Tobacco (Nicotiana tabacum L.) is one of the most widely cultivated and economically important cash crops worldwide (Tong et al., 2016; Liu P. et al., 2020). However, tobacco root rot disease, caused by soilborne fungal pathogens such as Fusarium solani f sp. eumartii and Phytophthora nicotianae, has become a major threat to the tobacco industry, leading to significant yield losses (3–5%) and economic damage (Romberg and Davis, 2007; D’Ippólito et al., 2010). Root rot disease can cause wilting, stunting, and death of tobacco plants, with the infected roots turning brown or black and then rotting, ultimately leading to plant death (Bodah, 2017). Research has been conducted to understand the causal agents, epidemiology, and management strategies for tobacco root rot disease. Several studies have identified Fusarium species as the primary causal agent of root rot disease in tobacco plants, while others have reported the involvement of Phytophthora and Rhizoctonia species (Tan et al., 2021, 2022). The incidence and severity of disease are affected by a variety of factors, including soil pH, moisture, temperature, and the presence of other soilborne pathogens (Burdon and Zhan, 2020; Pokhrel, 2021). Additionally, recent research, such as the findings reported in ‘Legume Root Rot Control Through Soil Management for Sustainable Agriculture’ (Naseri, 2019), highlights the interaction of F. solani with agro-ecological factors on a larger scale.

In the context of addressing this challenge, understanding the interactions between various nutritional factors and soilborne pathogens is crucial. Nutrient availability, including nitrogen (N), phosphorus (P), potassium (K), and organic matter (OM), has been shown to profoundly affect soil microbial communities and subsequently influence plant health (Niu et al., 2017). To date, many studies have investigated the differences between healthy and diseased plants by examining bacterial and fungal communities (Hu et al., 2018; Tan et al., 2021; Gu et al., 2022). However, the current body of research lacks a comprehensive understanding of the regulatory mechanisms underlying environmental factors, such as the impact of different nutritional levels, particularly the effect of nutrient improvement, on disease suppression in the context of F. solani (Guo et al., 2022). Therefore, we intend to further explore and elucidate how soil nutrient levels, including N, P, K, and others, interact with F. solani populations both in the soil and within plant systems. Recognizing the significance of comprehending the interplay between nutrients and microbial communities for sustainable disease management in agriculture, our study distinguishes itself from prior research on F. solani and soil NPK interactions (Naseri and Hamadani, 2017). We aim to contribute novel insights by investigating these interactions in the specific context of our study system. Through this approach, we strive to elucidate the unique dynamics between nutrients and microbial communities, thereby providing valuable implications for the development of targeted strategies for disease suppression in agricultural systems.

Understanding the role of microbial communities in root rot disease is crucial for developing effective management strategies to reduce crop losses (Lareen et al., 2016; Li et al., 2021). Within the microbial community, bacteria, fungi, and protists play significant roles in the prevention and outbreak of root rot disease (Du et al., 2022). For example, changes in the relative abundance of specific taxa from bacterial and fungal communities have been shown to be associated with the healthy and diseased states of roots (Robbins et al., 2018; Liu X. et al., 2020). Many studies have also investigated the impact of microbial communities on root health, and some have shown promising results in reducing the incidence of root rot disease by manipulating microbial community composition (Lareen et al., 2016; Trivedi et al., 2020). For instance, the use of biocontrol agents, such as Bacillus spp. and Trichoderma spp., has been shown to promote plant growth and reduce root rot disease by suppressing the growth of pathogenic fungi (Fira et al., 2018; Kalantari et al., 2018). Protists, unicellular eukaryotic organisms, play pivotal roles in soil microbial dynamics (Bonkowski, 2004). Notably, some protists, such as those from the Cercozoa group, have emerged as key regulators of interactions between bacteria and fungi in the soil (Geisen et al., 2016). Their presence and activities could potentially influence the occurrence and severity of root rot diseases, emphasizing the need to delve deeper into their biocontrol potential against pathogens like Fusarium root rot (Deveau et al., 2018; Guo et al., 2022). Furthermore, recent studies have emphasized the critical role of environmental factors in determining the behavior and pathogenicity of F. solani (Gamboa-Becerra et al., 2021). Specifically, soil pH, organic matter, temperature, and moisture have been found to significantly influence the interactions of F. solani with microbial communities and plants (Yan and Nelson, 2020; Wang et al., 2023). The composition and structure of microbial communities can be altered by changes in these factors, which in turn can impact the incidence of root rot disease. In summary, the roles of bacteria, fungi, and protists in root rot disease prevention and outbreak are critical and interdependent. However, despite significant progress in understanding the role of microorganisms in root rot disease, much remains unknown about the specific mechanisms involved.

Network analysis methods have become increasingly popular in ecological studies, as they provide a useful framework for understanding the complex relationships between the different organisms in a community (Deng et al., 2016). Random matrix theory (RMT) has been applied in network analysis to examine the structure of random networks and compare them to real-world ecological networks (Deng et al., 2012). Meanwhile, the identification and evaluation of dual networks (IDEN) has been used to study bipartite networks in ecological systems, which involve two different biological domains, such as bacteria, fungi, and protists (Feng et al., 2022). In addition to their application in ecological research, network analysis methods have also been used to study the stability and complexity of ecological communities (Herren and McMahon, 2017; Yuan et al., 2021). Robustness and cohesion are two important aspects of community stability and complexity, respectively. Robustness refers to the ability of a community to maintain its structure and function in the face of perturbations, such as disturbances or species loss (Mumby et al., 2014; Yan et al., 2022). Cohesion, on the other hand, refers to the degree of interconnectedness between species within a community, and is a measure of how tightly integrated the community is (Hernandez et al., 2021). Network analysis methods such as RMT random networks and IDEN bipartite networks can be used to quantify these aspects of community stability and complexity (Deng et al., 2012; Feng et al., 2022). By using these methods, researchers can gain insights into the factors that contribute to the robustness and cohesion of ecological communities, and identify the potential strategies that maintain their stability and complexity in the face of environmental change (Yuan et al., 2021). Overall, network analysis methods have become an increasingly important tool for studying ecological systems (Xing and Fayle, 2021). By characterizing the complex patterns of interactions between species in these systems, network analysis can provide insights into the structure and function of ecological communities, as well as the processes that govern their dynamics (Tobias et al., 2014; Wang, 2018). Furthermore, network analysis methods can be used to investigate the stability and complexity of ecological communities, providing valuable information for conservation and management efforts (Yuan et al., 2021). As such, network analysis methods have the potential to contribute significantly to our understanding of ecological systems and inform efforts to protect and manage them.

The objective of this study was to shed light on the characteristics of prokaryotic, fungal, and protistan communities in the soil of healthy tobacco plants and those infected by F. solani. Specifically, we aimed to achieve the following: (i) use molecular ecological network analysis to reveal the associations among species of prokaryotic, fungal, and protistan communities; (ii) explore the associations between fungi and other microbial groups, including prokaryotes and protists, through interdomain ecological network (IDEN) analysis; (iii) study the associations between the pathogen F. solani and prokaryotes and predatory protists through sub-network analyses; and (iv) investigate how environmental factors regulate these mutual relationships. Through the accomplishment of these objectives, we aim to offer a novel strategy and theoretical foundation for enriching the investigation of microbial resources in tobacco soil. Additionally, we aim to explore antagonistic microbial resources that specifically target F. solani within the context of our well-defined pathosystem.

In the context of the existing literature on Fusarium solani root rot, this study sought to elucidate the intricate interactions between soil microbial communities and the incidence of this pathogenic infection in tobacco fields. Our objectives were guided by the need to address gaps in our understanding of how various soil factors influence the proliferation of F. solani and how microbial communities respond to the presence of this pathogen. By accomplishing these objectives, we aimed to provide a new strategy and theoretical support for enriching the study of tobacco soil microbial resources and for exploring antagonistic microbial resources targeting F. solani. The current research builds upon prior investigations that have examined the relationships between soil properties, microbial communities, and disease incidence. A careful review of the literature reveals a scarcity of research on the regulatory mechanisms of environmental factors such as different nutritional levels, particularly the impact of nutrient improvement on disease suppression (Guo et al., 2022). Additionally, while there are studies that have explored the impact of soil properties on the occurrence of F. solani (Tan et al., 2021, 2022), little attention has been given to the intricate network of interactions involving other microorganisms, such as bacteria, fungi, and protists, in the context of this pathogenic invasion.

Our findings have brought to light significant disparities in soil attributes between healthy and diseased plants within the same field. The soil hosting the infection exhibited notably higher levels of organic matter, total organic carbon, total nitrogen, total phosphorus, available potassium, available phosphorus, nitrate nitrogen, and ammonium nitrogen compared to the healthier soil. These pronounced differences underscore the existence of substantial heterogeneity in soil texture and nutrient content across the field, a phenomenon commonly referred to as “soil patchiness” (Eviner and Firestone, 2007; He et al., 2012). Soil patchiness can stem from various sources, including soil type, water distribution, chemical gradients, and topography (Laekemariam et al., 2017; Lefebvre et al., 2020). These variations can translate into discrepancies in plant growth (Wang et al., 2021; Cao et al., 2023). Importantly, soil patchiness plays a multifaceted role in shaping disease dynamics. In environments characterized by varied soil attributes, different regions of the same field may provide varying levels of vulnerability to pathogenic organisms (Tan et al., 2021). The localized enrichment of nutrients in certain patches might create more favorable conditions for the proliferation of pathogens such as F. solani. Conversely, patches with less favorable attributes may hinder pathogen growth. This dynamic interplay between soil patchiness and disease dynamics underscores the complexity of microbial interactions in the soil ecosystem and has important implications for disease management strategies.

The establishment of pathogenic F. solani in soil demonstrates significant repercussions on the soil’s microbial community (Tan et al., 2021). Our research underscores that the microbial community of afflicted plants contrasts starkly with that of their healthy counterparts, as evidenced by reduced microbial diversity and richness (Supplementary Figure S3 and Supplementary Table S1). This corroborates prior studies that indicate the presence of pathogenic fungi can curtail microbial diversity and reshape community structure (Mendes et al., 2018; Qu et al., 2020; Tan et al., 2022). This decline in diversity could emanate from the competitive edge that pathogenic fungi gain, possibly through the release of toxins or competition for resources (Bais et al., 2003; Fu et al., 2017). Furthermore, alterations in microbial community structure might stem from shifts in soil conditions induced by pathogenic intrusion, such as changes in pH or nutrient availability (Zhang et al., 2016). Our findings underscore that pathogenic fungal incursions exert a substantial influence on soil microbial communities, yielding diminished diversity and richness. These outcomes possess crucial implications for plant well-being and agricultural yield, considering the pivotal role of soil microbial communities in nutrient cycling, soil structure, and plant-microbe interplay. Further explorations are essential to unravel the mechanisms orchestrating these effects and to devise strategies for managing plant diseases borne in the soil.

This study casts light on the sway of environmental factors upon the composition and structure of microbial communities within tobacco soils (Wang et al., 2022). The results of our study demonstrate that soil pH, TOC, NO₃⁻-N, and NH₄⁺-N are key environmental factors that influence the composition and structure of microbial communities in tobacco soils. Our findings are consistent with previous studies that have identified soil pH as a major driver of microbial community structure (Christian et al., 2009; Fierer et al., 2013). Moreover, our results indicate that the influence of soil nutrients on microbial communities may vary depending on the specific microbial group under consideration (Zhou et al., 2021). For example, TOC and NH₄⁺-N have a greater impact on fungal communities than on prokaryotic or protistan communities, while NO₃⁻-N has a stronger effect on fungal and protistan communities than on prokaryotic communities. Our study also suggests that environmental factors account for a substantial, but incomplete, portion of the variation in microbial community structure (Chen and Gu, 2022). Variance partitioning analysis revealed that only a portion of the variation in microbial communities could be explained by environmental factors, indicating the potential importance of neutral or stochastic processes in community aggregation. These findings are consistent with previous studies that have suggested that neutral or stochastic processes may play a significant role in shaping microbial community structure (Nemergut et al., 2016; Zhou and Ning, 2017). Overall, our study highlights the intricate interplay between environmental factors and microbial communities in tobacco soils. Our findings suggest that soil pH, TOC, NO₃⁻-N, and NH₄⁺-N may be pivotal in shaping the microbiome of this ecosystem, and emphasize the need for further research to elucidate the underlying mechanisms driving these relationships.

This study also expounds on the transformative influence of pathogenic F. solani on the arrangement and dynamics of soil microbial communities (Tan et al., 2021, 2022). The contraction of network scale, indicated by diminished nodes and edges, subsequent to infection signifies a reduced complexity in microbial communities (Figure 2), a phenomenon echoed in earlier studies (Urich et al., 2008; Banerjee et al., 2018). Furthermore, the decline in keystone species numbers within prokaryotic and protistan community networks portends a diminution in their robustness and stability, given the crucial roles played by keystone species in sustaining ecosystem function and resilience (Deng et al., 2012; Banerjee et al., 2018). In contrast, the augmentation in keystone species numbers within fungal community networks could denote a response to pathogenic invasion, with these key species potentially stifling F. solani growth or facilitating the establishment of other beneficial fungal strains (Tan et al., 2022). Noteworthy examples, such as Monacrosporium thaumasium and Rhizophlyctis rosea isolate, could emerge as pivotal contributors in the microbial ecosystem of infected soils, offering novel strategies for curtailing fungal root rot outbreaks (Hu et al., 2020; Tan et al., 2022). Additionally, cohesion analysis unveils an intensified fungal competition in healthy soils, juxtaposed with heightened protist cooperation in infected soils (Figure 3A). This shift implies altered community interactions, manifesting as changes in microbial community composition and structure (Chapelle et al., 2016). Further insights arise from ANOVA results, pointing to the superior robustness of prokaryotic and fungal communities in healthy soils relative to infected ones (Figure 3B), potentially indicating the deleterious impact of F. solani on the stability of these microbial communities (Yan and Nelson, 2020; Saengchan et al., 2022). However, the augmented robustness of protistan communities in healthy soils may mirror their adeptness at withstanding pathogenic incursions and upholding stability (Guo et al., 2022).

The soil, a multifaceted ecosystem, houses a plethora of microorganisms, including bacteria and fungi, pivotal in nutrient cycling and plant prosperity (Effmert et al., 2012). Endophytic microorganisms further inhabit plant tissues, potentially conferring disease resistance or nutrient access (Bamisile et al., 2018). The interactions among fungi, bacteria, and protists within this milieu assume critical roles in microbial communication networks, integral to the equilibrium and functioning of these habitats (Deng et al., 2012). Our study spotlights the notable role of inter-domain interactions, interweaving prokaryotes, fungi, and protists, paramount to soil ecosystem dynamics. The encroachment of pathogenic fungi, exemplified by F. solani, disrupts these interactions, exerting transformative effects (Figures 4, 5). Investigation through the IDEN approach discerns intricate bacterial-fungal associations in healthy soils, while infection enriches F. solani’s connections with prokaryotic species (Figures 4C,D). This phenomenon suggests that pathogenic fungi might stimulate the growth of specific bacterial species, possibly fostering or suppressing fungal growth—a key insight into the regulation of F. solani’s expansion and virulence (Tan et al., 2021). Our discoveries corroborate earlier findings showcasing the vital role of prokaryotes in soil health by controlling fungal pathogens (Zahir et al., 2003; Niu et al., 2017). Notably, our study unveils that F. solani invasion escalates Cercozoa numbers, triggering predatory protists’ predation on fungal communities (Figures 5C,D). Remarkably, the predation of pathogenic F. solani declines in infected soil (Figures 5E,F), hinting at reduced survival pressure on the pathogen. These trends align with previous research underlining the influential role of predatory protists in steering soil microbial communities (Rønn et al., 2002; Geisen et al., 2015). Collectively, our study accentuates the indispensability of accounting for inter-domain interactions when scrutinizing soil microbial communities, particularly under the impact of pathogenic fungi.

Several studies have shown that soil physical and chemical factors play crucial roles in regulating the interactions between predators and prey in the soil ecosystem (Bastida et al., 2021; Ding et al., 2022). For instance, pH, nutrient availability, and organic matter content have been found to be important drivers of protistan-fungal interactions in the soil (Zhou et al., 2021; Guo et al., 2022). The pH of the soil can affect both the growth and activity of protists and fungi, thereby influencing the strength and direction of their interactions (Örmälä-Odegrip et al., 2015; Rocca et al., 2022). Nutrient availability, particularly the availability of nitrogen (N) and phosphorus (P), has been found to be a key determinant of protistan-fungal interactions, with high N:P ratios favoring fungal growth and low ratios favoring protistan growth (Bonkowski, 2004; Gellner and McCann, 2016). In addition, organic matter content has been shown to influence the diversity and composition of soil microbial communities, which can in turn affect the interactions between predators and prey (Wall et al., 2015; Martin et al., 2023). Our results provide further evidence that these factors also influence the connectivity of bipartite networks (Table 1), which in turn can affect the structure and function of the soil ecosystem. For example, in healthy protistan-fungal networks, connectivity was significantly influenced by TK, AP, and AK. These findings suggest that increasing soil nutrient availability could promote the growth and activity of both protists and fungi, potentially leading to a more efficient transfer of energy and nutrients within the soil food web. In contrast, in infected soil, connectivity was significantly influenced by TOC, AK, and NH₄⁺-N. This suggests that managing soil carbon and nitrogen availability could be particularly important for maintaining the stability and function of protistan-fungal networks in degraded soils. Overall, these findings highlight the complex nature of the interactions between predators and prey in the soil, and the importance of understanding the roles of soil physical and chemical factors in regulating these interactions. By identifying the specific soil variables that influence these interactions, we can develop more effective soil management practices that promote a healthy and diverse soil ecosystem.

Collectively, our study advances the understanding of the intricate interactions within soil microbial communities and their responses to the invasion of pathogenic fungi like F. solani. By elucidating the roles of different microbial groups, we provide insights into the dynamics of tobacco root rot disease. This knowledge holds significant implications for disease management strategies. The identification of potential key players within the microbial ecosystem, including both beneficial and pathogenic species, opens avenues for targeted interventions. Leveraging the biotic regulatory role of predatory protists and the intricate microbial interactions revealed in our study could lead to innovative approaches for controlling root rot outbreaks. Furthermore, understanding how environmental factors influence these interactions underscores the importance of maintaining optimal soil conditions to mitigate disease incidence. Overall, our findings offer practical insights that may contribute to the development of sustainable and effective strategies for managing root rot disease in tobacco cultivation.

Overall, this study demonstrates the complex interplay between soil physicochemical properties, microbial diversity, and plant health. The results suggest that the presence of pathogenic fungi can significantly alter the soil microbial community and increase soil nutrient levels. Additionally, the study highlights the importance of considering multiple factors, including pH and soil nutrients, in understanding the interactions between different microbial groups. The findings have implications for the development of sustainable agricultural practices that can promote soil health and prevent disease outbreaks in crops. Further research is needed to investigate the specific mechanisms underlying these complex microbial interactions and their effects on plant health.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

PL, TX, QH, YZ, and WM designed the experiments. TX, YY, ZW, XD, BW, and WL took samples and performed all data measurement. SG and PL contributed to the data analysis and wrote the paper. All authors contributed to the article and approved the submitted version.

This research was supported by the Foundations for Tobacco Science of Wenshan Tobacco Company of Yunnan Province (2021530000241033) and Zhuzhou Tobacco Company of Hunan Province (22-004) of China.

PL was employed by the Wenshan Tobacco Company of Yunnan Province.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.