Fungal communities are vital for soil health and crop productivity. However, the effects of continuous cropping, particularly on flue-cured tobacco, remain poorly understood. In southwest China's mountainous regions, continuous cropping of flue-cured tobacco is prevalent due to limited land and a lack of sustainable cropping practices. This practice leads to nutrient depletion, particularly nitrogen, phosphorus, and potassium imbalances, and decreased soil enzyme activity, resulting in reduced yield and quality. Soil microorganisms, particularly fungi, are critical for nutrient cycling, organic matter decomposition, and plant health. Some soil fungi are plant pathogens, while others are biocontrol agents. Soil fungal communities are influenced by soil physico-chemical properties, and agricultural practices significantly impact their structure. In continuous cropping systems, consistent root exudates from the same species can increase plant pathogens and exacerbate soil-borne diseases. This study aimed to investigate (1) the effects of continuous flue-cured tobacco cropping on fungal communities and soil properties and (2) the correlation between soil properties and soil fungal communities under continuous cropping systems. The researchers hypothesized that different continuous cropping durations would lead to distinct soil fungal community selection based on varying soil physico-chemical properties.

Previous research has shown that continuous cropping of various crops (eggplant, cotton, soybeans) leads to increased pathogen numbers. However, most studies on microbial diversity under continuous flue-cured tobacco cropping focused on bacterial communities. Limited research exists on the effect of continuous flue-cured tobacco cropping on soil fungal community structure. Studies have demonstrated the impact of cropping systems on soil fungal abundance and diversity. The influence of soil physico-chemical properties, such as pH and enzyme activity, on rhizosphere fungal communities is well-documented. Research has also shown that continuous cropping can reduce soil enzyme activity and alter the abundance of Ascomycota and Basidiomycota. The role of Mortierellomycota in solubilizing mineral phosphorus and its correlation with continuous cropping is a subject of ongoing research.

The study was conducted at the Panzhihua Flue-cured Tobacco Experimental Station in Sichuan Province, China. Three treatments were established: (1) flue-cured tobacco continuously cropped for 3 years (3ys), (2) continuously cropped for 5 years (5ys), and (3) a control (CK) with flue-cured tobacco cropped for 1 year. A randomized block design with three replicates was used. Soil samples were collected from the 0-20 cm soil layer, 65 days after transplanting. Soil pH, total nitrogen (TN), ammonium-nitrogen (NH₄⁺-N), nitrate-nitrogen (NO₃⁻-N), soil organic carbon (SOC), available phosphorus (Ava-P), available potassium (Ava-K), sucrase, urease, and catalase activities were determined. Total DNA was extracted from the soil samples, and fungal ITS gene copy numbers were measured using quantitative PCR (qPCR). High-throughput sequencing of the fungal ITS2 region was performed using Illumina NovaSeq platform. Bioinformatics analysis was performed using UPARSE, RDP Classifier, and various R packages to assess alpha and beta diversity, correlations between soil properties and fungal communities, and co-occurrence networks. Statistical analysis included one-way ANOVA, t-tests, Mantel tests, and redundancy analysis (RDA).

Soil physico-chemical properties differed significantly among treatments. SOC and NO₃⁻-N were lower under continuous cropping (3ys and 5ys) than in CK. Ava-P increased with cropping years, while Ava-K decreased. Urease and catalase activities were also lower under continuous cropping. Fungal abundance increased with continuous cropping years, with the highest abundance in 5ys. Shannon and Simpson diversity indices also showed significantly higher diversity in 5ys compared to 3ys and CK. Ascomycota, Basidiomycota, and Mortierellomycota were dominant phyla across all treatments. The relative abundance of Ascomycota and Basidiomycota decreased with increasing cropping years, while Mortierellomycota increased significantly in 5ys. At the order level, Sordariales was dominant in CK, while Hypocreales and Mortierellales were dominant in 5ys. Beta diversity analysis (NMDS and PCOA) showed distinct separation of fungal communities among the three treatments. Mantel tests revealed significant correlations between fungal community structure and TN, Ava-P, Ava-K, and catalase activity. RDA showed Ava-K and Ava-P as the most influential factors shaping fungal community structure. Co-occurrence network analysis showed that the 5ys treatment had the most complex and stable network, with OTU65 (*Cylindrocarpon* sp.) and OTU246 (Geminibasidium sp.) identified as keystone taxa.

The increased Ava-P in 5ys, despite lower SOC and NO₃⁻-N, suggests that long-term continuous cropping may enhance microbial nutrient cycling, consistent with previous findings. The decrease in Ava-K is likely due to high potassium uptake by flue-cured tobacco. The increased fungal abundance and diversity in 5ys might be due to a more stable soil environment and increased nutrient availability, potentially from root exudates. The dominance of Ascomycota aligns with previous research. The increase in Mortierellomycota and its correlation with Ava-P support its role in phosphorus solubilization. The shift in dominant fungal orders (from Sordariales in CK to Hypocreales and Mortierellales in 5ys) reflects changes in substrate availability and potentially increased pathogenic risk. The more complex and stable fungal network in 5ys suggests enhanced microbial interactions under long-term continuous cropping. The identified keystone taxa highlight the importance of both beneficial (Geminibasidium sp. in phosphorus cycling) and potentially harmful (Cylindrocarpon sp. as a potential pathogen) fungi in shaping the soil ecosystem.

Continuous flue-cured tobacco cropping significantly altered soil properties and increased fungal abundance and diversity. The increase in Mortierellales and Hypocreales, along with a more complex fungal network in long-term continuous cropping, highlights the intricate responses of soil fungal communities. Differences in Ava-K and Ava-P were major drivers of fungal community structure. Future research should focus on the roles of keystone taxa, particularly those with pathogenic potential, and strategies for improving soil health under long-term continuous cropping systems.

The study was conducted at a single location, limiting the generalizability of findings to other geographical regions or soil types. The sampling time point might not capture the full temporal dynamics of fungal community responses to continuous cropping. Further investigations are needed to explore the specific mechanisms underlying the observed changes in fungal community structure and function, and how these changes impact crop health and yield.