Effects of reductive soil disinfestation on microbiological and physicochemical properties of continuous cropping soils in karst areas of Guizhou Province

Abstract

Purpose

The soil-borne diseases have limited the development of agricultural production in Guizhou Province of southwest China which was caused by long-term continuous cropping of crops. To reduce the limit factors of continuous cropping of corps has become an urgent problem.

Methods

Reductive soil disinfestation (RSD) is an environmentally friendly soil amendment technology. In this study, high-throughput sequencing was used to investigate the mechanisms of RSD technology to improve long-term continuous cropping soil health. The examination focused on discerning how RSD influences the composition and structure of the rhizosphere microbial community.

Result

The results demonstrated that: (1) RSD treatment increased the content of soil organic matter (SOM), alkaline hydrolyzed nitrogen (AN), available phosphorus (AP), available potassium (AK) and pH; (2) RSD changed the fungal and bacterial community structure and the relative abundance of pathogenic microorganisms (e.g., Fusarium) was reduced, while the beneficial microorganisms (e.g., Trichoderma and Penicillium) was increased. (3) AN and pH had a greater impact on the bacterial community in the rhizosphere soil than on the fungal community. (4) RSD treatment improved the agronomic traits of tobacco and reduced the disease incidence of root rot disease.

Conclusion

Our results revealed that RSD treatment improved the physicochemical properties of continuous cropping tobacco soil and maintain the soil nutrient balance, resulting in the effective alleviation of continuous cropping barriers.

Introduction

Continuous cropping has become a popular cropping pattern for intensive agricultural production under the context of decreased available arable lands and increasing demands for food and cash crops driven by a growing population (Tan et al. 2021). Guizhou Province, located in southwest China, is marked by mountainous terrain and poor soils. Limited arable land and unsustainable planting practices lead to continuous tobacco cropping in most areas (Wang et al. 2020a, b). The long-term cropping pattern has resulted in highly severe soil replant disease. Long-term continuous cropping caused effective nutrient reduction and soil acidification due to the selective uptake of crops (Beretta-Blanco et al. 2019; Li et al. 2024). Long-term continuous cropping also reduced soil microbial diversity and increased pathogenic dominant species (Li et al. 2022). Tobacco is one of the largest cash crops grown in Guizhou Province and has a great impact on soil health. Our previous study found that the “barrel effect” of soil physical and chemical properties and the imbalance of soil microbial communities were important causes of tobacco continuous cropping obstacles (Gong et al. 2024). In this sense, it is urgent to develop a green remediation technology to improve the soil physicochemical properties and soil microbial community structure of continuous cropping soils.

The reductive soil disinfestation (RSD) method is a green and eco-friendly soil amendment measure, which was innovatively proposed by Shinmura and Dutch (Blok et al. 2000; Shinmura 2000). Considerable studies have reported that RSD exerted a noticeable sterilization effect and was effective against bacterial wilt, root rot, root-knot nematode, verticillium wilt, phytophthora infestation, etc., and it could effectively kill pathogenic bacteria, such as Phytophthora, Pythium, and Fusarium (Luo et al. 2023; Masahiko et al. 2009; Momma et al. 2006; Wen et al. 2016). Meanwhile, RSD also regulated pH, increased organic fractions, and improved soil nutrients and structure (Luo et al. 2023; Zhan et al. 2021). RSD treatment created an anaerobic and high-temperature conditions, which cause significant changes in microbial metabolism. In anoxic soil conditions, the growth of some aerobic pathogens is significantly slowed (Huang et al. 2016). Under RSD treatment, the toxic and harmful substances generated during the degradation of organic materials are key to sterilization. Volatile fatty acids, as key products of anaerobic fermentation, with the accumulation of acetic acid and butyric acid, can inhibit the survival of Fusarium and Ralstonia (Huang et al. 2019). RSD significantly benefits soil health by both preventing soil degradation and enhancing microbial activity and community structure (Yin et al. 2023). The karst area has formed a unique ecosystem including discontinuous soil development, and fragile carbonate rock formations (Zhu et al. 2022). The karst area was exposed to strong degradation stress from continuous cropping activities and soil conservation was the important target. However, the potential of RSD to prevent soil degradation in the karst area has not yet been demonstrated. Here, we investigated the effects of RSD on soil physicochemical properties and microbial community composition. Our research emphasizes the role of RSD in combating soil-borne crop diseases and advocates for actively restoring rhizosphere communities.

Materials and methods

Experimental site

The experimental site was located in the Long-term Positioning Experimental Area, Bijie City, Guizhou Province, P. R. China (105° 20′ E, 26° 52′ N). The region has a typical karst plateau landform and an average sea level of 1520 m with a mean annual temperature of 13.5 °C. The frost-free period lasts for an average of 250 days annually. On average, the annual sunshine hours and precipitation reached 1,495 h and 1,200 mm, respectively. The basic physical and chemical properties of this experimental site are shown in Supplementary Table S1.

Experimental design

Three different treatments as follows: CK, conventional fertilization (The amount of base fertilizer applied: N: 90 kg·ha^− 1, P₂O₅: 90 kg·ha^− 1, and K₂O: 120 kg·ha^− 1. Topdressing was performed every 30 days two times with N: 25 kg·ha^− 1, P₂O₅: 25 kg·ha^− 1, and K₂O: 50 kg·ha^− 1); T1, 20% reduction in the conventional fertilizer amount; T2, 20% reduction in the conventional fertilizer amount plus 40% organic fertilizer. The amount of reduced chemical fertilizer and organic fertilizer were calculated as the pure nitrogen content (vinasse, total carbon: 280.41 g/kg, total nitrogen: 7.80 g/kg; C/N: 35.95). The experiment details were shown in Supplementary Table S2. Before the experiment began, the T2 treatment was covered with plastic film to keep 80% soil relative water content and anaerobic environment for 20d. The area of each treatment plot was 70m² and each treatment had three independent replicate plots. Field practices of management were consistent for each treatment.

Soil sample collection

Soil samples were collected on days 20, 50, 80, 110, and 140 after tobacco transplantation to the experimental field, respectively. We used a five-point sampling method to collect soil samples. Remove dead branches and small stones and packed in sterile bags. The soil samples were divided into two parts: one was air-dried for physicochemical properties analysis, and the other was stored at -80 °C for DNA extraction.

Physical and chemical properties of the field experiment

Determination of soil physical and chemical properties according to according to Soil and Agricultural Chemistry Analysis (Bao 2000). The pH was determined in suspensions with a soil-water (w/w) ratio of 1:2.5. Soil organic matter (SOM) was determined by the potassium dichromate oxidation external heating method. Alkali hydrolyzable nitrogen (AN) was determined using the alkaline diffusion method. The available phosphorus (AP) was determined by the molybdenum antimony antibody colorimetric method. The available potassium (AK) was determined with flame photometry.

Agronomic traits and disease incidence of plants

On the 80 d after tobacco transplantation, the plant height, stem diameter, numbers of effective leaves, and maximum leaf area from each plot (15 plants total per plot) were recorded. A bioassay for disease incidence was performed during the harvest season based on observations of typical wilt symptoms, including necrosis and leaf drooping. According to “Tobacco Pest and Disease Classification and Survey Methods” (GB/T 23222 − 2008), the disease incidence was investigated based on observations of typical wilt symptoms of leaves on the 110 d.

Analysis of soil microbial communities

Total soil DNA was extracted from soil (0.5 g) and subjected to amplification and sequencing The V3-V4 regions of the16S rRNA genes were amplified by the bacterial primers 338 F (5’- ACTCCTACGGGGAGGCAGCAG-3’) and 806R (5’- GGACTACHVGGGTWTCTAAT-3’), universal primers ITS1F (5’-CTTGGTCATTTAGAGGAAGTAA-3’) and ITS2R (5’-GCTGCGTTCTTCATCGATGC-3’) were used to amplify fungal ITS1 region. Then, the purified amplicons were employed for library construction using the Illumina Miseq PE300 platform. Raw data was filtered primarily through Trimmomatic v0.33 (Edgar 2013). The primer sequences were identified and removed with cute-adapt v1.9.1 (Callahan et al. 2016). The PE reads obtained previously were assembled using USEARCH v10 (Segata et al. 2011), and then inlay removal was performed with UCHIME v8.1 (Quast et al. 2012). These high-quality sequences were clustered into operational taxonomic units (OTUs) based on a 97% similarity threshold using UPARSE (Edgar 2013). The annotation of OTU classification was based on the Naive Bayesian classifier in QIIME2 (Bolyen et al. 2019) using the SILVA database (release 132) (Quast et al. 2012) with a confidence threshold of 70%. Alpha diversity was calculated by employing QIIME2 and R software. Beta diversity was calculated using QIIME to measure the similarity of microbial communities between samples.

Statistical analysis

A one-way ANOVA was performed to determine the significance of soil properties and microbial community structures by SPSS. A P-value < 0.05 was considered to be statistically significant (Amenu and Bacha 2023). Principal coordinate analysis (PCoA) based on the Bray-Curtis distance algorithm was used to explore the differences in bacterial and fungal community composition. Based on the soil physicochemical properties and microbial community, the Spearman correlation coefficient and its significance were calculated using the R package. Origin 2021 software was used for plotting.

Results

Analysis of agronomic traits and disease incidence of plants

The effect of RSD treatment on agronomic traits of tobacco was shown in Supplementary Table S3. The effective numbers of leaves among all treatments were met the requirements of agricultural production. The plant height, stem diameter, and maximum leaf area of T1 were significantly lower than those of CK, indicating that a 20% reduction in the conventional fertilizer amount had a significant impact on the growth of the tobacco plant. The root rot disease was dominant in the experimental site (Supplementary Table S4). The incidence rate and disease index of CK and T1 were similar. Compared to CK, the incidence rate and disease index in T2 was decreased by 38.45% and 29.13%, respectively. Overall, RSD treatment reduced pathogen damage to plants.

Effect of RSD treatment on soil physicochemical properties

The physicochemical properties of soil including alkaline nitrogen (AN), available phosphorus (AP), available potassium (AK), organic matter (SOM), and pH were shown in Fig. 1. RSD treatment increased the available nutrients in the soil. The content of AN, AP, and AK in T2 were significantly higher than those in CK at 50 d and 80 d, while the content of AN, and AP were significantly higher than those in CK and T1 at 110d and 140d. These results revealed the effective nutrients of the soil could be stored in RSD treatment.

Fig. 1

Effect of RSD treatment on the physicochemical properties of soil. (a) Soil organic matter (SOC), (b) Alkaline hydrolyzed nitrogen (AN), (c) Available phosphorus (AP), (d) available potassium (AK), and (e) pH. Data are presented as the mean ± standard error (n = 3), with significant differences indicated by different letters in the same column at P < 0.05

Effect of RSD treatment on soil microbial diversity

In this study, the microbial diversity of soil was assessed by the Chao1 and Shannon indexes, respectively (Table 1). At 20 d, both the Shannon and Chao1 indices for fungi and bacteria in T2 were lower than those in CK. At 80d, the Shannon index for fungi and bacteria in T2 were higher than that in CK and T1. This result suggested that the RSD treatment increased soil microbial community diversity.

Table 1 Alpha diversity index of bacterial and fungal at 20 days and 80 days

Effect of RSD treatment on soil microbial communities

To investigate the differences in soil microbial communities, principal coordinate analysis (PCoA) was utilized to compare the variability of soil fungal and bacterial community structure among different treatments based on the Bary-Curtis algorithm (P < 0.05). As shown in Fig. 2, the PC1 and PC2 coordinates of all samples were more than 50%, highlighting the overall differences and indicating reasonable soil sampling. In T2, significant differences among treatment groups were observed in fungi and bacteria, suggesting that soil microbial community structure was significant changes by RSD.

Fig. 2

PCoA analysis based on the Bary-Curtis distance algorithm for the fungal (a, c) and bacterial (b, d) communities (P < 0.05)

For fungi, at 20d, Ascomycota, Basidiomycota, and Mortierellomycota were the dominant fungal phyla, accounting for 97% of all sequences (Fig. 3a). The relative abundance of Ascomycota and Basidiomycota in T1 was higher than that in CK. Ascomycota relative abundance of the T2 group increased, and the relative abundance of Basidiomycota and Mortierellomycota was lower than CK at 80d (Fig. 3c). At 20d, Ascomycota (57.48–70.55%) was the dominant phyla in all samples (Fig. 3b). The relative abundance of Ascomycota and Chytridiomycota was higher in T1 than in CK. In contrast, the relative abundance of Basidiomycota and Mortierellomycota in T1 were lower than that in CK. For bacteria, the relative abundance of Ascomycota in T2 was higher than that in CK, but the relative abundance of Basidiomycota and Mortierellomycota showed the opposite trend. At 80d, Proteobacteria, Actinobacteriota, and Chloroflexi were the dominant bacterial communities, with relative abundance accounting for more than 70% of all sequences (Fig. 3d). In T2, the relative abundance of Proteobacteria and Actinobacteriota was higher than that in CK. In addition, the relative abundance of Acidobacteriota and Patescibacteria was lower than that in CK.

Fig. 3

The phylum-level composition of fungal (a, c) and (b, d) bacterial communities. Phyla representing less than 1% of the total reads were grouped as “Others.”

At the genus level, the relative abundance of Saitozyma, Mortierella, and Fusarium in T2 were lower than that in CK at 20d, while the relative abundance of Trichoderma and Penicillium were higher than that in CK (Supplementary Figure S1a). At 80d, the relative abundance of Saitozyma, Fusarium, Mortierella, Penicillium, Coniochaeta and Chaetomium were decreased compared with CK (Supplementary Figure S1c). For bacteria, the relative abundance of Sphingomonas was higher than that in CK and the remaining species were lower than that in CK (Supplementary Figure S1b). At 80d, Rhodanobacter (8.22–12.03%), Sphingomonas (2.61–3.74%), Chujaibacter (1.19–3.09%), Bryobacter (1.51–1.91%) were the dominant species (Supplementary Figure S1d). The relative abundance of Rhodanobacter, Sphingomonas, and Chujaibacter in T2 exhibited higher than that of CK, and the remaining species decreased.

Correlation analysis between soil microbial communities and soil physicochemical properties

Pearson correlation analysis was applied to explore the relationships between physicochemical properties and OUT reads. At 20d, Gemmatimonadota significantly positively correlated with pH (P < 0.001) and AN (P < 0.01) (Fig. 4b). AN was significantly positive correlation with Actinobacteriota (P < 0.01) and Bacteroidota(P < 0.001), and negative correlation with Chloroflexi (P < 0.001), Firmicutes (P < 0.01), Planctomycetota (p < 0.01)and Acidobacteriota(P < 0.05). AP was significantly positively correlated with Actinobacteriota (P < 0.05) and Bacteroidota (P < 0.05) and negatively correlated with Chloroflexi (P < 0.01). At 80d, Olpidiomycota (P < 0.05) had a significantly positive correlation with AP (Fig. 4c).

Fig. 4

Correlation heatmap of the top 10 fungal (a, c) and (b, d) bacterial genera with soil properties. R values are indicated on the right side of the legend with different colors. * P < 0.05, ** P < 0.01, and *** P < 0.001

Soil microbial function prediction

FUNGuild functional prediction was used to clarify the functional changes in soil fungal communities among treatment groups. At 20d, the relative abundance of T2 Undefined Saprotroph and Plant Pathogen significantly increased, while the relative abundance of Fungal Parasite-Undefined Saprotroph decreased (Fig. 5a). At 80d, the relative abundance of Fungal Parasite- Undefined Saprotroph significantly decreased in T2 compared with CK and the relative abundance of Endophyte was increased (Fig. 5c).

Fig. 5

Changes in functional groups on fungal (a, c) and (b, d) bacterial OTU data in different treatments

FAPROTAX functional prediction was utilized to analyze the functional changes in the bacterial community that were subjected to different treatments. As shown in Fig. 5, T2 and CK had similar bacterial functional composition. At 20d, the relative abundance of chemoheterotrophy and aerobic chemoheterotrophy accounting for more than 70%, representing the dominant function of the bacteria (Fig. 5b). Compared to CK, the relative abundance of nitrate reduction and ureolysis was increased in T2, indicating that the relative abundance of functional bacteria involved in nitrogen cycling process was increased in RSD treatment. There was no significant difference in the functional composition of bacteria among the treatment samples in the vigorous period, and the bacterial functions tended to be consistent (Fig. 5d).

Discussion

Analysis of agronomic traits and disease incidence of plants

Long-term continuous cropping reduces crop agronomic traits and impedes crop growth and development, thereby affecting crop dry matter accumulation and reducing yield (Xu et al. 2022). The agronomic traits of tobacco at the topping stage are closely related to yield. Our result showed that RSD treatment enhanced plant height, stem girth, and maximum leaf area of tobacco.

Continuous cropping of tobacco exacerbates disease occurrence, limiting crop growth and development, thereby reducing tobacco yield (Jin et al. 2004). The organic acids and metal ions produced by RSD treatment can kill pathogenic bacteria such as Fusarium, which causes black rot of tobacco roots (Momma et al. 2011). Under the experimental conditions in this research, the relative abundance of Fusarium in the soil subjected to RSD treatment was decreased. In addition, the incidence of tobacco black root rot dropped by 38.45%, and the disease index experienced a reduction of 29.13%. These results indicated that the RSD treatment could decrease the incidence and the degree of disease in tobacco. Overall, RSD treatment reduced the incidence condition of tobacco black root rot disease.

Effect of RSD treatment on soil physicochemical properties

Soils with harmonized and sufficient nutrient levels are important for the production of crops. Previous studies have demonstrated that RSD treatment can significantly enhance soil nutrients and improve the physical and chemical properties of soil (Luo et al. 2023; Zhan et al. 2021). Soil pH affects the transformation and effectiveness of soil nutrients (Barrow and Hartemink 2023). The present study revealed that RSD treatment significantly increased soil pH, consistent with the pot experimental results of Qin et al. (Qin et al. 2023). SOM, AN, AP and AK are the key indexes to characterize soil fertility. The present study showed that RSD treatment significantly increased SOM, AN, AP and AK, which was helpful to improve the fertility level of the soil, which was similar to the results of our study (Qin et al. 2023; Teng et al. 2022). The large amount of easily decomposed organic matter added to RSD treatment contributed to the proliferation of soil microorganisms (Zhu and Wang 2020). This directly resulted in a significant increase in SOM and C/N ratio of soil, promoting the mineralization of organic nitrogen into inorganic form and increasing the content of alkali-hydrolyzed nitrogen. The organic acids produced by RSD can drive phosphorus mineralization (Damon et al. 2014), increasing the effective phosphorus content in the soil. From the completion of the RSD treatment to the maturity periods of tobacco, the soil effective nutrients in the RSD treatment groups were higher than those of the control, indicating a better holding period of the soil nutrients in treated samples.

Effect of RSD treatment on soil microbial community structure

RSD is exceptionally effective in controlling soil continuous cropping obstacles, creating a strong anaerobic-strongly reducing environment in a short period. This process produces volatile gases, organic acids (Huang et al. 2016; Masahiko et al. 2009; Messiha et al. 2007), low-valent heavy metals (Fakih et al. 2008; Momma et al. 2011), and other toxic substances to inhibit or kill pathogenic microorganisms.

Regarding the structural changes of the soil fungal community, Alpha diversity analysis demonstrated that the abundance of the fungal community increased, and the diversity decreased in the RSD treatment. According to the results of the PCoA analysis, significant differences arose in species composition between the RSD treatment and the control. The analysis at the fungal phylum level demonstrated that Ascomycota, Basidiomycota, and Mortierellomycota were the dominant fungal species in RSD samples. In addition, the relative abundance of Ascomycota in in T1 and T2 was increased compared to CK, and the relative abundance of Mortierellomycota was decreased. Ascomycota and Basidiomycota served as the main saprophytic fungal communities in the treated soil. The former mainly degrades unstable parts of plant residues at the early stage of decomposition, while the latter primarily decomposes difficult-to-degrade organic matter at the later stage (Francioli et al. 2016; Ma et al. 2013). Notably, Ascomycota is a complex soil microbial population containing both harmful and beneficial fungi. Furthermore, the proliferation of pathogenic fungi may occur in Ascomycota, e.g., an increase in Plant Pathogen (Fig. 5). Most Mortierellomycota are aerobic decomposers of soils. Some specific periphytic Mortierella sp. are able to improve the availability of phosphorus in the soil (Osorio and Habte 2014). The reduced relative abundance of these species may be due to the strongly anaerobic conditions during the RSD process. The analysis at the taxonomic level of fungal genera showed that RSD-treated soils experienced a decrease in the relative abundance of Fusarium, a pathogenic fungus of tobacco root rot (Wu et al. 2018). Our experimental results revealed that RSD treatment significantly controlled the disease caused by Fusarium. The relative abundance of Trichoderma was increased in RSD. Trichoderma has a preventive and therapeutic effect on pathogens, such as Fusarium and Phytophthora infestans (Li et al. 2020; Yao et al. 2022). The increase of the abundance of Trichoderma may explain the decrease in the relative abundance of Fusarium. Penicillium is a heat-tolerant saprophytic fungus involved in soil soluble phosphorus conversion (Wakelin et al. 2004), and an increase in its relative abundance could improve soil nutrient status.

For bacteria, Alpha diversity analysis showed that the abundance of the bacterial community increased and diversity decreased in RSD treatment. PCoA analysis demonstrated that the difference in bacterial species composition between the RSD treatment and the control was significant. The relative abundance of Actinobacteriota, Acidobacteriota, Bacteroidota, and Gemmatimonadota was increased, while Chloroflexi and Firmicutes exhibited decreased. As multifunctional microorganisms in the soil, Actinobacteriota can grow under various stress conditions, participate in the soil nitrogen cycle, release cellulases and other enzymes to promote organic matter decomposition, and produce phytohormones (e.g., IAA) to promote plant growth; it can also increase the availability of soil phosphate and generate iron carriers to inhibit soil-borne pathogens (Grover et al. 2016; Mitra et al. 2022), improving nutrient availability and plant resilience of soils. Acidobacteriota is closely associated with the soil nitrogen cycle (Eichorst et al. 2018) and is an important group of bacteria involved in cellulose degradation and Fe cycling in acidic soil environments (Wang et al. 2016). Its reduced relative abundance may be a result of competition with other microorganisms. Bacteroidota is beneficial in degrading soil insoluble cellulose, producing acids that directly inhibit the proliferation of pathogenic bacteria, and mineralizing organic nitrogen into ammonium nitrogen or nitrate nitrogen to provide nutrients for plants (Lydell et al. 2004). Chloroflexi are autotrophic anaerobic bacteria that contribute to soil carbon, nitrogen, and sulfur cycling, and some of them can decompose and adsorb soil toxic substances (Xian et al. 2020). The decrease in its relative abundance may be due to the inhibitory effect of organic acids produced by RSD treatments. Gemmatimonadota is an aerobic or partially anaerobic bacterium and serves as an important microorganism for denitrification of the soil nitrogen cycle (Jia et al. 2019). Firmicutes are mostly aerobic or partially anaerobic bacteria involved in soil microbial nitrogen fixation and cellulose degradation (Ravenschlag et al. 1999), and their decreased relative abundance may be due to the inhibition of aerobic Firmicutes in RSD treatment. Rhodanobacter became the dominant species after 80d. Rhodanobacter has been reported to antagonize root rot disease and also denitrify under low nutrient and acidic conditions (Huo et al. 2018; Ghosh et al. 2022).

Effect of RSD treatment on soil microbial community and physicochemical properties correlation

Previous studies have shown that plant growth is closely related to physicochemical properties and microbial community structure of soils (Liu et al. 2021; Song et al. 2019). As a major group of saprophytic fungi in the soil, Ascomycota increases soil nutrients by decaying plant, animal residues, etc. (Ma et al. 2013). In RSD, the community abundance was significantly higher than that in CK (Fig. 3a), corresponding to the better physical and chemical properties of T2 (Fig. 1a). Basidiomycota was significantly negatively correlated with the physical and chemical properties of the soil. Specifically, it exhibited a significant negative correlation with SOM and AP.

For bacteria, Actinobacteriota (P < 0.05), Bacteroidota (P < 0.05), and Gemmatimonadota (P < 0.001) were significantly positively correlated with pH at 20d. The increase in pH was consistent with changes in their relative abundance, suggesting that pH altered these communities. Actinobacteriota (P < 0.01), Bacteroidota (P < 0.001), and Gemmatimonadota (P < 0.01) were significantly positive correlation with AN, indicating that the involvement of these bacteria in nitrogen fixation or nitrification and denitrification to increase AN.

Soil microbial function prediction

According to FUNGuild functional prediction analysis, the relative abundance of Fungal Parasite-Undefined Saprotroph decreased in RSD compared to CK, indicating that the RSD had an inactivating effect on parasitic pathogenic fungi. In addition, the relative abundance of Undefined Saprotroph and Plant Pathogens in RSD increased significantly. Such increases are closely related to the elevation in the abundance associated with the Ascomycota community, which contains considerable plant pathogenic fungi (Paungfoo-Lonhienne et al. 2015). The relative abundance of Fungal Parasite- Undefined Saprotroph decreased significantly in the RSD treatment at 80d. This observation suggested that the RSD treatment combined with reduced fertilizer application exerted a preventive effect against parasitic pathogenic fungi under natural field conditions. The relative abundance of Endophyte increased significantly in RSD treatment, indicating that the RSD treatment incorporated with reduced fertilizer application increased the beneficial fungi under field cultivation conditions. Notably, combined with the results of FUNGuild, RSD treatment decreased the incidence and disease index of tobacco black root rot disease. In conclusion, Plant Pathogen fungal communities did not significantly negatively impact tobacco.

Based on the FAPROTAX function prediction analysis results, chemoheterotrophy and aerobic chemoheterotrophy were the dominant functions of bacteria. The two basic functions of microorganisms enable the degradation of organic matter and energy acquisition to maintain life activities. The relative abundance changes of nitrogen fixation, nitrate reduction, and ureolysis indicated that RDS stimulated the activities of nitrogen cycle-related bacteria in soils (Huang et al. 2016), such as the increase in the relative abundance of Acidobacteriota (Fig. 2). Acidobacteriota is closely related to the soil nitrogen cycle (Eichorst et al. 2018) and is an important class of bacteria involved in cellulose degradation in acidic soil environments.

Amplicon sequencing has become a standard tool for assessing soil microbial diversity. However, the full spectrum of microbial communities present in the soil is not covered by the particular primers. The rare taxa that were missed by low-depth sequencing will be the focus of our future research.

Conclusion

In this study, we demonstrated that RSD treatment increased soil physicochemical properties in karst areas including SOC, AN, AP, AK and pH. The variation of bacterial and fungal community was closely correlated with the changes of soil properties. The disease incidence of tobacco was reduced by the synergistic increase of beneficial microorganisms under the RSD treatment. This work advanced our understanding of the complex RSD treatment-soil interactions and provided ecological support for the targeted manipulation of the microbiome in karst agricultural systems.