At the species level (Fig. 5 ), throughout the entire fermentation stage, unclassified_g_Paraglomus dominated, showing an increasing-then-decreasing trend, with its relative abundance at the end of fermentation being lower than in the pre-fermentation stage. The second most abundant species was unclassified_c_Glomeromycetes , whose relative abundance increased rapidly in the early stages of fermentation, exhibiting a wave-like trend. By the end of fermentation, its relative abundance reached a peak, significantly higher than in the pre-fermentation stage. This phenomenon is related to the dynamic regulation of fermentation environmental factors. Studies have shown that the community structure of AMF is jointly influenced by changes in temperature, humidity, and the physicochemical properties of substrates (Lenoir et al., 2016 ). The high temperature and humidity at the beginning of fermentation provided a brief, suitable growth environment for Paraglomus , but as metabolic products accumulated during fermentation, the survival pressure increased, leading to a decline in its abundance. The wave-like trend of Glomeromycetes may be related to its adaptation to the substrates (such as cellulose and lignin degradation products) at different fermentation stages. Its final peak abundance reflects its dominant position in functional metabolism during the later stages of fermentation. Notably, the dynamic changes of Glomus-group-B-Glomus-sp.-VTX00279 and Glomus-group-B-Glomus-lamellosu-VTX00193 were particularly unique. The former occupied 19.67% of the relative abundance before fermentation but rapidly decreased to 0.23% in the early fermentation stages and gradually declined until it completely disappeared. In contrast, the latter was undetectable in both the pre-fermentation and early fermentation stages, but its relative abundance rapidly increased to 15.3% in the later stages. These unusual phenomena are related to multiple factors. In the early stages of fermentation, the rapid degradation of carbohydrates in the tobacco leaves generates a large amount of simple carbon sources, such as glucose, which triggers intense competition within the microbial community (Banožić et al., 2020 ; Ma et al., 2024 ). Glomus-group-B-Glomus-sp.-VTX00279 may have a lower carbon source utilization efficiency, preventing it from competing with rapidly growing bacteria (such as Proteobacteria ) or functional fungi, leading to a sharp decline in its abundance. In contrast, Glomus-group-B-Glomus-lamellosu-VTX00193 may possess the ability to efficiently degrade complex organic matter. During the later stages of fermentation, cellulose and hemicellulose in the tobacco leaves are gradually broken down into more stable lignin derivatives, and Glomus-lamellosu-VTX00193 may gain a competitive advantage by secreting specific extracellular enzymes, such as laccase and peroxidase, to utilize these substrates. Additionally, studies have indicated that some species within the Glomus genus can enhance heat and salt tolerance by accumulating compatible solutes like trehalose, and environmental stress in the later stages of fermentation may select for strains with such stress-resistance mechanisms (Ocón et al., 2007 ). The decline of Glomus-sp.-VTX00279 and the rise of Glomus-lamellosu-VTX00193 may represent a "functional relay" within the AMF community at different stages of fermentation. The former may participate in the early mobilization of carbon sources before fermentation, while the latter dominates the conversion of complex organic matter in the later stages. This successional pattern is closely related to key metabolic pathways involved in the formation of cigar tobacco leaf quality (e.g., terpene synthesis) (Liu et al., 2024 ; Jiang et al., 2024 ). Previous studies have highlighted the dominant role of Saccharomyces cerevisiae and Thermoascus fungi in flavor formation (He et al., 2022 ), while this study suggests that Glomus in AMF may indirectly influence the generation of flavor precursors through substrate pre-conversion, indicating that the functional roles of AMF communities in fermentation remain to be further explored. Dynamic changes of key chemical components during cigar tobacco fermentation Figure 6 shows the changes in the total nitrogen (Total Nitrogen), total sugar (Total Sugar), reducing sugar (Reducing Sugar), and total alkaloids (Total Alkaloids) content for YX-1, YX-2, and YX-6 at different fermentation times. Overall, with the extension of fermentation time, all samples showed varying degrees of decrease in total nitrogen, total sugar, reducing sugar, and total alkaloids, but the decline trends differed between varieties. Specifically, for total nitrogen and total sugar, YX-1 exhibited a more gradual decline compared to YX-2 and YX-6. YX-6 showed more fluctuation in the decline trend, particularly in the mid-fermentation stage, between the second and third weeks, with the highest content observed before fermentation for both of the latter. YX-2 consistently had the lowest total sugar content throughout the fermentation process. This result indicates that the decomposition or transformation of total nitrogen and total sugar during fermentation is a common phenomenon, related to the microbial metabolism of proteins and sugars. By degrading nitrogen sources and sugars, microbes create more favorable growth conditions for themselves (Nord et al., 1926; Hu et al., 2022 ). The overall trend of reducing sugar content showed no significant differences in its decline. YX-2 had significantly higher reducing sugar content in the early stages compared to YX-1 and YX-6, but by the end of fermentation, YX-2's reducing sugar content was significantly lower than that of YX-1. This suggests that YX-2 may have undergone more extensive enzymatic hydrolysis during fermentation, resulting in a more pronounced reduction in reducing sugar content. The reduction in reducing sugars is typically associated with microbial sugar degradation, a process that helps improve the flavor of cigars by reducing sweetness and enhancing complexity, indicating that AMF plays a key role in sugar degradation (Su et al., 2024 ). In the case of total alkaloids, YX-1 exhibited a slower decline in alkaloid content throughout fermentation, while YX-6 showed the most significant changes in alkaloid content, with a noticeable increase at the end of fermentation compared to the previous week. This phenomenon may be related to changes in the microbial community or adjustments in metabolic pathways, as some microorganisms may begin to resynthesize or transform alkaloid compounds. Particularly at the end of fermentation, due to changes in the fermentation environment, the metabolic direction of the microbial community might shift, leading to the rebound in alkaloid content (Ponomarova et al., 2015). This phenomenon was observed only in YX-6, suggesting that YX-6 cigar tobacco may contain specific compounds or exhibit stronger microbial activity, causing greater fluctuations in the later stages of fermentation. A linear regression model was used to further quantify the dynamic relationship between key chemical components and the Sobs index of AMF during the fermentation of Yunxue series cigar tobacco (Fig. 7 ). Total nitrogen (TN), total sugar (TS), and reducing sugar (RS) all showed significant negative correlations with the Sobs index, indicating that the degradation of these components during fermentation promotes an increase in AMF community diversity. This may be because the reduction in sugars and nitrogen sources inhibits the rapid proliferation of certain dominant microbial populations, allowing the AMF community to develop quickly. Although total alkaloids (TA) showed a certain negative correlation with the Sobs index, the correlation was weaker, suggesting that the impact of total alkaloids on AMF community diversity is more complex and may be regulated by other environmental factors. These results suggest that during the fermentation of cigar tobacco, properly regulating the diversity of the AMF community helps to precisely control the content of key chemical components, which in turn influences the quality and flavor of the final product. Using relatively rare AMF strains as cigar fermentation agents could provide a new approach to improving cigar quality and open new research directions for the optimization of fermentation processes. Correlation analysis of key chemical components and AMF community diversity Based on the species-species correlation, a species correlation network diagram was constructed (Fig. 8 A). The results show that the diversity of the AMF community exhibits complex species interactions. Paraglomus species occupy a central position in the community, with s_unclassified_g_Paraglomus showing the highest relative abundance and a significant negative correlation with s_unclassified_c_Glomeromycetes , which has the second-highest relative abundance. The relative abundances of both fluctuate during fermentation. Additionally, s_unclassified_g_Paraglomus was also negatively correlated with s_unclassified_g_Glomus_f_Glomeraceae and s_Paraglomus-Glom-1B.13-VTX00308 , though these correlations were not significant. These three negative correlations were the only ones observed in the entire correlation network diagram. The fermentation environment significantly influenced the interactions between these species, with some species displaying strong adaptability under these conditions, while others were affected by competition or resource limitations. The positive and negative interactions within the AMF community reveal their potential ecological roles during the tobacco fermentation process. To further understand the mechanisms through which these interactions affect the quality of tobacco fermentation, a correlation heatmap (Fig. 8 B) was constructed, and a Mantel test analysis was conducted. The results show that the correlations within the AMF community under different fermentation pile sample conditions follow a complex pattern. Glomus-MO-G16-VTX00072 was significantly positively correlated with total nitrogen, total sugar, reducing sugar, and total alkaloids, suggesting that Glomus-MO-G16-VTX00072 may enhance sweetness and optimize cigar flavor by promoting carbohydrate metabolism and increasing the total sugar and reducing sugar content in tobacco leaves. At the same time, it may influence the secondary metabolism of tobacco, raising the total alkaloid levels, thereby enhancing the strength and smoking experience of the cigar (Hu et al., 2024 ; Hu et al., 2022 ). This microorganism could be an important beneficial fermentation strain within the AMF community. Certain species, such as unclassified_g__Paraglomus , showed significant correlations with major sugars (total sugar, reducing sugar), suggesting potential for development. On the other hand, unclassified_o__Archaeosporales exhibited negative correlations with all four quality indicators measured, indicating that its high abundance may negatively impact the flavor of the cigar. The abundance of this species should be appropriately controlled during fermentation to prevent it from diminishing the cigar's flavor and reducing its complexity. The Mantel test further revealed the dynamic correlation between chemical components and AMF community diversity during cigar tobacco fermentation (Fig. 8 C). Total nitrogen (TN) showed a positive correlation with AMF diversity throughout the fermentation process, but the correlation was not significant. Total sugar (TS) was positively correlated with AMF diversity in the early fermentation stages (F₁-F₂), but as fermentation progressed into the mid- and late stages, it became negatively correlated, suggesting that the sugar resources were rapidly consumed or microbial competition intensified in the earlier stages. Reducing sugar showed a negative correlation with AMF diversity in the mid-fermentation stage, while it was positively correlated in the early and later stages, confirming the ecological strategy of AMF to prioritize easily metabolizable carbon sources (Wu et al., 2013 ). Total alkaloids (TA), due to their antimicrobial properties, significantly inhibited the AMF community, possibly leading to the elimination of sensitive strains, and became negatively correlated with AMF diversity as soon as the fermentation stage began. Overall, the AMF community exhibited complex interaction patterns during cigar tobacco fermentation, with different species having varying impacts on tobacco leaf quality. Among these, Glomus-MO-G16-VTX00072 , as a potential beneficial fermentation strain, can optimize the sugar-alkaloid ratio and enhance cigar flavor, while certain species such as unclassified_o__Archaeosporales may negatively affect quality and should be controlled. These findings provide new directions for regulating AMF communities and optimizing fermentation processes, helping to improve the quality stability and flavor profile of cigar tobacco. The analytical methods employed in this study primarily emphasize the correlation between community structure and physicochemical components. However, the precise metabolic functions and mechanisms of arbuscular mycorrhizal fungi (AMF) in cigar fermentation have not been conclusively established through isolation culture, metagenomic, or macrotranscriptome techniques. The existing functional evidence remains relatively limited. Subsequent investigations will enhance sample representativeness, bolster functional verification, and conduct in-depth mechanism analyses to comprehensively elucidate the role of AMF in the fermentation microecosystem. This research aims to support the practical application of AMF in tobacco fermentation and other related industries. Discussion The discovery of dynamic and structured arbuscular mycorrhizal fungal (AMF) communities during the fermentation of cured (non-living) cigar tobacco presents a fascinating ecological paradox. Traditionally, AMF are considered obligate biotrophic symbionts dependent on living plant hosts for carbon and survival (Velásquez et al., 2025 ; Samanta et al., 2025 ). Their persistence and successional dynamics throughout the 35-day fermentation process of processed tobacco leaves suggest a significant ecological plasticity or a previously underappreciated saprotrophic capability in certain taxa within the Glomeromycota phylum. This study, combined with insights from recent literature, suggests that these fungi likely originate from pre-existing colonization within the tobacco leaves during growth and the rhizosphere soil (Liu et al., 2025 ; Zhang et al., 2023 ). The curing process may not completely eradicate these robust fungi, whose spores and hyphal fragments remain viable. During fermentation, the changing microenvironment—characterized by shifting nutrient availability, moisture, temperature, and microbial competition—appears to drive a functional transition in these communities, allowing them to participate in the decomposition and transformation of tobacco organic matter. The inverse abundance patterns observed between Paraglomus (e.g., OTU217) and unclassified Glomeromycetes , along with the dramatic species-level succession (e.g., the decline of Glomus-group-B-Glomus-sp.-VTX00279 and the rise of Glomus-group-B-Glomus-lamellosu-VTX00193 ), suggest a "functional relay" within the AMF community. This indicates that different AMF taxa may adapt to utilize distinct nutritional niches at various fermentation stages. The sustained dominance of OTU217 ( Paraglomus ) highlights its potential role as a core functional unit, likely contributing to the degradation of complex organic compounds like cellulose, hemicellulose, and lignin derivatives—processes crucial for releasing precursors for flavor development (Wu et al., 2024 ; Zhang et al., 2023 ). This is supported by studies in other systems showing AMF capabilities in organic matter decomposition and nutrient cycling (Liu et al., 2025 ). Furthermore, the significant negative correlations between AMF community diversity (Sobs index) and the degradation of total nitrogen, total sugar, and reducing sugar underscore the potential impact of AMF on cigar tobacco quality. The negative correlations suggest that as these nutrients are metabolized, competitive pressure on the microbial community may shift, allowing for greater AMF diversity and activity. This aligns with the concept that nutrient depletion can reduce the dominance of fast-growing r-strategists, creating opportunities for more K-strategist or specialist fungi like AMF. The strong correlations between specific AMF taxa and key chemical components, as revealed by the Mantel test and correlation network analysis, further support their functional contributions. For instance, the positive correlation between Glomus-MO-G16-VTX00072 and total sugar, reducing sugar, and total alkaloids suggests that certain AMF taxa may actively participate in modulating the sugar-alkaloid balance, ultimately influencing the sensory profile of the final cigar product (Carpenter et al., 2007 ; Hu et al., 2023 ). This is consistent with findings in other agricultural products, where AMF have been shown to influence secondary metabolite accumulation. Conversely, the negative correlation between unclassified_o__Archaeosporales and all quality indicators suggests that some AMF taxa may have detrimental effects, possibly through the production of off-flavor compounds or inefficient resource competition (Ran et al., 2022 ). The presence of active AMF communities in fermenting cigar tobacco, a non-living substrate, can be explained by several factors. First, the fermentation environment—characterized by elevated temperatures, limited oxygen availability, and rich in complex organic compounds—may selectively favor AMF taxa with robust saprotrophic capabilities or those that can enter a dormant, spore-forming state until conditions improve. AMF spores are known for their resilience and can survive in harsh environments (Nie et al., 2024 ; Carrillo-Saucedo et al., 2018 ). Second, the initial chemical composition of the tobacco leaf, particularly the content of macromolecules such as proteins, starch, and pectin, varies between varieties (Yunxue No. 1, No. 2, No. 6) and likely provides a diverse nutrient base that can be utilized by AMF with specific enzymatic capacities. The variety-specific successional patterns observed in this study support this notion (Bisht et al., 2022). Finally, the interactions between AMF and other microbes (bacteria and other fungi) within the fermentation pile likely play a crucial role in determining AMF activity. For example, bacteria may pre-degrade complex polymers into simpler compounds that AMF can assimilate, or AMF may provide access to otherwise inaccessible nutrients through their hyphal networks (Wipf et al., 2019 ). This study offers a new perspective on AMF, suggesting that they may be more than just remnants of a past symbiotic relationship and could potentially act as active participants in the cigar tobacco fermentation process. The variety-specific successional patterns indicate that the initial chemical composition of the tobacco leaf, influenced by genotype and growth conditions, might be used to predict and guide microbial succession toward desired outcomes. These findings open potential avenues for developing novel fermentation strategies, such as using tailored microbial consortia that include AMF, to possibly improve fermentation efficiency, consistency, and final product quality. For example, inoculating fermentation piles with specific AMF taxa that appear beneficial could help accelerate the breakdown of undesirable compounds, promote the formation of favorable aroma precursors, and enhance the overall smoking experience. Alternatively, managing fermentation conditions (e.g., temperature, humidity, turning frequency) to suppress the growth of potentially unfavorable taxa like unclassified_o__Archaeosporales might also be a useful strategy. The analytical methods employed in this study primarily emphasize the correlation between community structure and physicochemical components. However, the precise metabolic functions and mechanisms of arbuscular mycorrhizal fungi (AMF) in cigar fermentation have not been conclusively established through isolation culture, metagenomic, or macrotranscriptome techniques. The existing functional evidence remains relatively limited. To fully elucidate the functional roles of AMF in cigar tobacco fermentation, future studies should prioritize the isolation and cultivation of dominant taxa for in vitro functional validation, combined with multi-omics and stable isotope probing to characterize their in situ activity and nutrient metabolism. Furthermore, manipulative experiments using inoculation or suppression of specific AMF, along with expanded sampling across varieties and fermentation conditions, will be essential to establish causal relationships and enhance the generalizability of the findings. Conclusions This study is the first to reveal the dynamic succession patterns of the arbuscular mycorrhizal fungi (AMF) community during cigar tobacco fermentation and its association with tobacco leaf quality. The AMF community exhibited significant variety-specific and stage-dependent succession characteristics during fermentation. A total of 524 OTUs were identified, with Paraglomus being the absolute dominant genus. Its metabolic activity is closely linked to its adaptability to the high-temperature, nutrient-rich environment, and it dominates the fermentation process through carbon-nitrogen cycling. Meanwhile, unclassified_c__Glomeromycetes showed an increase in abundance in the later stages, possibly involved in the degradation of residual substances. The dynamic differences in AMF diversity between varieties were significant, with Yunxue No. 1 displaying wave-like fluctuations in the Shannon index due to competitive microbial fluctuations, Yunxue No. 6 experiencing a surge in rare species in the late stages as indicated by the Chao index, and Yunxue No. 2 showing a decrease in the Sobs index in the early stages due to the difficulty in degrading certain substrates. The key species Glomus-group-B-Glomus-lamellosu-VTX00193 experienced a sharp increase in abundance to 15.3% in the later stages, possibly through the secretion of extracellular enzymes to degrade lignin derivatives, forming a functional relay with Glomus-sp.-VTX00279 . Chemical component analysis showed significant negative correlations between total nitrogen, total sugar, and AMF abundance, suggesting that AMF promotes community diversity through the degradation of macromolecular substances. Glomus-MO-G16-VTX00072 was positively correlated with sugars, indicating its potential to optimize flavor. The study confirms that AMF can utilize residual carbon sources from plant remains to maintain metabolic activity and survive in the cigar tobacco fermentation system. The community succession of AMF is regulated by both the physicochemical properties of the cigar variety and the fermentation environment. The core functions of Paraglomus and the specific metabolism of Glomus species provide a theoretical basis for the targeted regulation of fermentation quality. Declarations Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Conflict of Interest The authors declare no competing interests. Funding This study work was supported by the Science and Technology Plan Project of the China National Tobacco Corporation, Yunnan Provincial Company (2023530000241002). Author Contribution HZ reviewed and edited the manuscript. XRS, QZ, YMY, YY, JLZ, YHC, LDB and YBS conducted the investigation. YBS acquired funding and reviewed the manuscript. All authors read and approved the final manuscript. Data Availability All data supporting the conclusions of this manuscript have been presented in the manuscript and are publicly accessible. Regarding the raw sequencing data of this study, they have not yet been deposited in public data repositories due to the signed confidentiality agreement. For further inquiries, please contact the corresponding author at [email protected] to obtain the data. 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Front Microbiol 15:1425553. https://doi.org/10.3389/fmicb.2024.1425553 Zhang G, Zhao L, Li W, Yao H, Lu C, Zhao G, Wu Y, Li Y, Kong G (2023) Changes in physicochemical properties and microbial community succession during leaf stacking fermentation. AMB Express 13:132. https://doi.org/10.1186/s13568-023-01642-8 Zhang M, Guo D, Wang H, Wu G, Shi Y, Zhou J, Zhao E, Zheng T, Li X (2024) Analyzing microbial community and volatile compound profiles in the fermentation of cigar tobacco leaves. Appl Microbiol Biot 108:243. https://doi.org/10.1007/s00253-024-13043-3 Zhang Q, Huang Y, An H, Yang S, Lei J, Wang Y, Li P, Zhang H, Cai W, Jia Y, Pang Y, Li D (2024) The impact of gradient variable temperature fermentation on the quality of cigar tobacco leaves. Front Microbiol 15:1433656. https://doi.org/10.3389/fmicb.2024.1433656 Zhang Q, Kong G, Zhao G, Liu J, Jin H, Li Z, Zhang G, Liu T (2023) Microbial and enzymatic changes in cigar tobacco leaves during air-curing and fermentation. Appl Microbiol Biot 107:5789–5801. https://doi.org/10.1007/s00253-023-12663-5 Zhao S, Li Y, Liu F, Song Z, Yang W, Lei Y, Tian P, Zhao M (2024) Dynamic changes in fungal communities and functions in different air-curing stages of cigar tobacco leaves. Front Microbiol 15:1361649. https://doi.org/10.3389/fmicb.2024.1361649 Zheng T, Zhang Q, Wu Q, Li D, Wu X, Li P, Zhou Q, Cai W, Zhang J, Du G (2022) Effects of inoculation with Acinetobacter on fermentation of cigar tobacco leaves. Front Microbiol 13:911791. https://doi.org/10.3389/fmicb.2022.911791 Additional Declarations No competing interests reported. Supplementary Files floatimage1.png Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7416527","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":511889667,"identity":"e8c677ad-5569-47db-8f7a-c3ce5f2b3f84","order_by":0,"name":"Hui Zhang","email":"","orcid":"","institution":"Yunnan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Zhang","suffix":""},{"id":511889668,"identity":"f5d81b99-032e-43c4-b592-0019fef9266e","order_by":1,"name":"Xueru Song","email":"","orcid":"","institution":"Yunnan Tobacco Company Yuxi City Corporation","correspondingAuthor":false,"prefix":"","firstName":"Xueru","middleName":"","lastName":"Song","suffix":""},{"id":511889669,"identity":"cdeb1c71-5232-48e1-b0a9-3c8492032c8d","order_by":2,"name":"Qi Zhou","email":"","orcid":"","institution":"Yunnan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Zhou","suffix":""},{"id":511889670,"identity":"59039f64-7580-491c-af30-d7debc469488","order_by":3,"name":"Yuming Yin","email":"","orcid":"","institution":"Yunnan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yuming","middleName":"","lastName":"Yin","suffix":""},{"id":511889671,"identity":"aa427d58-c708-4221-babc-9b327aaa4a64","order_by":4,"name":"Ying Yang","email":"","orcid":"","institution":"Yunnan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Yang","suffix":""},{"id":511889672,"identity":"2974cf1b-de88-49c3-86cf-1ceffcd0d4ba","order_by":5,"name":"Jilai Zhang","email":"","orcid":"","institution":"Yunnan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jilai","middleName":"","lastName":"Zhang","suffix":""},{"id":511889673,"identity":"30da5597-c714-4cb6-9b81-9f8162d07303","order_by":6,"name":"Yonghe Cui","email":"","orcid":"","institution":"Yunnan Tobacco Company Yuxi City Corporation","correspondingAuthor":false,"prefix":"","firstName":"Yonghe","middleName":"","lastName":"Cui","suffix":""},{"id":511889674,"identity":"20298ac8-55d3-4cda-958c-f6298f576dbb","order_by":7,"name":"Lingduo Bu","email":"","orcid":"","institution":"Yunnan Tobacco Company Yuxi City Corporation","correspondingAuthor":false,"prefix":"","firstName":"Lingduo","middleName":"","lastName":"Bu","suffix":""},{"id":511889675,"identity":"80422459-2343-4436-898a-fb49ffc6ef84","order_by":8,"name":"Yulong Su","email":"","orcid":"","institution":"Yunnan Tobacco Company Yuxi City Corporation","correspondingAuthor":false,"prefix":"","firstName":"Yulong","middleName":"","lastName":"Su","suffix":""},{"id":511889676,"identity":"d7f2be3e-158b-4906-8375-9daf6a6a365b","order_by":9,"name":"Youbo Su","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqElEQVRIiWNgGAWjYHACNhAhx8befoA0LcZ8PGcSSNOSOE/CwYA49QbHe8wefPhzOL1NgiGB4UfFNiK0nDljbjiz7XBum3TjAcaeM7eJ0HIjx0yatwGoReZAAjNjG7FaeIAOY5NIMCBFC9vhBOK1SJ45Vg70S7phGzCQDxLlF77jzduAIWYtL9/efvDBjwoitCgcQOIcwKEIFcg3EKVsFIyCUTAKRjQAAA8zPPWGTJ+XAAAAAElFTkSuQmCC","orcid":"","institution":"Yunnan Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Youbo","middleName":"","lastName":"Su","suffix":""}],"badges":[],"createdAt":"2025-08-20 10:38:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7416527/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7416527/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91209529,"identity":"96a1d455-ee1f-4efd-b32e-3c5d6590e549","added_by":"auto","created_at":"2025-09-12 17:34:52","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":74691,"visible":true,"origin":"","legend":"\u003cp\u003eMicrobial community OTU distribution and composition analysis of cigar tobacco samples from three major varieties at different fermentation stages. The Venn diagram (upper part) shows the changes in OTU numbers before and after fermentation for all three varieties. The community bar chart (lower part) illustrates the dynamic changes in the OTU community abundance of AMF across fermentation stages.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7416527/v1/deb4a2f583709edd44f3cdbe.jpg"},{"id":91209530,"identity":"9a70923c-5505-4e05-952b-cc7ad842a200","added_by":"auto","created_at":"2025-09-12 17:34:52","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":71474,"visible":true,"origin":"","legend":"\u003cp\u003eAlpha diversity analysis of AMF community OTUs during cigar fermentation. (A) Sobs index, (B) Shannon index, and (C) Chao index display the changes in the three sample groups (YX-1, YX-2, YX-6) across different fermentation weeks (0-5).\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7416527/v1/09cc298ecec76ecdedaeacab.jpg"},{"id":91210399,"identity":"5a6ab195-8139-4ed2-a863-d1ea5355b77e","added_by":"auto","created_at":"2025-09-12 17:42:52","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":78379,"visible":true,"origin":"","legend":"\u003cp\u003eAbundance changes of AMF communities at the genus level during cigar fermentation.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7416527/v1/171cf465c0d3e33adb623126.jpg"},{"id":91209532,"identity":"f7c9afd7-6f95-4d1b-ac60-ffa09aeda319","added_by":"auto","created_at":"2025-09-12 17:34:52","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":55817,"visible":true,"origin":"","legend":"\u003cp\u003eDynamic changes in the relative abundance of core AMF communities at different fermentation stages.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7416527/v1/fedd6f42b432ff16b96438f6.jpg"},{"id":91209531,"identity":"ead3b2b0-09d8-488d-b1eb-40a0078e6390","added_by":"auto","created_at":"2025-09-12 17:34:52","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":45240,"visible":true,"origin":"","legend":"\u003cp\u003eDynamic changes of AMF communities at the species level during the fermentation of the three major cigar varieties.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7416527/v1/3dc72d50c3881d165d3bd3e5.jpg"},{"id":91209535,"identity":"bd8f43af-33a9-4d36-8a9f-58405e27b787","added_by":"auto","created_at":"2025-09-12 17:34:52","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":59772,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in chemical components of three cigar tobacco samples at different fermentation times.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7416527/v1/f0e51e9bdfcf7472e08e19ac.jpg"},{"id":91209537,"identity":"c392ba5f-60b4-497b-bcbd-7e13903a3c06","added_by":"auto","created_at":"2025-09-12 17:34:52","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":52623,"visible":true,"origin":"","legend":"\u003cp\u003eLinear regression relationship between cigar tobacco chemical components and community richness at different fermentation stages, and sample distribution.\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7416527/v1/38150bf05fe28988a8a012c7.jpg"},{"id":91209542,"identity":"6de45a33-1b8f-4a3c-87ea-f65ae042a8da","added_by":"auto","created_at":"2025-09-12 17:34:52","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":53576,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogeny, correlation network, and statistical parameter analysis of AMF taxa. (A) Core AMF taxa correlation network; (B) Correlation heatmap of tobacco leaf physicochemical components and the AMF community; (C) Mantel test statistical parameters and correlation strength.\u003c/p\u003e","description":"","filename":"Picture8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7416527/v1/13b709aa9cf06de394848746.jpg"},{"id":93370674,"identity":"79de3c31-6369-4ee7-8737-927b79e56cc3","added_by":"auto","created_at":"2025-10-13 06:32:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1384580,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7416527/v1/5353af2c-560c-4f80-8565-555197e9bedf.pdf"},{"id":91210786,"identity":"048783e5-8fd7-4290-8418-4fd1a227899f","added_by":"auto","created_at":"2025-09-12 17:50:52","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1375278,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7416527/v1/3dbb7fb1040b4e559571bf33.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dynamic changes and potential contributions of arbuscular mycorrhizal fungi in cigar tobacco fermentation","fulltext":[{"header":"Key Points","content":"\u003cul start=\"50\"\u003e\n \u003cli\u003e\u003cem\u003eThe dynamic survival of arbuscular mycorrhizal fungi (AMF) during cigar tobacco fermentation and their functional roles are revealed for the first time.\u003c/em\u003e\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eParaglomus is identified as the dominant AMF genus, influencing tobacco chemical composition through modulation of carbon and nitrogen metabolism.\u003c/em\u003e\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eAMF community diversity varies significantly across tobacco cultivars, with Yunxue No. 6 exhibiting the highest richness of rare species, while Glomus-lamellosu-VTX00193 increases markedly in late fermentation stages, potentially contributing to lignin degradation and flavor enhancement.\u003c/em\u003e\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe fermentation of cigar tobacco is a critical step in enhancing tobacco leaf quality. Its primary objectives are to improve aroma, optimize the smoking experience, and ensure product consistency and safety (Liu et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Fermentation of tobacco leaves via microbial metabolism serves to degrade impurities and irritants, resulting in a purer aroma and a smoother taste. This process also facilitates the conversion of carbohydrates and amino acids, leading to the production of various aromatic compounds like ketones, alcohols, and terpenes. These transformations contribute to the distinctive aroma and multifaceted flavor profile characteristic of cigars (Zhang et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Additionally, fermentation improves the physical properties of the tobacco leaves, enhancing their flexibility and combustion performance. More importantly, the fermentation process effectively reduces the content of harmful substances such as nicotine and nitrosamines, thus improving the health quality of the cigars. In conclusion, fermentation not only determines the aroma and taste of cigars but also ensures their high quality and market competitiveness (Zhang et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe study of microbial community changes during cigar tobacco fermentation has become a key focus of current research. It not only helps to reveal the mechanisms behind the generation of aromatic compounds but also facilitates the improvement of cigar quality, consistency, and safety through the regulation of fermentation conditions. This makes it a crucial aspect of enhancing cigar production and quality control (Su et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). During the fermentation of cigar tobacco leaves, the microbial community exhibits significant dynamic changes, which can be divided into three main stages. In the early stage, the diversity of bacteria predominates, with the major phyla being \u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003eProteobacteria\u003c/em\u003e. Studies have shown that \u003cem\u003eCorynebacterium\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, and \u003cem\u003eStaphylococcus\u003c/em\u003e genera exhibit high abundance in this stage. These microorganisms are involved in the breakdown of carbohydrates in the tobacco leaves, such as starch, pectin, and cellulose. In the mid-fermentation stage, after rehydration, microbial communities begin to undergo significant shifts as humidity and total acidity increase. \u003cem\u003eAerococcus\u003c/em\u003e dominates during this stage, utilizing reducing sugars and organic acids (such as malic acid and citric acid) in the tobacco leaves. This leads to increased temperature and pH in the fermentation pile, promoting the growth of other microorganisms. At the same time, the structure of the fungal community remains relatively stable, with \u003cem\u003eAspergillus\u003c/em\u003e, \u003cem\u003eAlternaria\u003c/em\u003e, and \u003cem\u003eCladosporium\u003c/em\u003e continuing to dominate. In the final fermentation stage, the drying phase, microbial diversity decreases. \u003cem\u003eFirmicutes\u003c/em\u003e continues to dominate, while \u003cem\u003eAspergillus\u003c/em\u003e remains the primary fungal genus. At this stage, the dynamic changes in the microbial community gradually stabilize, accompanied by an increase in volatile compounds (such as ketones, aldehydes, and terpenes), which intensifies the aroma (Guo et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Si et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Xue et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). It can be observed that throughout the fermentation process, microorganisms such as \u003cem\u003eAerococcus\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eStaphylococcus\u003c/em\u003e, and \u003cem\u003eAspergillus\u003c/em\u003e play key roles. They are not only responsible for the transformation of chemical components in the tobacco leaves but are also closely associated with the generation of various volatile organic compounds (VOCs). In particular, \u003cem\u003ePseudomonas\u003c/em\u003e is positively correlated with several ketones and aldehydes (such as isophorone and acetophenone), which significantly influence the aroma characteristics of cigars, imparting them with a rich, sweet, and roasted fragrance (Liu et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Additionally, \u003cem\u003eAspergillus\u003c/em\u003e and other fungi in the community are related to the generation of terpenes and aromatic compounds (such as indole), which contribute to the unique floral and fruity characteristics of cigars (Zhang et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eArbuscular Mycorrhizal Fungi (AMF), belonging to the phylum \u003cem\u003eGlomeromycota\u003c/em\u003e, are obligate symbiotic fungi (Willis et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). They form mutualistic symbiotic relationships with over 80% of terrestrial plants by penetrating plant roots to form arbuscules, which serve as the core structures for nutrient exchange between the host plant and the fungi. This symbiotic relationship significantly enhances the plant's ability to absorb essential mineral elements such as phosphorus, nitrogen, and zinc. In particular, in poor or arid soils, the fungal mycelial network of AMF can greatly extend the root system\u0026rsquo;s ability to absorb nutrients (Gosling et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Furthermore, AMF secretes glomalin (a sticky glycoprotein) to improve soil aggregation, enhancing water retention and organic matter content. Its ecological functions have been widely applied in the fields of sustainable agriculture and soil remediation (Singh et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Purin et al., 2007). However, traditional views hold that AMF is highly dependent on living plant roots, and its survival is strictly limited by the active carbon sources and cellular signals provided by the host. Consequently, AMF is generally considered unable to survive once the plant tissue dies or during processing, leading to its potential role in food and tobacco fermentation being largely overlooked, with relevant research being nearly nonexistent. It is noteworthy that recent studies have found that AMF may maintain metabolic activity through spore dormancy or by utilizing residual carbon sources in plant remains, providing new insights into understanding the ecological adaptability of AMF in non-symbiotic environments (Pepe et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wei et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Gavito et al., 2003). However, whether these phenomena have functional significance remains unknown. In the current cigar tobacco fermentation process, the known and widely used strains mainly include pectinase-producing bacteria, protease-producing bacteria, lactic acid bacteria, yeasts, acetic acid bacteria, cellulose-degrading bacteria, and antioxidant bacteria. These strains improve tobacco leaf quality by degrading macromolecular organic compounds, generating flavor precursors, or regulating redox states (Su et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Ren et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Song et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zheng et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). While the utilization of current strains is well-established, their functional constraints pose a barrier to the advancement of fermentation strain innovation. This research represents the initial investigation into the dynamic shifts of the arbuscular mycorrhizal fungi (AMF) community within a cigar fermentation system. The findings validate that AMF can sustain metabolic activity via spore dormancy or residual carbon sources in plant remnants. The alterations in microbial communities during cigar fermentation are pivotal for the development of cigar aroma and excellence. Hence, delving into the functional importance and potential impact of AMF in the cigar fermentation process not only elucidates the microbial mechanisms underlying cigar aroma formation from a novel perspective but also introduces fresh concepts and a theoretical framework for enhancing cigar production techniques and elevating cigar quality. This study aimed to elucidate the role of arbuscular mycorrhizal fungi (AMF) in tobacco degradation, flavor development, and stress response. Additionally, it sought to assess the suitability of AMF as a novel fermentation agent and propose a novel approach for targeted modulation of the cigar fermentation microbiome. Correlation analysis was conducted to investigate the relationship between physicochemical constituents and fungal populations during tobacco leaf fermentation. By elucidating the intricate interplay between fungi and the physicochemical characteristics of tobacco leaves, the study unveiled the potential impact of fungi on cigar fermentation.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCigar tobacco fermentation management and sample collection\u003c/h2\u003e\u003cp\u003eThe fermentation site was located at the Ede Cigar Drying and Fermentation Management Workshop in Mosha Town, Xinping County, Yunnan Province, Yuxi City, China, at an altitude of 493 meters. The selected cigar varieties included Yunxue No. 1, Yunxue No. 2, and Yunxue No. 6. The fermentation facility measured 10m x 8m x 5m, employing the fermentation method with each fermentation pile weighing 500kg. Each of the three cigar varieties was processed in a separate fermentation plant, totaling three plants. The fermentation process maintained a temperature of 35\u0026deg;C and a humidity level of 75%. Fermentation lasted for 35 days, with sampling conducted weekly, removing 500g of tobacco leaves from each fermentation pile using a 5-point sampling technique. The tobacco samples were promptly stored at -80\u0026deg;C in an ultra-low temperature refrigerator.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDetermination of physicochemical components\u003c/h3\u003e\n\u003cp\u003eThe collected samples were ground into powder using a mortar in liquid nitrogen and stored in a \u0026minus;\u0026thinsp;80\u0026deg;C ultra-low temperature freezer. Subsequently, the total nitrogen, total sugar, reducing sugar, total alkaloids, and other physicochemical components of the tobacco leaves were measured. The standards for the determination methods are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(Fu et al., 2018; Jia et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Ren et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMethods and equipment for testing physicochemical composition of cigar\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhysicochemical composition\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDetection method follows the standard\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal nitrogen (TN)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTobacco and tobacco products-Determination of total nitrogenContinuous flow method (YC/T 161\u0026ndash;2002)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal alkaloid (TA)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTobacco and tobacco products-Determination of total alkaloids-Continuous flow (potassium thiocyanate) method (YC/T 468\u0026ndash;2013)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal sugar (TS)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDetermination of water-soluble sugars in tobacco and tobacco products by continuous flow method (YC/T 159\u0026ndash;2019)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eReducing sugar (RS)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDetermination of reducing sugar in sugar beet root (NY/T 1751\u0026ndash;2009)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eTobacco leaf DNA extraction and PCR amplification\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was extracted and detected using 1% agarose gel electrophoresis. Based on the specified sequencing regions, specific primers with barcodes were synthesized. Low cycle number amplification was used to ensure consistent amplification cycle numbers across all samples. A pre-experiment randomly selected representative samples to ensure that, under low cycle numbers, most samples generated products of appropriate concentration. PCR amplification was performed using TransGen AP221-02 TransStart Fastpfu DNA Polymerase, with an ABI GeneAmp\u0026reg; 9700 PCR instrument. The amplification primers used were AML1F-AML2R and AMV4-5NF-AMDGR (Ma et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In the formal experiment, each sample was run in triplicate, and the mixed products were detected using 2% agarose gel electrophoresis. The PCR products were recovered from the gel using the AxyPrep DNA Gel Extraction Kit, eluted with Tris-HCl, and rechecked using 2% agarose gel electrophoresis. Based on the preliminary quantification results from electrophoresis, PCR products were quantified using the QuantiFluor\u0026trade;-ST blue fluorescence quantification system from Promega. The samples were then mixed in proportions according to sequencing requirements.\u003c/p\u003e\n\u003ch3\u003eLibrary construction and sequencing\u003c/h3\u003e\n\u003cp\u003eIn Illumina sequencing, linker sequences were initially incorporated at both ends of the target region using PCR technology. Subsequently, the PCR products were fragmented and isolated utilizing gel extraction kits, eluted with Tris-HCl buffer, and assessed for recovery efficiency via 2% agarose gel electrophoresis. The resulting product was then denatured with sodium hydroxide to yield single-stranded DNA fragments, which were preserved using the TruSeq\u0026trade; DNA Sample Prep Kit. Upon progression to the sequencing phase, the linker sequences at the fragment ends could hybridize with complementary bases immobilized on the chip surface, thereby anchoring the fragment to the chip. Utilizing the immobilized DNA fragment as a template, the chip-bound sequence served as a primer for bridge PCR amplification, facilitating the synthesis of a new complementary strand. Subsequent denaturation and annealing allowed the other end of the newly synthesized strand to randomly hybridize with an adjacent primer, forming a \"bridge\" structure. Numerous DNA clusters were generated through iterative bridge amplification and subsequently linearized into single strands. During sequencing, a specialized DNA polymerase and dNTPs containing fluorescent labels and termination groups were utilized, with the introduction of only one base per reaction cycle. A laser scanner identified the fluorescently labeled base incorporated into each DNA template during synthesis, followed by the removal of the fluorescent and termination groups to reactivate the 3' end for the subsequent cycle of base addition. Ultimately, the precise nucleotide sequence of the desired DNA fragment was determined through statistical analysis of the fluorescence signals from each sequencing cycle.\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis of tobacco leaf physicochemical components and sequencing data was performed using Excel 2019 and SPSS 26.0 software. One-way ANOVA was used to evaluate the differences in the changes of physicochemical components and AMF microbial diversity during cigar tobacco fermentation. Pearson correlation analysis was conducted to explore the potential correlation between the AMF microbial community and the physicochemical components of tobacco leaves. Alpha diversity, based on Sobs, Shannon, and Chao indices, was used to study the dynamic changes in species diversity during fermentation. The relevant calculations and visualizations for AMF community analysis were performed in R software (version 4.1.1, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.r-project.org\u003c/span\u003e\u003cspan address=\"https://www.r-project.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and its \u0026ldquo;vegan,\u0026rdquo; \u0026ldquo;phyloseq,\u0026rdquo; \u0026ldquo;DESeq2,\u0026rdquo; and \u0026ldquo;picante\u0026rdquo; packages(Aci, et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAll data are presented as \"mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error,\" and graphs were generated using Excel 2019, GraphPad Prism 8.0, and MATLAB 2019 software.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eDynamic succession patterns and variety-specificity of the AMF community OTUs\u003c/h2\u003e\u003cp\u003eThe species annotation results of the AMF community in this cigar tobacco fermentation study encompass species quantities across different taxonomic levels. A total of one phylum and one class were identified, further subdivided into five orders, five families, five genera, and 22 species, resulting in the detection of 524 Operational Taxonomic Units (OTUs). Through OTU analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), it is clear to observe and record the dominance of specific AMF communities at particular stages during the fermentation of cigar tobacco and how these communities change over time. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, both shared and unique OTUs between varieties were evident before and after fermentation, demonstrating the diversity and similarity of microbial communities. During fermentation, the number of unique OTUs in Yunxue No. 6 and the shared OTUs among all three varieties increased significantly, while the shared OTUs between Yunxue No. 1 and Yunxue No. 6, as well as between Yunxue No. 2 and Yunxue No. 6, showed a downward trend. This suggests that the AMF communities of different cigar tobacco varieties exhibit significant differences in OTU numbers as fermentation progresses. The distribution of overlapping and unique OTUs highlights the impact of different fermentation conditions on the diversity of the AMF community. The number of OTUs in all three varieties showed different trends during fermentation, particularly from the early to mid-fermentation stages. Yunxue No. 2 and Yunxue No. 6 displayed a more stable OTU number, showing a trend of decrease followed by an increase, while Yunxue No. 1 exhibited significant fluctuations during the mid-fermentation stage. These fluctuations may reflect the competition and interactions among different AMF species, that is, the successional dynamics of AMF across different habitats and time scales, while also revealing the differences in physicochemical properties among the three cigar tobacco varieties (Hart et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Due to the differences in the content of macromolecules (such as proteins, starch, pectin, etc.) between tobacco leaf varieties, these physicochemical properties may significantly impact the composition and metabolic activity of the AMF community during fermentation, leading to significant fluctuations in the community structure of AMF throughout the fermentation process. This variability also suggests that the variety-specific chemical composition plays an important regulatory role in the adaptability and ecological succession of microorganisms. However, for all three varieties, the number of OTUs showed a clear increase from the early stages of fermentation to the end of fermentation (Wang et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDuring the 35-day fermentation process, the AMF communities of Yunxue No. 1, Yunxue No. 2, and Yunxue No. 6 exhibited dynamic changes. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e represents taxonomic statistics of species in different OTU sets. OTU217 consistently dominated across all varieties, and its significant presence indicates the core role of AMF fungi in the cigar tobacco fermentation process. During fermentation, certain saprophytic fungi may play an important role in the chemical composition changes of tobacco leaves. The sustained high abundance of OTU217 may suggest its potential positive impact on tobacco leaf quality through processes such as organic matter degradation and nutrient cycling during fermentation (Zhao et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). At the same time, the abundance changes of OTU217 and OTU88 were prominent across the three varieties, which aligns with the role of AMF as \"key mutualists\" in soil ecosystems, indicating that this phenomenon also exists in cigar tobacco fermentation (Yang et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In the later stages of fermentation, the presence of these OTUs will be related to the flavor and texture of the cigar tobacco, as studies have shown that any high-abundance specific fungi can alter the chemical composition in tobacco leaves during fermentation, thereby affecting the sensory properties of the final product (Zhang et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eOTU taxonomic statistics\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOrder\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFamily\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGenus\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOTU\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePercent\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003ePrevalence\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eo__Paraglomerales\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ef__Paraglomeraceae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eg__Paraglomus\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003es__unclassified_g__Paraglomus\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOTU217\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e176698\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.592819\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e96.15%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eo__Paraglomerales\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ef__Paraglomeraceae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eg__Paraglomus\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003es__unclassified_g__Paraglomus\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOTU88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e33198\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.111379\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e92.31%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eo__unclassified_c__Glomeromycetes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ef__unclassified_c__Glomeromycetes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eg__unclassified_c__Glomeromycetes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003es__unclassified_c__Glomeromycetes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOTU216\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e11678\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.03918\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e96.15%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eo__Paraglomerales\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ef__Paraglomeraceae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eg__Paraglomus\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003es__Paraglomus-Glom-1B.13-VTX00308\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOTU82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e4239\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.014222\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e26.92%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eo__Paraglomerales\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ef__Paraglomeraceae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eg__Paraglomus\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003es__unclassified_g__Paraglomus\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOTU76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e3458\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.011602\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e26.92%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eo__unclassified_c__Glomeromycetes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ef__unclassified_c__Glomeromycetes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eg__unclassified_c__Glomeromycetes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003es__unclassified_c__Glomeromycetes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOTU219\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2530\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.008488\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e61.54%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eo__unclassified_c__Glomeromycetes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ef__unclassified_c__Glomeromycetes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eg__unclassified_c__Glomeromycetes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003es__unclassified_c__Glomeromycetes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOTU24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1594\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.005348\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e3.85%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eo__unclassified_c__Glomeromycetes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ef__unclassified_c__Glomeromycetes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eg__unclassified_c__Glomeromycetes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003es__unclassified_c__Glomeromycetes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOTU265\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1657\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.005559\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e61.54%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eo__unclassified_c__Glomeromycetes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ef__unclassified_c__Glomeromycetes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eg__unclassified_c__Glomeromycetes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003es__unclassified_c__Glomeromycetes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOTU470\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e3047\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.010223\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e69.23%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eo__unclassified_c__Glomeromycetes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ef__unclassified_c__Glomeromycetes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eg__unclassified_c__Glomeromycetes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003es__unclassified_c__Glomeromycetes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOTU260\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1614\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.005415\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e50%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAMF microbial diversity index analysis\u003c/h3\u003e\n\u003cp\u003eThe Sobs index represents the number of observed species and reflects the trend in community richness (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Before fermentation, the richness of the three cigar tobacco varieties was relatively low, showing a relatively small number of OTUs. By the second week, the Sobs index of Yunxue No. 1 and Yunxue No. 6 increased rapidly, with a significant rise in richness, peaking in the third week. Afterward, it showed a declining trend, reaching its lowest point in the fifth week, indicating that these two varieties may share a similar AMF community succession pattern. In contrast, Yunxue No. 2 showed a decrease in the second week and did not exhibit a continuous upward trend. This suggests that the chemical components in Yunxue No. 2 may be more difficult for AMF to utilize directly in the early fermentation stages, or that the available substrates for AMF are limited. For example, if certain macromolecules in Yunxue No. 2 are difficult to degrade, the growth of AMF may be inhibited. This is consistent with the relationship between AMF and substrate availability observed in the study by Gryndler et al. (2022). During fermentation, microbial communities must continuously adapt to the changing chemical environment. The chemical composition of Yunxue No. 2 may lead to fluctuations in its AMF community's adaptability, resulting in a decrease during the second week. The succession pattern of AMF in the fermentation process of all three cigar tobacco varieties followed a \"rapid growth\u0026mdash;peak\u0026mdash;gradual decline\" trend, indicating that the AMF succession pattern is similar to that of typical microbial community succession. Despite differences in the physicochemical properties of these varieties, the succession of the AMF community during fermentation is driven by similar external factors, such as nutrient depletion and environmental changes. This phenomenon suggests that the microbial dynamics during fermentation in different cigar tobacco varieties may follow a common pattern, which is of significant importance for the standardization and optimization of fermentation processes (Michele et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Si et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe Shannon index (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) shows the microbial community diversity of the three cigar tobacco varieties, with significant differences in the trends observed during fermentation. Yunxue No. 1 displayed a wavy trend, with the highest diversity occurring in the third week. Yunxue No. 2 and Yunxue No. 6 followed a \"decrease followed by increase\" trend, with Yunxue No. 6 reaching the highest microbial diversity in the first week. Yunxue No. 1 may have introduced more competitive microorganisms during fermentation, which dynamically competed with AMF and other microorganisms at different fermentation stages (Atta et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For example, in the early fermentation stage, certain microbial communities may grow rapidly, causing the Shannon index to decline; later, other microorganisms reoccupy ecological niches, leading to an increase in diversity. This frequent competitive replacement process results in the wavy fluctuations of the Shannon index. For Yunxue No. 2 and Yunxue No. 6, the competitive effect may be weaker, mainly due to the gradual depletion of nutrients by the early dominant microorganisms, followed by the recovery of secondary microorganisms, showing a single \"decrease then increase\" pattern.\u003c/p\u003e\u003cp\u003eThe Chao index (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) also shows significant fluctuations during fermentation for all three varieties, with clear differences in the patterns observed. Notably, the highest and lowest points for each variety's index do not align. For Yunxue No. 1, the richness of rare species peaked in the second week and was lowest in the fifth week; for Yunxue No. 2, it was highest in the fifth week and lowest in the third week; and for Yunxue No. 6, it was highest in the fourth week and lowest in the first week. The Chao index for the three cigar tobacco varieties exhibited different fluctuation patterns during fermentation, particularly the differences between the highest and lowest points. This may be due to differences in the physicochemical components and nutritional substrates of each variety, which cause rare species in the microbial community to respond differently to specific nutrients at different fermentation stages (Li et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Little et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Specifically, the composition of Yunxue No. 1 promotes the rapid growth of rare species in the early fermentation stage, resulting in the highest richness in the second week. In contrast, the composition of Yunxue No. 2 and Yunxue No. 6 favors the continued colonization and growth of rare species in the later stages of fermentation, leading to peak richness in the fourth and fifth weeks, respectively. These differences in substrate composition may affect the adaptability and colonization dynamics of rare species, causing them to reach their peak richness at different stages of fermentation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eDynamic differences of AMF at the genus level\u003c/h2\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, during fermentation, the AMF communities of the three varieties exhibited significant differences at the genus level. \u003cem\u003eParaglomus\u003c/em\u003e was the absolute dominant genus, with the highest relative abundance across all varieties and fermentation stages, indicating its strong adaptability and stability in the fermentation environment. This may be closely related to the high metabolic activity and competitive ability of \u003cem\u003eParaglomus\u003c/em\u003e in the high-temperature, low-oxygen, and nutrient-rich fermentation environment, allowing it to dominate and become the core functional group in the fermentation process. Furthermore, the dominance of \u003cem\u003eParaglomus\u003c/em\u003e may play an important role in key metabolic functions, such as carbon-nitrogen cycling and organic matter degradation (Gosling et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Mello et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). \u003cem\u003eUnclassified_c__Glomeromycetes\u003c/em\u003e ranked second to \u003cem\u003eParaglomus\u003c/em\u003e and showed a gradual increase in relative abundance towards the end of fermentation, reflecting its adaptation to the environmental changes in the later fermentation stages. This phenomenon is related to its specific metabolic functions, such as the degradation of residual materials or participation in nutrient cycling, thereby reflecting the dynamic balance and functional division within the microbial ecosystem (Oehl et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In the later stages of fermentation, the environmental changes caused by nutrient depletion and the accumulation of metabolic products likely provided a new ecological niche for this genus, promoting its increased abundance. Additionally, an interesting observation was made that the AMF community of Yunxue No. 2 exhibited relatively high diversity in the first week of fermentation, with a significant presence of \u003cem\u003eGlomus_f__Glomeraceae\u003c/em\u003e and a small amount of \u003cem\u003eunclassified_o__Archaeosporales\u003c/em\u003e. However, as fermentation progressed, the community structure gradually stabilized and became more consistent with the other two varieties. This initial high diversity reflects the more complex microbial ecosystem of Yunxue No. 2 in the early stages of fermentation, which may be related to the unique metabolic demands or greater microbial adaptability under specific conditions of this variety. The fluctuations in the microbial community observed in the early fermentation stages could be linked to the initial phase of material degradation and the rapid response of the community to environmental changes (Zhang et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Overall, these findings suggest that environmental selection pressures during fermentation drive the concentration of dominant AMF populations, while also revealing the functional division of different microbial communities at various stages of fermentation. These results provide important insights into the potential role of AMF in tobacco fermentation and suggest that future efforts could optimize community functions by regulating fermentation conditions, thereby improving fermentation quality.\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e illustrates the changes in the relative abundance of the two most abundant species, \u003cem\u003eParaglomus\u003c/em\u003e and \u003cem\u003eunclassified_c__Glomeromycetes\u003c/em\u003e, across the three varieties. \u003cem\u003eParaglomus\u003c/em\u003e consistently dominated throughout the fermentation process but exhibited significant fluctuations in abundance across the different varieties. In Yunxue No. 1, the relative abundance of \u003cem\u003eParaglomus\u003c/em\u003e followed a \"decrease, increase, then decrease\" trend, with the highest abundance occurring in the mid-fermentation phase. This may suggest that the \u003cem\u003eParaglomus\u003c/em\u003e community has slightly weaker adaptability to the early fermentation environment, but its metabolic activity increases as fermentation progresses. However, by the end of fermentation, its abundance likely decreased again due to nutrient depletion or the inhibitory effects of secondary metabolites. For Yunxue No. 2, the fluctuations of \u003cem\u003eParaglomus\u003c/em\u003e were more pronounced, with a significant decrease in abundance during the third week, and the highest abundance observed in the pre-fermentation stage. This reflects that the initial microbial activity in this variety was high, but during the mid-fermentation stage, the community faced greater environmental pressures, possibly influenced by pH fluctuations or nutrient competition (Wu et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In contrast, the fluctuations of \u003cem\u003eParaglomus\u003c/em\u003e in Yunxue No. 6 were smaller, with abundance gradually increasing after fermentation began, stabilizing in the mid-fermentation phase, and decreasing as fermentation neared its end. This suggests that the AMF community in Yunxue No. 6 was more stable, with its AMF population possibly better adapted to the changes in the fermentation environment. Overall, the relative abundance of \u003cem\u003eParaglomus\u003c/em\u003e at the end of fermentation was generally lower than in the pre-fermentation stage, indicating the presence of limiting factors that restricted its growth during fermentation. Meanwhile, the abundance trend of \u003cem\u003eunclassified_c__Glomeromycetes\u003c/em\u003e was the opposite of \u003cem\u003eParaglomus\u003c/em\u003e, with the two genera showing reciprocal fluctuations in abundance during fermentation. In the early stages of fermentation, \u003cem\u003eParaglomus\u003c/em\u003e was dominant, leading the community diversity, while the abundance of \u003cem\u003eunclassified_c__Glomeromycetes\u003c/em\u003e was relatively low. This may be related to the nutrient breakdown demands and the competitive ability of \u003cem\u003eParaglomus\u003c/em\u003e in the early fermentation phase (Wang et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Song et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, in the later stages of fermentation, the abundance of \u003cem\u003eunclassified_c__Glomeromycetes\u003c/em\u003e increased significantly, suggesting that it may play specific metabolic roles in the later stages, such as breaking down residual fermentation materials or utilizing metabolic products generated in mid-fermentation. At the same time, its abundance rebound reflects the dynamic balance of the microbial ecosystem, especially in the later stages of fermentation, where non-dominant microbial groups may play compensatory roles in nutrient cycling or environmental regulation (Yang et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eCommunity succession of AMF at the species level and functional potential analysis\u003c/h2\u003e\u003cp\u003eAt the species level (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), throughout the entire fermentation stage, \u003cem\u003eunclassified_g_Paraglomus\u003c/em\u003e dominated, showing an increasing-then-decreasing trend, with its relative abundance at the end of fermentation being lower than in the pre-fermentation stage. The second most abundant species was \u003cem\u003eunclassified_c_Glomeromycetes\u003c/em\u003e, whose relative abundance increased rapidly in the early stages of fermentation, exhibiting a wave-like trend. By the end of fermentation, its relative abundance reached a peak, significantly higher than in the pre-fermentation stage. This phenomenon is related to the dynamic regulation of fermentation environmental factors. Studies have shown that the community structure of AMF is jointly influenced by changes in temperature, humidity, and the physicochemical properties of substrates (Lenoir et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The high temperature and humidity at the beginning of fermentation provided a brief, suitable growth environment for \u003cem\u003eParaglomus\u003c/em\u003e, but as metabolic products accumulated during fermentation, the survival pressure increased, leading to a decline in its abundance. The wave-like trend of \u003cem\u003eGlomeromycetes\u003c/em\u003e may be related to its adaptation to the substrates (such as cellulose and lignin degradation products) at different fermentation stages. Its final peak abundance reflects its dominant position in functional metabolism during the later stages of fermentation. Notably, the dynamic changes of \u003cem\u003eGlomus-group-B-Glomus-sp.-VTX00279\u003c/em\u003e and \u003cem\u003eGlomus-group-B-Glomus-lamellosu-VTX00193\u003c/em\u003e were particularly unique. The former occupied 19.67% of the relative abundance before fermentation but rapidly decreased to 0.23% in the early fermentation stages and gradually declined until it completely disappeared. In contrast, the latter was undetectable in both the pre-fermentation and early fermentation stages, but its relative abundance rapidly increased to 15.3% in the later stages. These unusual phenomena are related to multiple factors. In the early stages of fermentation, the rapid degradation of carbohydrates in the tobacco leaves generates a large amount of simple carbon sources, such as glucose, which triggers intense competition within the microbial community (Banožić et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ma et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). \u003cem\u003eGlomus-group-B-Glomus-sp.-VTX00279\u003c/em\u003e may have a lower carbon source utilization efficiency, preventing it from competing with rapidly growing bacteria (such as \u003cem\u003eProteobacteria\u003c/em\u003e) or functional fungi, leading to a sharp decline in its abundance. In contrast, \u003cem\u003eGlomus-group-B-Glomus-lamellosu-VTX00193\u003c/em\u003e may possess the ability to efficiently degrade complex organic matter. During the later stages of fermentation, cellulose and hemicellulose in the tobacco leaves are gradually broken down into more stable lignin derivatives, and \u003cem\u003eGlomus-lamellosu-VTX00193\u003c/em\u003e may gain a competitive advantage by secreting specific extracellular enzymes, such as laccase and peroxidase, to utilize these substrates. Additionally, studies have indicated that some species within the \u003cem\u003eGlomus\u003c/em\u003e genus can enhance heat and salt tolerance by accumulating compatible solutes like trehalose, and environmental stress in the later stages of fermentation may select for strains with such stress-resistance mechanisms (Oc\u0026oacute;n et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The decline of \u003cem\u003eGlomus-sp.-VTX00279\u003c/em\u003e and the rise of \u003cem\u003eGlomus-lamellosu-VTX00193\u003c/em\u003e may represent a \"functional relay\" within the AMF community at different stages of fermentation. The former may participate in the early mobilization of carbon sources before fermentation, while the latter dominates the conversion of complex organic matter in the later stages. This successional pattern is closely related to key metabolic pathways involved in the formation of cigar tobacco leaf quality (e.g., terpene synthesis) (Liu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Jiang et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Previous studies have highlighted the dominant role of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e and \u003cem\u003eThermoascus\u003c/em\u003e fungi in flavor formation (He et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), while this study suggests that \u003cem\u003eGlomus\u003c/em\u003e in AMF may indirectly influence the generation of flavor precursors through substrate pre-conversion, indicating that the functional roles of AMF communities in fermentation remain to be further explored.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eDynamic changes of key chemical components during cigar tobacco fermentation\u003c/h2\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows the changes in the total nitrogen (Total Nitrogen), total sugar (Total Sugar), reducing sugar (Reducing Sugar), and total alkaloids (Total Alkaloids) content for YX-1, YX-2, and YX-6 at different fermentation times. Overall, with the extension of fermentation time, all samples showed varying degrees of decrease in total nitrogen, total sugar, reducing sugar, and total alkaloids, but the decline trends differed between varieties. Specifically, for total nitrogen and total sugar, YX-1 exhibited a more gradual decline compared to YX-2 and YX-6. YX-6 showed more fluctuation in the decline trend, particularly in the mid-fermentation stage, between the second and third weeks, with the highest content observed before fermentation for both of the latter. YX-2 consistently had the lowest total sugar content throughout the fermentation process. This result indicates that the decomposition or transformation of total nitrogen and total sugar during fermentation is a common phenomenon, related to the microbial metabolism of proteins and sugars. By degrading nitrogen sources and sugars, microbes create more favorable growth conditions for themselves (Nord et al., 1926; Hu et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The overall trend of reducing sugar content showed no significant differences in its decline. YX-2 had significantly higher reducing sugar content in the early stages compared to YX-1 and YX-6, but by the end of fermentation, YX-2's reducing sugar content was significantly lower than that of YX-1. This suggests that YX-2 may have undergone more extensive enzymatic hydrolysis during fermentation, resulting in a more pronounced reduction in reducing sugar content. The reduction in reducing sugars is typically associated with microbial sugar degradation, a process that helps improve the flavor of cigars by reducing sweetness and enhancing complexity, indicating that AMF plays a key role in sugar degradation (Su et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In the case of total alkaloids, YX-1 exhibited a slower decline in alkaloid content throughout fermentation, while YX-6 showed the most significant changes in alkaloid content, with a noticeable increase at the end of fermentation compared to the previous week. This phenomenon may be related to changes in the microbial community or adjustments in metabolic pathways, as some microorganisms may begin to resynthesize or transform alkaloid compounds. Particularly at the end of fermentation, due to changes in the fermentation environment, the metabolic direction of the microbial community might shift, leading to the rebound in alkaloid content (Ponomarova et al., 2015). This phenomenon was observed only in YX-6, suggesting that YX-6 cigar tobacco may contain specific compounds or exhibit stronger microbial activity, causing greater fluctuations in the later stages of fermentation.\u003c/p\u003e\u003cp\u003eA linear regression model was used to further quantify the dynamic relationship between key chemical components and the Sobs index of AMF during the fermentation of Yunxue series cigar tobacco (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Total nitrogen (TN), total sugar (TS), and reducing sugar (RS) all showed significant negative correlations with the Sobs index, indicating that the degradation of these components during fermentation promotes an increase in AMF community diversity. This may be because the reduction in sugars and nitrogen sources inhibits the rapid proliferation of certain dominant microbial populations, allowing the AMF community to develop quickly. Although total alkaloids (TA) showed a certain negative correlation with the Sobs index, the correlation was weaker, suggesting that the impact of total alkaloids on AMF community diversity is more complex and may be regulated by other environmental factors. These results suggest that during the fermentation of cigar tobacco, properly regulating the diversity of the AMF community helps to precisely control the content of key chemical components, which in turn influences the quality and flavor of the final product. Using relatively rare AMF strains as cigar fermentation agents could provide a new approach to improving cigar quality and open new research directions for the optimization of fermentation processes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eCorrelation analysis of key chemical components and AMF community diversity\u003c/h2\u003e\u003cp\u003eBased on the species-species correlation, a species correlation network diagram was constructed (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). The results show that the diversity of the AMF community exhibits complex species interactions. \u003cem\u003eParaglomus\u003c/em\u003e species occupy a central position in the community, with \u003cem\u003es_unclassified_g_Paraglomus\u003c/em\u003e showing the highest relative abundance and a significant negative correlation with \u003cem\u003es_unclassified_c_Glomeromycetes\u003c/em\u003e, which has the second-highest relative abundance. The relative abundances of both fluctuate during fermentation. Additionally, \u003cem\u003es_unclassified_g_Paraglomus\u003c/em\u003e was also negatively correlated with \u003cem\u003es_unclassified_g_Glomus_f_Glomeraceae\u003c/em\u003e and \u003cem\u003es_Paraglomus-Glom-1B.13-VTX00308\u003c/em\u003e, though these correlations were not significant. These three negative correlations were the only ones observed in the entire correlation network diagram. The fermentation environment significantly influenced the interactions between these species, with some species displaying strong adaptability under these conditions, while others were affected by competition or resource limitations. The positive and negative interactions within the AMF community reveal their potential ecological roles during the tobacco fermentation process. To further understand the mechanisms through which these interactions affect the quality of tobacco fermentation, a correlation heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB) was constructed, and a Mantel test analysis was conducted. The results show that the correlations within the AMF community under different fermentation pile sample conditions follow a complex pattern. \u003cem\u003eGlomus-MO-G16-VTX00072\u003c/em\u003e was significantly positively correlated with total nitrogen, total sugar, reducing sugar, and total alkaloids, suggesting that \u003cem\u003eGlomus-MO-G16-VTX00072\u003c/em\u003e may enhance sweetness and optimize cigar flavor by promoting carbohydrate metabolism and increasing the total sugar and reducing sugar content in tobacco leaves. At the same time, it may influence the secondary metabolism of tobacco, raising the total alkaloid levels, thereby enhancing the strength and smoking experience of the cigar (Hu et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Hu et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This microorganism could be an important beneficial fermentation strain within the AMF community. Certain species, such as \u003cem\u003eunclassified_g__Paraglomus\u003c/em\u003e, showed significant correlations with major sugars (total sugar, reducing sugar), suggesting potential for development. On the other hand, \u003cem\u003eunclassified_o__Archaeosporales\u003c/em\u003e exhibited negative correlations with all four quality indicators measured, indicating that its high abundance may negatively impact the flavor of the cigar. The abundance of this species should be appropriately controlled during fermentation to prevent it from diminishing the cigar's flavor and reducing its complexity. The Mantel test further revealed the dynamic correlation between chemical components and AMF community diversity during cigar tobacco fermentation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Total nitrogen (TN) showed a positive correlation with AMF diversity throughout the fermentation process, but the correlation was not significant. Total sugar (TS) was positively correlated with AMF diversity in the early fermentation stages (F₁-F₂), but as fermentation progressed into the mid- and late stages, it became negatively correlated, suggesting that the sugar resources were rapidly consumed or microbial competition intensified in the earlier stages. Reducing sugar showed a negative correlation with AMF diversity in the mid-fermentation stage, while it was positively correlated in the early and later stages, confirming the ecological strategy of AMF to prioritize easily metabolizable carbon sources (Wu et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Total alkaloids (TA), due to their antimicrobial properties, significantly inhibited the AMF community, possibly leading to the elimination of sensitive strains, and became negatively correlated with AMF diversity as soon as the fermentation stage began.\u003c/p\u003e\u003cp\u003eOverall, the AMF community exhibited complex interaction patterns during cigar tobacco fermentation, with different species having varying impacts on tobacco leaf quality. Among these, \u003cem\u003eGlomus-MO-G16-VTX00072\u003c/em\u003e, as a potential beneficial fermentation strain, can optimize the sugar-alkaloid ratio and enhance cigar flavor, while certain species such as \u003cem\u003eunclassified_o__Archaeosporales\u003c/em\u003e may negatively affect quality and should be controlled. These findings provide new directions for regulating AMF communities and optimizing fermentation processes, helping to improve the quality stability and flavor profile of cigar tobacco. The analytical methods employed in this study primarily emphasize the correlation between community structure and physicochemical components. However, the precise metabolic functions and mechanisms of arbuscular mycorrhizal fungi (AMF) in cigar fermentation have not been conclusively established through isolation culture, metagenomic, or macrotranscriptome techniques. The existing functional evidence remains relatively limited. Subsequent investigations will enhance sample representativeness, bolster functional verification, and conduct in-depth mechanism analyses to comprehensively elucidate the role of AMF in the fermentation microecosystem. This research aims to support the practical application of AMF in tobacco fermentation and other related industries.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe discovery of dynamic and structured arbuscular mycorrhizal fungal (AMF) communities during the fermentation of cured (non-living) cigar tobacco presents a fascinating ecological paradox. Traditionally, AMF are considered obligate biotrophic symbionts dependent on living plant hosts for carbon and survival (Vel\u0026aacute;squez et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Samanta et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Their persistence and successional dynamics throughout the 35-day fermentation process of processed tobacco leaves suggest a significant ecological plasticity or a previously underappreciated saprotrophic capability in certain taxa within the \u003cem\u003eGlomeromycota\u003c/em\u003e phylum. This study, combined with insights from recent literature, suggests that these fungi likely originate from pre-existing colonization within the tobacco leaves during growth and the rhizosphere soil (Liu et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The curing process may not completely eradicate these robust fungi, whose spores and hyphal fragments remain viable. During fermentation, the changing microenvironment\u0026mdash;characterized by shifting nutrient availability, moisture, temperature, and microbial competition\u0026mdash;appears to drive a functional transition in these communities, allowing them to participate in the decomposition and transformation of tobacco organic matter.\u003c/p\u003e\u003cp\u003eThe inverse abundance patterns observed between \u003cem\u003eParaglomus\u003c/em\u003e (e.g., OTU217) and unclassified \u003cem\u003eGlomeromycetes\u003c/em\u003e, along with the dramatic species-level succession (e.g., the decline of \u003cem\u003eGlomus-group-B-Glomus-sp.-VTX00279\u003c/em\u003e and the rise of \u003cem\u003eGlomus-group-B-Glomus-lamellosu-VTX00193\u003c/em\u003e), suggest a \"functional relay\" within the AMF community. This indicates that different AMF taxa may adapt to utilize distinct nutritional niches at various fermentation stages. The sustained dominance of OTU217 (\u003cem\u003eParaglomus\u003c/em\u003e) highlights its potential role as a core functional unit, likely contributing to the degradation of complex organic compounds like cellulose, hemicellulose, and lignin derivatives\u0026mdash;processes crucial for releasing precursors for flavor development (Wu et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This is supported by studies in other systems showing AMF capabilities in organic matter decomposition and nutrient cycling (Liu et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Furthermore, the significant negative correlations between AMF community diversity (Sobs index) and the degradation of total nitrogen, total sugar, and reducing sugar underscore the potential impact of AMF on cigar tobacco quality. The negative correlations suggest that as these nutrients are metabolized, competitive pressure on the microbial community may shift, allowing for greater AMF diversity and activity. This aligns with the concept that nutrient depletion can reduce the dominance of fast-growing r-strategists, creating opportunities for more K-strategist or specialist fungi like AMF. The strong correlations between specific AMF taxa and key chemical components, as revealed by the Mantel test and correlation network analysis, further support their functional contributions. For instance, the positive correlation between \u003cem\u003eGlomus-MO-G16-VTX00072\u003c/em\u003e and total sugar, reducing sugar, and total alkaloids suggests that certain AMF taxa may actively participate in modulating the sugar-alkaloid balance, ultimately influencing the sensory profile of the final cigar product (Carpenter et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Hu et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This is consistent with findings in other agricultural products, where AMF have been shown to influence secondary metabolite accumulation. Conversely, the negative correlation between \u003cem\u003eunclassified_o__Archaeosporales\u003c/em\u003e and all quality indicators suggests that some AMF taxa may have detrimental effects, possibly through the production of off-flavor compounds or inefficient resource competition (Ran et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe presence of active AMF communities in fermenting cigar tobacco, a non-living substrate, can be explained by several factors. First, the fermentation environment\u0026mdash;characterized by elevated temperatures, limited oxygen availability, and rich in complex organic compounds\u0026mdash;may selectively favor AMF taxa with robust saprotrophic capabilities or those that can enter a dormant, spore-forming state until conditions improve. AMF spores are known for their resilience and can survive in harsh environments (Nie et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Carrillo-Saucedo et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Second, the initial chemical composition of the tobacco leaf, particularly the content of macromolecules such as proteins, starch, and pectin, varies between varieties (Yunxue No. 1, No. 2, No. 6) and likely provides a diverse nutrient base that can be utilized by AMF with specific enzymatic capacities. The variety-specific successional patterns observed in this study support this notion (Bisht et al., 2022). Finally, the interactions between AMF and other microbes (bacteria and other fungi) within the fermentation pile likely play a crucial role in determining AMF activity. For example, bacteria may pre-degrade complex polymers into simpler compounds that AMF can assimilate, or AMF may provide access to otherwise inaccessible nutrients through their hyphal networks (Wipf et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis study offers a new perspective on AMF, suggesting that they may be more than just remnants of a past symbiotic relationship and could potentially act as active participants in the cigar tobacco fermentation process. The variety-specific successional patterns indicate that the initial chemical composition of the tobacco leaf, influenced by genotype and growth conditions, might be used to predict and guide microbial succession toward desired outcomes. These findings open potential avenues for developing novel fermentation strategies, such as using tailored microbial consortia that include AMF, to possibly improve fermentation efficiency, consistency, and final product quality. For example, inoculating fermentation piles with specific AMF taxa that appear beneficial could help accelerate the breakdown of undesirable compounds, promote the formation of favorable aroma precursors, and enhance the overall smoking experience. Alternatively, managing fermentation conditions (e.g., temperature, humidity, turning frequency) to suppress the growth of potentially unfavorable taxa like \u003cem\u003eunclassified_o__Archaeosporales\u003c/em\u003e might also be a useful strategy.\u003c/p\u003e\u003cp\u003eThe analytical methods employed in this study primarily emphasize the correlation between community structure and physicochemical components. However, the precise metabolic functions and mechanisms of arbuscular mycorrhizal fungi (AMF) in cigar fermentation have not been conclusively established through isolation culture, metagenomic, or macrotranscriptome techniques. The existing functional evidence remains relatively limited. To fully elucidate the functional roles of AMF in cigar tobacco fermentation, future studies should prioritize the isolation and cultivation of dominant taxa for in vitro functional validation, combined with multi-omics and stable isotope probing to characterize their in situ activity and nutrient metabolism. Furthermore, manipulative experiments using inoculation or suppression of specific AMF, along with expanded sampling across varieties and fermentation conditions, will be essential to establish causal relationships and enhance the generalizability of the findings.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study is the first to reveal the dynamic succession patterns of the arbuscular mycorrhizal fungi (AMF) community during cigar tobacco fermentation and its association with tobacco leaf quality. The AMF community exhibited significant variety-specific and stage-dependent succession characteristics during fermentation. A total of 524 OTUs were identified, with \u003cem\u003eParaglomus\u003c/em\u003e being the absolute dominant genus. Its metabolic activity is closely linked to its adaptability to the high-temperature, nutrient-rich environment, and it dominates the fermentation process through carbon-nitrogen cycling. Meanwhile, \u003cem\u003eunclassified_c__Glomeromycetes\u003c/em\u003e showed an increase in abundance in the later stages, possibly involved in the degradation of residual substances. The dynamic differences in AMF diversity between varieties were significant, with Yunxue No. 1 displaying wave-like fluctuations in the Shannon index due to competitive microbial fluctuations, Yunxue No. 6 experiencing a surge in rare species in the late stages as indicated by the Chao index, and Yunxue No. 2 showing a decrease in the Sobs index in the early stages due to the difficulty in degrading certain substrates. The key species \u003cem\u003eGlomus-group-B-Glomus-lamellosu-VTX00193\u003c/em\u003e experienced a sharp increase in abundance to 15.3% in the later stages, possibly through the secretion of extracellular enzymes to degrade lignin derivatives, forming a functional relay with \u003cem\u003eGlomus-sp.-VTX00279\u003c/em\u003e. Chemical component analysis showed significant negative correlations between total nitrogen, total sugar, and AMF abundance, suggesting that AMF promotes community diversity through the degradation of macromolecular substances. \u003cem\u003eGlomus-MO-G16-VTX00072\u003c/em\u003e was positively correlated with sugars, indicating its potential to optimize flavor. The study confirms that AMF can utilize residual carbon sources from plant remains to maintain metabolic activity and survive in the cigar tobacco fermentation system. The community succession of AMF is regulated by both the physicochemical properties of the cigar variety and the fermentation environment. The core functions of \u003cem\u003eParaglomus\u003c/em\u003e and the specific metabolism of \u003cem\u003eGlomus\u003c/em\u003e species provide a theoretical basis for the targeted regulation of fermentation quality.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis study work was supported by the Science and Technology Plan Project of the China National Tobacco Corporation, Yunnan Provincial Company (2023530000241002).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eHZ reviewed and edited the manuscript. XRS, QZ, YMY, YY, JLZ, YHC, LDB and YBS conducted the investigation. YBS acquired funding and reviewed the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data supporting the conclusions of this manuscript have been presented in the manuscript and are publicly accessible. Regarding the raw sequencing data of this study, they have not yet been deposited in public data repositories due to the signed confidentiality agreement. For further inquiries, please contact the corresponding author at [email protected] to obtain the data.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAci MM, Agosteo GE, Pelle G, Malacrin\u0026ograve; A, Schena L (2025) Environmental isolates of \u003cem\u003ePseudomonas\u003c/em\u003e spp. inhibit \u003cem\u003eArmillaria mellea\u003c/em\u003e and promote plant growth through microbiome-mediated effects. 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Front Microbiol 13:911791. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2022.911791\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2022.911791\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cigar Tobacco, Fermentation, Arbuscular Mycorrhizal Fungi, Physicochemical Components, Microbial Community","lastPublishedDoi":"10.21203/rs.3.rs-7416527/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7416527/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eArbuscular Mycorrhizal Fungi (AMF) are key species in plant-microbe interactions, and this study is the first to discover their dynamic survival in the fermentation system of cigar tobacco. To explore the functional significance of AMF in cigar tobacco fermentation, this study focused on the Yunxue variety of cigar tobacco. We combined multi-time point sampling over a 35-day fermentation process and used Internal Transcribed Spacer (ITS) gene high-throughput sequencing to analyze the AMF community structure. Diversity indices, species correlation networks, and Mantel tests were employed to explore the relationship between AMF and chemical components. The study revealed a significant dynamic succession within the arbuscular mycorrhizal fungi (AMF) community throughout the fermentation process, identifying 22 species (comprising 524 operational taxonomic units [OTUs]), with Paraglomus being the predominant species. Core functional flora included OTU217 and OTU88, whose abundance variations aligned with the generation of volatile flavor compounds. AMF diversity peaked during the mid-fermentation stage and exhibited a negative correlation with total nitrogen (TN), total sulfur (TS), and reducing sugars (RS), indicating that sugar and nitrogen metabolism were driving factors in the reorganization of the AMF community. Notably, \u003cem\u003eGlomus-group-B-Glomus-lamellosu-VTX00193\u003c/em\u003e demonstrated a marked increase in abundance towards the end of fermentation, suggesting its crucial role in the degradation of complex organic compounds. Analysis specific to different tobacco varieties revealed a significant increase in the number of OTUs unique to Yunxue 6, with fluctuations in total acidity (TA) content significantly associated with changes in AMF abundance. The findings highlight the regulatory role of AMF in modulating the chemical composition of tobacco leaves through carbon and nitrogen metabolism, with \u003cem\u003eParaglomu\u003c/em\u003es and \u003cem\u003eGlomus\u003c/em\u003eidentified as core functional flora. These results offer a foundational framework for targeted manipulation of AMF communities and the design of innovative fermentation processes.\u003c/p\u003e","manuscriptTitle":"Dynamic changes and potential contributions of arbuscular mycorrhizal fungi in cigar tobacco fermentation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-12 17:34:48","doi":"10.21203/rs.3.rs-7416527/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9c8eda8a-a0d6-4516-8143-3781d7db2c89","owner":[],"postedDate":"September 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-13T06:24:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-12 17:34:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7416527","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7416527","identity":"rs-7416527","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}