Response of soil fungal communities to continuous cropping of flue-cured tobacco

Flue-cured tobacco is generally intolerant to continuous cropping, yet land limitations and practices in mountainous areas of southwest China often lead to continuous monoculture. Repeated cropping can cause soil nutrient loss and imbalance (N, P, K), decreased soil enzyme activity, and reduced yield and quality. Soil microorganisms are key to nutrient cycling, organic matter decomposition, and plant health, and microbial community structure is a sensitive indicator of soil quality. Root exudates and altered soil properties under continuous cropping can shift microbial biomass and diversity and promote soil-borne diseases. Fungal communities, strongly influenced by soil physicochemical properties (e.g., pH, enzymes), include both plant pathogens and beneficial taxa. Agricultural management, including continuous cropping, can enrich plant pathogens via persistent root exudation and disrupt agroecosystem health. The study hypothesized that different continuous cropping durations would select distinct soil fungal communities due to differences in soil physicochemical properties. Objectives were: (1) to determine the effects of continuous flue-cured tobacco cropping on soil properties and fungal communities (abundance, diversity, composition), and (2) to examine correlations between soil properties and fungal communities under continuous cropping systems.

Prior research shows continuous cropping in various crops (eggplant, cotton, soybean) increases pathogen prevalence. In tobacco, most work has focused on microbial quantity and bacterial communities, leaving fungal community responses less explored. Fungal community structure is shaped by soil chemistry, pH, and enzyme activity, and management practices (tillage, rotations, continuous monoculture) significantly affect fungal abundance and diversity. Continuous cropping can reduce enzyme activity and alter the balance among dominant fungal phyla. Some fungal groups (e.g., Mortierellomycota) are linked to phosphorus solubilization, while others (e.g., Hypocreales including Fusarium and Cylindrocarpon) include potential pathogens that may proliferate under monoculture. Studies in other systems (soybean, banana, strawberry) report that long-term monoculture can increase certain available nutrients and restructure microbial networks, sometimes yielding complex co-occurrence patterns with keystone taxa that are not necessarily the most abundant.

Experimental site: Long-term field experiment at Panzhihua Flue-cured Tobacco Experimental Station, Panzhihua City, Sichuan, China (27°04′N, 101°45′E; 2080 m a.s.l.), subtropical dry-hot valley climate, mean annual precipitation 1065 mm (June–October), 2307 h sunshine, mean annual temperature 19.2 °C. Soil classified as Alumi-Ferric Alisols; field flat with uniform fertility. Fertilization: 750 kg ha−1 compound fertilizer (N:P2O5:K2O = 1:1.5:3) annually. Variety: Yunyan87. Experimental design and sampling: Three treatments: continuous cropping for 3 years (3ys), for 5 years (5ys), and cropping for 1 year (CK). Randomized block design with three replicates; plot size 330 m² (33 m × 10 m). Tobacco transplanted May 1 to August 31 annually; fields left fallow otherwise. On July 5, 2018 (65 days after transplanting), five cores (0–20 cm) from rhizosphere zone per plot were composited, large debris removed (2 mm sieve). Nine composite samples (3 treatments × 3 reps) were obtained. Samples were split: −80 °C for DNA; 4 °C for soil properties and enzyme assays. Soil properties and enzyme assays: pH measured per MOD ISO 10390:2005 using a pH meter. Total nitrogen (TN), ammonium (NH4+-N), and nitrate (NO3−-N) following Singh et al. Soil organic carbon (SOC), available phosphorus (Ava-P), and available potassium (Ava-K) per Zhu et al. Sucrase, urease, and catalase activities per Wang et al. DNA extraction and qPCR: DNA extracted from 0.5 g fresh soil (triplicate extractions) using FastDNA SPIN Kit for Soil. DNA quantified by NanoDrop 2000; integrity by 1% agarose gel. Fungal ITS gene copy number quantified by absolute qPCR (QuantStudio 7). Primers ITS1F/ITS2R amplified ~300 bp ITS region. Reaction mix (20 μL): 10 μL 2× TB Green Premix Ex Taq II, 1.0 μL each primer (10 μM), 1 μL BSA, 0.4 μL ROX II, 4.6 μL nuclease-free water, 2 μL template (10–50 ng). Cycling: 95 °C 5 min; 40 cycles of 95 °C 60 s, 51 °C 60 s, 72 °C 60 s; melt curve. Standard curves R² > 0.99; efficiency 75.41%. Illumina sequencing: Fungal ITS2 amplified with primers 2045F (5′-GCATCGATGAAGAACGCAGC-3′) and 2390R (5′-TCCTCCGCTTATTGATATGC-3′). Amplicons purified, pooled equimolarly, and sequenced (paired-end) on Illumina NovaSeq (Realbio Genomics, Shanghai). Sequences deposited to NCBI SRA: SRP241330. Bioinformatics and statistics: Raw fastq demultiplexed and quality-filtered by Trimmomatic; merged with FLASH. ITS2 tags truncated if average quality < Q20 over 500-bp sliding window; no more than three ambiguous bases. OTUs clustered at 97% similarity using UPARSE; chimeras removed with USEARCH v7. Representative sequences taxonomically assigned using RDP Classifier (confidence 0.8) against RDP database. Alpha diversity: Shannon and Simpson indices. Beta diversity: weighted and unweighted UniFrac distance matrices; NMDS for visualization. PCoA based on Bray–Curtis distances to test separation among treatments. Mantel tests assessed correlations between soil properties and community structure. Redundancy analysis (RDA) evaluated relationships between soil properties and fungal OTUs. Co-occurrence networks constructed in R with WGCNA (Spearman’s rank; edges for |ρ| > 0.6 and P < 0.01) and visualized in Gephi; network topology calculated; keystone taxa identified by high betweenness centrality. ANOVA and Tukey’s tests run in SPSS v19.0; multivariate analyses in R (vegan). Significance at P < 0.05.

- Soil properties: Continuous cropping significantly altered soil chemistry and enzymes. SOC and NO3−-N were lower in continuous cropping (3ys, 5ys) than CK (SOC: CK 29.802 g kg−1 vs 3ys 16.024, 5ys 15.609). Ava-P increased with years (5ys highest, 31.656 mg kg−1), while Ava-K decreased with years (CK 186.752 mg kg−1; 5ys lowest, 133.041 mg kg−1). Urease and catalase activities decreased under continuous cropping, lowest in 5ys (urease 1.019 mg kg−1; catalase 1.287 mg kg−1). pH was slightly lower under longer cropping (3ys 6.02, 5ys 6.13, CK 6.20). - Fungal abundance and alpha diversity: Fungal ITS gene abundance increased with cropping duration; 5ys had the highest (4.87 × 10^4 g−1 dry soil). Diversity increased with duration: Shannon’s H highest in 5ys (5.54) vs CK (4.83); Simpson’s index highest in 5ys (0.94). - Community composition: Across samples, dominant phyla were Ascomycota, Basidiomycota, and Mortierellomycota (together 74.70–88.21%). Ascomycota and Basidiomycota declined with cropping duration (Ascomycota in 5ys decreased by 19.93% vs CK; Basidiomycota: CK 17.58%, 3ys 16.44%, 5ys 7.86%). Mortierellomycota increased markedly; in 5ys it was 5.58- and 5.73-fold higher than CK and 3ys, respectively. At order level, CK was dominated by Sordariales (18.84%), which declined in 3ys (13.39%) and 5ys (6.56%). 3ys was dominated by Hypocreales (18.62%) and Agaricales (13.94%). 5ys was dominated by Hypocreales (26.57%) and Mortierellales (22.07%). - Beta diversity: NMDS (weighted and unweighted UniFrac) and PCoA (Bray–Curtis; PC1 58.19%, PC2 31.07%; P=0.005) showed clear separation among CK, 3ys, and 5ys, indicating distinct fungal communities by treatment. - Soil–community relationships: Mantel tests identified TN (r=0.532, P=0.023), Ava-P (r=0.752, P=0.011), Ava-K (r=0.780, P=0.011), and catalase activity (r=0.411, P=0.025) as significant correlates of fungal beta diversity. RDA indicated Ava-K had the strongest influence on community structure, with Ava-P also significant. - Co-occurrence networks: Network complexity and size increased with cropping duration. 5ys network had the most nodes and edges (nodes: 136; edges: 377), highest average degree (5.800), and longest average path length (2.317), indicating greater complexity/stability than 3ys (nodes 120, edges 336) and CK (nodes 119, edges 331). Keystone taxa under continuous cropping included OTU65 (Cylindrocarpon sp., potential pathogen) and OTU246 (Geminibasidium sp., heat- and xerotolerant basidiomycete with potential to release inorganic P).

The study demonstrated that continuous cropping of flue-cured tobacco significantly restructures soil fungal communities, increasing both abundance and alpha diversity while shifting taxonomic composition. The decline in Ascomycota and Basidiomycota alongside a strong rise in Mortierellomycota with longer monoculture suggests selection for saprotrophic taxa that can solubilize phosphorus, consistent with the observed accumulation of available P. Conversely, enrichment of Hypocreales (which includes plant pathogens such as Fusarium and Cylindrocarpon) indicates an elevated risk of soil-borne diseases under long-term monoculture. Beta diversity analyses confirmed distinct community assemblies by cropping duration, supporting the hypothesis that altered soil physicochemical conditions under continuous cropping drive fungal community differentiation. Mantel and RDA results implicate available K and P (and to a lesser extent TN and catalase activity) as primary environmental drivers, aligning with tobacco’s high K demand and the observed depletion of Ava-K in long-term plots. The more complex and larger co-occurrence network in 5-year fields suggests a more interconnected and potentially resilient fungal community, with keystone taxa not necessarily being the most abundant. The identification of Geminibasidium (nutrient-releasing potential) and Cylindrocarpon (pathogenic potential) as keystones underscores a trade-off between beneficial nutrient-cycling functions and heightened disease risk in long-term monocultures. Overall, these findings address the research question by linking continuous cropping–induced soil chemical changes, especially in K and P, to predictable shifts in fungal community diversity, composition, and interaction networks.

Continuous cropping of flue-cured tobacco significantly altered soil properties and reshaped fungal communities, increasing fungal abundance and diversity and shifting composition toward higher Mortierellales and Hypocreales. Long-term monoculture fostered more complex and stable fungal co-occurrence networks, with keystone taxa including both beneficial (Geminibasidium sp.) and potentially pathogenic (Cylindrocarpon sp.) fungi. Available potassium and phosphorus emerged as key drivers of community structure. These results highlight how nutrient dynamics under continuous cropping mediate fungal community assembly and potential plant health outcomes. Future research should focus on functional roles of identified keystone taxa, strategies to manage K and P to steer microbial communities toward beneficial configurations, and practices (e.g., rotations, amendments, biocontrol) to mitigate pathogen risks while maintaining soil health in long-term tobacco systems.

The study was conducted at a single experimental station on a specific soil type (Alumi-Ferric Alisols) with one tobacco cultivar (Yunyan87) and a single in-season sampling timepoint (65 days after transplanting), which may limit generalizability across regions, soils, cultivars, and seasons. Replication was limited to three blocks per treatment (nine composite samples total). Amplicon sequencing of ITS2 provides relative, not absolute, taxon abundances and lacks direct functional resolution; co-occurrence network inferences are correlative and do not prove interactions or causality. Enzyme activities and soil properties were measured at one timepoint, so temporal dynamics were not captured.