The effect of flue-curing and redrying on the diversity of fungal communities in tobacco leaves

Abstract

Flue-curing and redrying are important processing stages before tobacco fermentation, closely linked to microbial actions that influence the fermentation process. It is necessary to investigate the effects of flue-curing and redrying on diversity and succession of tobacco fungal communities. It was shown that a total of 9 phyla, 33 classes, 94 orders, 266 families, 646 genera, and 6,396 amplicon sequence variants (ASVs) were identified in the fungi communities of 36 samples from different processing stages (before flue-curing, after flue-curing, before redrying and after redrying) based on high-throughput sequencing technology. Dominant genera shared by tobacco leaves at different stages were Alternaria and Sampaiozyma. About 80% of fungi in stored tobacco leaves after redrying originated from fresh tobacco leaves before flue-curing, while the rest were primarily enriched in the post-harvest processing environment. After flue-curing, major molds like Aspergillus and Penicillium were notably enriched. The distribution of fungal communities suggested that the flue-curing and redrying had a significant impact on fungal composition. Functional annotation of fungal communities at the guild level exhibited differences during processing stages. Main fungal functional groups were identified. In summary, our study elucidated dynamic changes in the composition of fungal communities and highlighted key stages in mold enrichment during tobacco leaf processing, laying groundwork for mildew prevention and control during tobacco leaf fermentation.

Background

Tobacco (Nicotiana tabacum L.) is one of the most widely cultivated non-food commercial crops globally [1]. Flue-curing and redrying are important links in the production and processing of tobacco leaves in the tobacco industry. The fresh tobacco leaves are harvested from the field and then flue-cured in baking barns [2]. Tobacco flue-curing involves three main stages: yellowing, color-fixing, and stem-drying [3]. The process includes seven stages: Stage 1 (dry-bulb temperature of 38 °C) for 8–10 h, Stage 2 (dry-bulb temperature of 40 °C) for 20–22 h, Stage 3 (dry-bulb temperature of 42 °C) for 8–10 h, Stage 4 (dry-bulb temperature of 45 °C) for 20–22 h, Stage 5 (dry-bulb temperature of 50 °C) for 8–10 h, Stage 6 (dry-bulb temperature of 54 °C) for 8–10 h, and Stage 7 (dry-bulb temperature of 68 °C) for 20–22 h [4]. During this period, moisture rapidly dissipates from the tobacco leaves, and the color of leaves changes from green to yellow. Studies shown that a relatively stable microbial ecosystem formed on tobacco leaves during the flue-curing process, with representative genera such as Alternaria, Cladosporium, and Symmetrospora [5, 6]. In the redrying process, the maximum temperature setting should not exceed 85℃ (typically maintained between 65–80℃), and redrying should last no less than 30 min [7]. Redrying involves further moisture removal from the leaves through heating and steam treatment until a specified moisture content (11.5-13.0%) is achieved. At the redrying stage, strong physical shearing and high temperature may disrupt the microbial community and inactivate many native microorganisms [8]. Ye et al. found that redrying significantly reduced the diversity of bacterial communities in tobacco leaves [8].

Freshly harvested tobacco leaves need to undergo flue-curing and redrying to improve their quality. These two processing steps are crucial for ensuring the safety of tobacco storage [9]. The fungal communities on the surface of tobacco leaves after redrying directly affect the quality of tobacco leaves during storage. Moldy tobacco leaves not only cause severe economic losses but also bring serious health risks [10]. At present, researches on microbial community structure and diversity of flue-cured tobacco leaves mostly focus on storage and fermentation stages. Therefore, analyzing fungal community diversity during processing stages is essential to identify the main sources of mold in tobacco storage and develop effective prevention measures against leaf mold. Researchers have isolated numerous molds from moldy tobacco leaves from different regions and identified several genera of molds present in stored moldy tobacco leaves. Aspergillus was the most commonly isolated fungus in mildew tobacco leaves, with Aspergillus repens, A. niger and A. ruber being predominant [11]. Aspergillus and Penicillium were dominant fungal genera in both moldy and healthy tobacco leaves [12]. Aspergillus, Penicillium, Rhizopus and Mucor were found to be the primary fungi causing tobacco mold [13]. The most common fungal species isolated on petioles and laminas in the color-fixing stage was Rhizopus oryzae, followed by saprotrophs, mostly Aspergillus spp [14].

Recently, high-throughput sequencing technology has been widely used in fungal research in tobacco leaves. Based on the high throughput sequencing platform, Meyerozyma, Alternaria, Aspergillus, and Emericella were identified as dominant genera in flue-cured tobacco leaves [14]. MiSeq high-throughput sequencing technology was used to analyze fungal communities of tobacco leaves from 12 regions, including Yunnan, Guizhou, Fujian and Henan provinces, revealing Ascomycota as the dominant fungal phylum [15]. Zhou et al. studied the composition, distribution, and functional group of fungal communities using Illumina HiSeq2500 high-throughput sequencing platform, and found that nourishment types of tobacco-inhabiting fungi were dominantly saprotrophic and pathotrophic [16]. In this study, high-throughput sequencing technology was employed to analyze the composition and diversity of fungal communities of tobacco leaves during tobacco processing. The successions of main molds in tobacco leaves were revealed. The key stage of molds enrichment during tobacco leaf processing was showed and a series of intervention measures can be taken during this period to reduce the possibility of contamination, to further ensure the quality of tobacco leaves.

Materials and methods

Materials

Samples were collected at different stages of processing, including before flue-curing, after flue-curing, before redrying and after redrying from tobacco planting fields in Lufeng (Yunnan province), Bozhou (Guizhou province), Ninghua (Fujian province), Huidong (Sichuan province), Guiyang (Hunan province), Mianchi (Henan province), and Feixian (Shangdong province), as described previously [17]. Tobacco plants were grown and harvested under normal conditions in various production areas. Leaves were flue-cured locally in baking barns and subsequently redried at local tobacco leaf redrying company. Samples before flue-curing were collected during the tobacco leaf ripening and harvesting. Samples after flue-curing were collected after the flue-curing in the baking barns. Samples before redrying were collected at the entrance of leaf threshing and redrying. And samples after redrying were collected at the end of the redrying process. Tobacco leaf samples included Yunyan 87, CB-1, Qinyan 96, and Zhongyan Texiang 301. 9 samples were selected randomly from different stages of tobacco processing. A total of 36 samples were obtained. For each sample, 10 leaves were collected randomly and their bases removed. The leaves were ground to fine powder under liquid nitrogen using a mortar, and the homogenized powder was used for DNA extraction.

ITS amplification and high-throughput sequencing

Total microbial genomic DNA was extracted from tobacco leaves using the CTAB method [18]. The purity and concentration of the extracted DNA were determined via agarose gel electrophoresis. The DNA concentration was subsequently diluted to 1 ng/µL with sterile water. Each PCR reaction consisted of 15 µL of Phusion^® High-Fidelity PCR Master Mix (New England Biolabs, United States), 0.2 µL of each primer (1 µM), and 10 ng of template DNA. For PCR amplification, the ITS 1 region was amplified by PCR under the following conditions: 98℃ for 1 min, 30 cycles at 98℃ for 10 s, 50 °C for 30 s, and 72 °C for 30 s, followed by a final extension at 72 °C for 5 min) using primers ITS1F (5’-CTTGGGTCATTTAGAGGAAGTAA-3’) and ITS2R (5’-GCTGCGTTTCTCTCGATCGATGC-3’) [19]. PCR products were purified using the the Qiagen Gel Extraction Kit (Qiagen, Germany). NEBNext^® Ultra™ IIDNA Library Prep Kit (New England Biolabs, United States) was used for library construction. After passing the quality control, the libraries were sequenced using NovaSeq6000 platform (Illumina, United States).

Bioinformation analysis

The raw data obtained from sequencing was spliced to obtain tags. These spliced tags were subjected to quality control to produce clean tags. Subsequently, chimera filtering was performed to obtain effective data that can be used for subsequent analysis. The effective tags of all samples were analyzed by DADA2 method of QIIME (version 2) to obtain amplicon sequence variables (ASVs) [20]. The obtained ASVs were then compared with the database using the classify-sklearn module in the QIIME2 (version 2) to obtain taxonomic information for each ASV. Alpha diversity indices, including Shannon, Simpson, Chao1 and Coverage, were calculated using QIIME (version 2) with the input data being absolute abundance data after normalization. The analysis of differences between exponential groups was based on corr.test function in psych package of R software (Version 4.4.1), which uses T test, Wilcoxon signed rank test, and Tukey test (T test and Wilcoxon signed rank test were performed when there were only two groups, and Tukey rank sum test was performed when the groups were greater than 2) to analyze whether the differences in fungal diversity between groups were significant. The adonis and anosim functions in QIIME (version 2) software were used to analyze the significance of community structure differences between groups. The significant biomarkers were identified using Linear discriminant analysis effect size (LEfSe) with a threshold of 3.0 on the logarithmic LDA score. FunGuild, an environmental functional database of fungi, was used for functional prediction of fungal communities [21]. The ecological functions of fungi were classified, and functional abundances of Tropic Mode and Guild were calculated based on FunGuild database annotations.

Results

High-throughput sequencing analysis

A total of 6,469,326 valid sequences were obtained from 36 samples, with an average of 179,704 sequences per sample. The average length of sequences was 234 bp (Table 1). The results showed that 9 phyla, 33 classes, 94 orders, 266 families, 646 genera, and 6,396 amplicon sequence variants (ASVs) were identified in tobacco leaf fungal communities.

Table 1 Sequence data analysis and diversity index of samples for fungal communities in tobacco leaves at different processing stage

Alpha diversity indices were shown in Table 1 and Supplementary Fig. 1. The Shannon and Simpson values, reflecting the fungi community diversity, ranged from 3.49 to 4.17 and 0.72 to 0.82, respectively. The diversity index of the fungal communities was the lowest (3.49) after flue-curing, increasing after redrying, which might be due to mechanical microorganisms attachment during redrying. Chao values, reflecting community richness, ranged from 472.30 to 598.77. The richness of fungal communities was lower (p value 0.0273) after flue-curing (472.30) compared to before flue-curing (577.63), indicating that flue-curing reduced the richness of fungal communities (Table S1). The dilution curves all reached the saturation phase with coverage more than 99%, indicating that Illumina sequencing was sufficiently deep to represent all fungal communities (Fig. 1).

Fig. 1

Dilution curves of sequenced tobacco leaf samples

Distribution of fungal community

As shown in Fig. 2, there were 48, 42, 28, and 44 unique fungal genera in tobacco leaves before flue-curing, after flue-curing, before redrying, and after redrying stages, respectively, indicating that new fungal groups enriched at different processing stages. A total of 107 genera present in fresh tobacco leaves were eliminated during flue-curing stage, including Metaphochonia, Kabatiella, Lachancea, and Phaeotremella. After redrying, tobacco leaves were stored in warehouse for extended fermentation periods, and the fungal composition thereafter closely related to that of the originally stored tobacco leaves. After redrying, 91 fungal genera including Botryobasidium, Chaetomella, Achaetomium and Tinctophorellus were eliminated, while 105 fungal genera were also introduced, such as Virosphaerella, Dialonectria, Stilbocrea and Heterophoma. Overall, about 80% of the fungal communities in tobacco leaves after redrying were consistent with those in fresh tobacco leaves, while approximately 20% resulted from post-harvest contamination.

Fig. 2

Venn diagrams show shared and unique fungal genera in tobacco samples from different processing stages. Numbers in the non-overlapping region indicate unique ASVs for the single sample; numbers in the overlapping region indicate shared ASVs for multi-samples. Bcuring, Before flue-curing; Acuring, After flue-curing; Bredry, Before redrying; Aredry, After redrying

Changes in the relative abundance of fungal communities at the phylum level during flue-curing and redrying processes of tobacco leaves were shown in Fig. 3a. The dominant fungal groups identified by clustering were similar, but their relative abundances varied at different processing stages. Ascomycota and Basidiomycota were the predominant phyla during tobacco processing, with other phyla sucn as Mucoromycota, Mortierellomycota, and Olpidiomycota showing relatively lower abundance. The relative abundance of Ascomycota increased from 54.04 to 77.33% after flue-curing compared to before flue-curing, then decreased to 64.61% after redrying. On the contrary, the relative abundance of Basidiomycota decreased from 45.33 to 18.83% after flue-curing compared to before flue-curing, then increased to 34.73% after redrying. The trends in relative abundance changes of Ascomycota and Basidiomycota during tobacco leaf processing were exactly opposite. Flue-curing and redrying altered the fungal phyla composition of tobacco leaves.

As shown in Fig. 3b, the fungal communities were dominated by Alternaria and Sampaiozyma at the genus level shared by four processing stages. The main genera listed in Fig. 3b belonged to Ascomycota, except for Sampaiozyma and Filobasidium, which belonged to Basidiomycota. There were differences in the dominant genera structure at different processing stages. Before flue-curing, the dominant genera were Alternaria (16.79%), Sampaiozyma (21.05%), and Filobasidium (11.67%). After flue-curing, before redrying, and after redrying, the dominant genera were consistently Alternaria, Aspergillus, and Sampaiozyma. The relative abundance of Aspergillus and Sampaiozyma significantly increased after flue-curing compared to before flue-curing. The relative abundance of Aspergillus maintained at a high level with the processing of tobacco leaves, making it one of the dominant genera in the later stages of processing. These findings indicated that the flue-curing had an impact on the fungal community composition of tobacco leaves.

Fig. 3

Relative abundance of fungal communities at the level of phylum (a) and genus (b) in tobacco leaves at different processing stage

Unique and shared ASVs analysis

A total of 6,396 ASVs were identified from all samples. Based on clustering results, shared and unique ASVs were analyzed among samples from different processing stages, as depicted in Fig. 4. The number of shared ASVs in tobacco leaves from different processing stages was 694 (10.85%). The number of ASVs decreased by 18.16% after flue-curing compared to before flue-curing, and decreased by 9.34% after redrying compared to before redrying. The number of unique ASVs in tobacco leaves before flue-curing, after flue-curing, before drying and after redrying was 1,156, 823, 1,142, and 935, respectively, with the highest number of unique ASVs present before flue-curing (Fig. 4a). Compared with the other three processing stages, the number of total ASVs and unique ASVs in tobacco leaves before flue-curing were the highest. Additionally, about 45.05% of ASVs after redrying were consistent with those in tobacco leaves before flue-curing (fresh tobacco leaves) (Fig. 4b). These results suggested that the fungal communities differed at different processing stages.

Fig. 4

Unique and shared ASVs of fungal communities at various processing stages. Numbers in the non-overlapping region indicate unique ASVs for the single sample; numbers in the overlapping region indicate shared ASVs for multi-samples. Bcuring, Before flue-curing; Acuring, After flue-curing; Bredry, Before redrying; Aredry, After redrying

Differential fungus analysis

The fungi exhibiting significant differences in abundance at different processing stages were analyzed using LEfSe. Only those with LDA score values greater than 3.5 were included (Fig. 5). The results indicated that before flue-curing, 3 fungal groups differed from other stages: Tremellomycetes at the class level, and Capnodiales and Tremellales at the order level. Tremellomycetes and Tremellales belong to Basidiomycota, whereas Capnodiales belonging to Ascomycota. After flue-curing, 6 fungal groups differed from other stages: Eurotiomycetes at the class level, Eurotiales at the order level, Aspergillaceae at the family level, Aspergillus at the genus level, and Aspergillus flavus and Aspergillus protuberus at the species level, all belonging to Ascomycota. Before redrying, 4 fungal groups differed from other stages: Coniothyriaceae at the family level, Coniothyrium at the genus level, and Aspergillus ruber and Aspergillus penicillioides at the species level, all belonging to Ascomycota. After redrying, 2 fungal groups differed from other stages: Glomerellales at the order level and Hypoxylon crocopelum at the species level, both belonging to Ascomycota.

Fig. 5

LEfSe analysis of fungal communities at different processing stages. (a) In the cladogram, the circle radiating from inside to outside represents the classification from phyla to species. (b) In the LDA score histogram, the lowercase letters denote different taxonomic level, of which “s” means species, “g” means genus, “f” means family, “o” means order and “c” means class. Bcuring, Before flue-curing; Acuring, After flue-curing; Bredry, Before redrying; Aredry, after redrying

Functional annotation of fungal communities

Functional annotation analysis showed that the fungal communities of tobacco leaves could be classified into 8 types: saprotroph, pathotroph-saprotroph-symbiotroph, pathotroph, pathotroph-saprotroph, pathotroph-symbiotroph, saprotroph-symbiotroph, pathogen-saprotroph-symbiotroph, and symbiotroph (Fig. 6). The trophic modes of fungal communities were dominated by saprotroph (17.55 -32.28%) and pathotroph-saprotroph-symbiotroph (16.24 -23.78%) during the four processing stages. The proportion of saprotroph-symbiotroph and pathotroph fungal groups in tobacco leaves increased, while the proportion of saprotroph, pathotroph-saprotroph-symbiotroph, and pathogen-saprotroph-symbiotroph fungal groups decreased after flue-curing compared to before flue-curing. The proportion of pathotroph-saprotroph and saprotroph fungal groups increased, while the proportion of pathotroph-symbiotroph and symbiotroph fungal groups decreased after redrying compared to before redrying. These results suggested that the main trophic modes of fungal communities of tobacco leaves were altered during processing.

Fig. 6

The trophic modes of fungal communities during different processing stages

Further functional annotation analysis of fungal communities was conducted using FunGuild (Fig. 7). The identified main groups include undefined saprotroph (17.36-32.09%), animal pathogen-endophyte-plant pathogen-wood saprotroph (15.27%-23.26), endophyte plant pathogen (8.13-12.55%), plant pathogen (4.49-6.55%), fungal parasite-undefined saprotroph (0.51-1.82%), and fungal parasite-plant pathogen (0.73-2.36%). The relative abundance of endophyte-undefined saprotroph, fungal parasite-undefined saprotroph, plant pathogen-wood saprotroph, fungal parasite-plant pathogen, lichenized, and animal pathogen decreased (p values 0.0385, 0.0391, 0.0391, 0.0039, 0.0355 and 0.0234, respectively) after flue-curing compared to before flue-curing. Compared to before redrying, the relative abundance of wood saprotroph, and endophyte-undefined saprotroph-wood saprotroph decreased (p values 0.0391 and 0.0343) after redrying (Table S3). During the processing of tobacco leaves, there were some differences in the functional annotation of fungal communities at the guild level.

Fig. 7

Functional annotation of fungal communities at the guild level during different processing stages

Discussion

The study of fungal community composition revealed a rich fungi composition of tobacco leaves at different processing stages, with some differences in community structure. Ascomycota and Basidiomycota were the dominant phyla in tobacco leaves at different processing stages (Fig. 3a). Ascomycota was a common endophytic group in plants. Chen et al. found that the dominant fungal group in 12 aged tobacco samples at the phylum level were Ascomycota [22]. Ascomycota and Basidiomycota were the main fungal groups in healthy tobacco after flue-curing [22]. Basidiomycota and Ascomycota were found to be the dominant phyla shared by the four processing stages of tobacco leaves [23]. The results of the above researches were similar to the results of our study. Additionally, at the genus level, Sampaiozyma and Alternaria were the dominant groups shared by four processing stages (Fig. 3b). Chen et al. found that the dominant genus of healthy tobacco after flue-curing was Alternaria, followed by Aspergillus [22]. Sampaiozyma was the dominant genus in tobacco leaves at the early stage of fermentation [24], which was consistent with the findings of our study.

The fungi on tobacco leaves were the main source of stored tobacco molds. Among the 646 identified fungal genera, over 40% (279) were genera commonly found in tobacco leaves at four processing stages, such as Alternaria, Aspergillus, Sampaiozyma, Filobasidium, Pseudopithomyces, Cladosporium and Septoria, indicating some similarity in fungal communities during processing (Fig. 2). Additionally, our study found that some fungi in stored tobacco leaves originated from fresh tobacco leaves. About 82% of the fungal groups in tobacco leaves after redrying were consistent with those in fresh tobacco leaves, while about 18% came from post-harvest pollution (Fig. 2). Therefore, the processing environment (including ambient air, machinery, etc.) influenced the fungal community composition of tobacco leaves. Humidity is a key factor affecting mold growth on tobacco leaves. In high humidity environments, most molds can grow and reproduce on tobacco leaves, but their community significantly decreased with decreasing humidity [25]. Moreover, compared to sterilized tobacco leaves, many microorganisms growed better on non-sterilized tobacco leaves [26], indicating that microbial interactions also had a certain effect on the growth of molds on tobacco leaves.

The composition of fungal communities after redrying is closely related to tobacco leaf molds during fermentation. Tobacco leaves after redrying are promptly packaged and transported to the tobacco storage warehouse for fermentation. Therefore, the microbial community composition of the tobacco leaves after redrying can be considered the same as that of stored tobacco leaves. Aspergillus was the main fungal group causing mildew in tobacco leaves [27]. Aspergillus and Penicillium were found to be the dominant genera in rotten tobacco leaves [22]. Some studies also identified Aspergillus as the main cause of tobacco leaf mold during tobacco storage [28, 29]. Rhizopus was the pathogen causing tobacco leaf mold during flue-curing [30, 31]. In this study, the relative abundance of Aspergillus in tobacco leaves increased from 1.22% before flue-curing to 24.15% after flue-curing (Fig. 3b). The relative abundance of Rhizopus in tobacco leaves increased from 0.04% before flue-curing to 3.31% after flue-curing. The relative abundance of Penicillium in tobacco leaves also increased from 0.02% before flue-curing to 0.08% after flue-curing (Table S1). LEfSe analysis further showed that Aspergillus and Aspergillus flavus exhibited significant differences during the after flue-curing stage (Fig. 5). Aspergillus and Penicillium were significantly enriched during the flue-curing stage and maintained a high relative abundance after redrying. Thus, it was suggested that the main molds in tobacco leaves were predominantly enriched during the flue-curing period. The air in the production workshop was identified as the main source of contamination in products such as dried-cured meat, bread and cheese [32,33,34]. During the flue-curing process, the temperature and humidity of the curing room rise rapidly, providing a suitable growth environment for saprophytic fungi such as Aspergillus, Rhizopus and Penicillium. These fungi accelerate the degradation of organic matter in tobacco leaves, potentially lead to mildew and adversely affecting the quality of tobacco leaves. Therefore, controlling molds during the flue-curing period can reduce the probability of tobacco leaf mildew during processing and aging, thereby minimizing the impact on tobacco leaf quality and yield. Effective measures include managing the tobacco processing environment, cleaning of machinery, and strictly controlling tobacco moisture and environmental humidity.

This study focused on the effects of flue-curing and redrying on the fungal composition of tobacco leaves and revealing that certain fungi may play an important role in influencing the quality of tobacco leaves. The chemical composition of tobacco leaves determines their quality. Therefore, we will analyse the changes in the chemical composition of tobacco leaves at different processing stages and establish a direct relationship between fungal community dynamics and tobacco quality.

Conclusions

The dynamic changes in fungal community composition during flue-curing and redrying stages were described using high-throughput sequencing technology. The high-temperature treatments of flue-curing reduced the richness of fungal communities and changed the diversity of the fungal communities, resulting in varying compositions of fungal communities at different processing stages. Additionally, the saprophytic fungi that cause tobacco leaf mold, such as Aspergillus and Penicillium, were significantly enriched during the flue-curing period. This study provides insights into the effects of flue-curing and redrying on fungal communities in tobacco leaves and provides scientific and theoretical guidance for prevening and controlling tobacco mildew, which is of great significance for improving tobacco quality in fermentation.