The rhizosphere, a critical zone of soil surrounding plant roots, serves as a dynamic interface for interactions between plants and a diverse array of microorganisms. These microorganisms, including bacteria, fungi, and archaea, play vital roles in enhancing plant growth, improving soil fertility, and promoting ecosystem stability. They are involved in various processes such as nutrient cycling, organic matter decomposition, and the regulation of plant stress responses (Bais et al., 2006; Zhalnina et al., 2022). The presence of beneficial microbes in the rhizosphere can improve plant health by enhancing nutrient uptake, providing protection against pathogens, and promoting plant growth through mechanisms like nitrogen fixation and phosphorus solubilization (Lugtenberg and Kamilova, 2009; Van der Heijden et al., 2015).

In addition to their direct benefits to plant health, rhizosphere microbes also interact with plant-parasitic nematodes (PPN’s), which are significant pests in agriculture. Nematodes, particularly those that feed on plant roots, cause substantial damage to crops by disrupting root function thereby affecting the plant growth that ultimately results in the yield loss. However, the rhizosphere is the niche to a wide variety of microorganisms that can influence nematode populations through several mechanisms. Beneficial bacteria and fungi in the rhizosphere can suppress nematode infestations by producing nematicidal compounds, competing for resources, or acting as biological control agents (Zhao et al., 2017; Singh et al., 2020).

Reniform nematodes (RN) is a devastating pest in agriculture due to their widespread distribution affecting several crop species and the ability to thrive in diverse soil conditions. Their infestations can significantly alter the microbial community structure within the rhizosphere, potentially leading to decreased microbial diversity and disrupted nutrient dynamics (van der Putten and Bakker, 2018). Studies have demonstrated that specific microbial taxa can improve plant health by suppressing nematode populations and enhancing nutrient availability (Latz et al., 2021). For instance, beneficial bacteria and fungi can establish symbiotic relationships with cotton roots, leading to improved nutrient uptake and overall plant vigor (Garbeva et al., 2004; Lugtenberg and Kamilova, 2009). Furthermore, these beneficial microbes in the rhizosphere can also produce bioactive compounds that directly inhibit hatching and development of nematodes (Prasad and De Vries, 2019). Identifying these microorganisms within cotton rhizospheres is crucial for developing innovative management strategies aimed at nematode control and soil nutrient enhancement, aiding in reducing the reliance on chemical pesticides (Hassan and Abo-Elyousr, 2019).

However, nematode infestations can significantly alter the structure and diversity of microbial communities in the rhizosphere (Liu et al., 2017; Naylor et al., 2021). Changes in microbial diversity, especially a reduction in beneficial bacteria and fungi, have been linked to increased nematode damage in crops such as cotton and soybean (Yuan et al., 2020a). Additionally, plant-parasitic nematodes (PPNs) can influence plant performance by altering root exudation patterns, which in turn modify the microbial composition of the rhizosphere and improve the availability of nitrogen (N) and phosphorus (P) to plants (Topalovic et al., 2020). Verschoor (2002) found that nematode feeding contributes to nutrient cycling through the excretion of ammonia (NH3), N defecation, and increased root exudation. Similarly, Xie et al. (2023) demonstrated that nematode infestations in rice altered microbial populations enhancing nutrient cycling, particularly by increasing nitrogen-fixing bacteria that support plant growth. Wang et al. (2022) reported that nematode feeding on wheat roots shifted microbial communities, favoring fungi that contribute to organic matter decomposition, thus enhancing soil nutrient availability. In another study, Patel et al. (2024) showed that nematode-induced changes in microbial diversity helped plants by promoting the activity of specific microbes involved in phosphorus cycling, supporting plant growth under nutrient-limited conditions. Increased nematode presence often correlates with a decline in beneficial microbes, disrupting the ecological balance and negatively impacting soil health (Zhao et al., 2018; Bhattacharyya and Jha, 2012). Therefore, understanding the interplay between nematodes and microbial communities is essential for fostering sustainable agricultural practices.

The interactions between nematodes and soil microorganisms are multifaceted, encompassing competition, predation, and mutualism (Cai et al., 2023). Beneficial microbes can suppress nematode populations through antagonistic mechanisms, while nematodes may alter microbial community dynamics by changing resource availability (Gomez et al., 2019). Recent studies have emphasized the role of certain bacterial phyla, such as Proteobacteria, Firmicutes, and Actinobacteria, in suppressing nematode populations and promoting plant health. For example, Proteobacteria has been shown to produce metabolites that can inhibit nematode development, while Firmicutes and Actinobacteria contribute to enhanced plant nutrient uptake and nematode resistance (Li et al., 2022; Zhang et al., 2020). A study proposed by Naylor and Gurevitch (2021) that nematode feeding can change the composition of these microbial communities, often favoring Ascomycota and Basidiomycota, which can either help control nematode populations or shift microbial balance in ways that may reduce plant vitality. Additionally, nematodes themselves can modulate the structure of these microbial communities, causing a decline in beneficial microbes, such as those from the Proteobacteria, which can have cascading effects on soil health and plant resilience (Shang and Wang, 2022). Some studies have reported shifts in microbial diversity and composition in response to nematode presence, with certain taxa thriving while others diminish (De Vries and Shade, 2013). Investigating these dynamics across varying infestation levels can provide insights into how nematodes impact microbial communities and their function.

Furthermore, understanding microbial shifts in response to nematode infestations can lead to the development of targeted microbial inoculants or soil amendments that enhance beneficial microbial populations (Luo et al., 2022). These strategies offer sustainable alternatives to chemical controls, promoting long-term soil health and resilience in cotton species (Wu et al., 2019). By fostering beneficial microbial communities, it may be possible to mitigate the adverse effects of nematodes on cotton production and improve overall soil health. Advancements in molecular techniques, specifically 16S rRNA and ITS2 sequencing, have revolutionized the study of rhizosphere microbial communities. The 16S rRNA gene serves as a universal marker for bacterial identification, while the ITS2 region is widely used for characterizing fungal diversity (Ranjan et al., 2020). Together, these sequencing techniques provide a comprehensive view of the microbiome, revealing complex interactions that can influence plant health and stress responses. Incorporating R and the Phyloseq package into data analysis allows for robust profiling of microbial communities derived from sequencing studies. Phyloseq offers an efficient framework for handling and visualizing complex ecological data, enabling in-depth analysis of microbial diversity, community composition, and potential functional roles within the rhizosphere (McGuire and Triplett, 2009). This approach is particularly useful for examining the influence of RN’s on microbial dynamics in cotton soils, facilitating a deeper understanding of how these interactions impact plant health and productivity.

This study aims to profile the rhizosphere microbiome of cotton soils infested with RN’s across various infestation levels in North Alabama. By employing 16S rRNA and ITS2 sequencing, combined with analyses in Phyloseq, we seek to explore the intricate relationships between nematodes and microbial communities. This investigation will help identify key microbial taxa associated with different infestation levels of RN, offering insights into potential indicators of soil health and crop resilience.

This study advances our understanding of the rhizosphere-associated microbiome to RN infestations by revealing bacterial and fungal richness and evenness shifts across varied infestation levels. Our analysis shows that nematode infestation significantly affects the composition and diversity of rhizospheric microbiota, with notable shifts across infestation levels. Specifically, Group A has the highest abundance of bacterial phyla Planctomycetes and fungal phyla Basidiomycota, Mucoromycota, and Ascomycota. Group D was characterized by the predominance of bacterial phyla Chloroflexi and fungal phyla Ascomycota. These shifts highlight the resilience and adaptability of the microbiome in response to RN, suggesting a complex interplay between microbial community dynamics and nematode presence. The observed alterations in microbial composition may enhance plant defense mechanisms, as specific microbial taxa can facilitate nutrient acquisition and promote plant growth during stress conditions (Zhang et al., 2020).

The analysis of alpha diversity metrics revealed distinct patterns in microbial community composition corresponding to varying reniform nematode (RN) infestation levels. Notable differences were observed in both bacterial and fungal richness and evenness. Group D showed higher bacterial richness and evenness, while Group A had lower bacterial richness. These results are consistent with those of Lundberg et al. (2020b), that reported increased pest incidence like nematode infestations, can lead to more diverse microbial communities due to improved nutrient availability during stress. Conversely, Group A’s fungal communities showed higher richness and evenness, indicating a more stable microbial community associated with healthy soil ecosystems, as reported by Yuan et al. (2020b). Moreover, Group B revealed elevated fungal richness in observed species, suggesting that even moderate nematode infestations can modify root exudation patterns that may subsequently benefit plant health, as Topalovic et al. (2020) indicated. Conversely, in Group D, both fungal richness and evenness were diminished, consistent with previous studies indicating that increased nematode infestation can disrupt the microbial balance, favoring opportunistic species and resulting in a decline in overall microbial diversity, as reported by Zhang et al. (2019).

The Shannon rarefaction curves generated in our study indicate that microbial diversity responds distinctly to varying levels of RN infestation for both bacterial and fungal communities. The observation that the Shannon index plateaued after reaching 30,000 sequences supports the notion that our sequencing efforts sufficiently captured the microbial diversity present in the samples, corroborating similar studies suggested by Olesen and Simmelsgaard (2019) and Chao et al. (2014), which suggest that adequate sampling depth is crucial for reliable diversity assessments. Group D exhibited higher microbial diversity, which aligned with previous reports showed that microbial diversity during nematode infestations (Huang et al., 2022; Zhang et al., 2023). In Group D, the values of the Shannon index showed a higher microbial diversity within bacterial communities suggesting a robust and resilient bacteria that is capable of sustaining functions that are critical for nutrient cycling and plant growth (Wang et al., 2023). Fungal communities showed distinct patterns, with Group A having the highest diversity, suggesting low RN infestation levels may favor a diverse fungal community, potentially enhancing plant health and nutrient uptake (Martínez-García et al., 2023). In contrast, lower fungal diversity indicates that biotic stress negatively impacts the dynamics of fungal communities (Liu et al., 2023).

The PERMANOVA analysis of weighted UniFrac distances (p < 0.01) showed differences in the microbial composition across all groups, suggesting that nematode-induced biotic stress is the primary factor driving microbial community shifts. As spatial variation could potentially influence microbial communities, our experimental design ensured that all samples were collected from similar environmental conditions, minimizing spatial variability. In addition, nematode behavior can also cause significant shifts in microbial communities, independent of spatial variation in the sampling environment (Zhang et al., 2019). In the principal coordinate analysis (PCoA), bacterial groups associated with different levels of RN infestation clustered distinctly from fungal groups ( Figures 3A, B ), a similar pattern reported by Raaijmakers et al., 2009; Zhao et al., 2018; Zhang et al., 2019. Interestingly, an overlap of fungal communities was observed among Groups A, C, and D, suggesting the functional stability of specific fungal taxa despite the fluctuations in biotic stress (Nielsen et al., 2023).

At the phylum level, microbial community composition reveals significant insights into the dynamics of bacterial and fungal populations in response to varying levels of RN infestation. Our results indicate that the bacterial phyla Actinobacteria, Proteobacteria, and Acidobacteria were highly abundant across all groups, consistent with earlier studies that highlighted their roles in nutrient cycling and plant growth promotion in soil ecosystems (Fierer et al., 2007). Specifically, Actinobacteria are known for their capacity to degrade organic matter and contribute to soil health (Jansson and Hofmockel, 2009). Notably, Planctomycetes were identified as abundant phyla in Group A, which may indicate their role in nitrogen cycling (Rao and Rao, 2016). Similarly, the higher relative abundance of Firmicutes in Group B, Verrucomicrobia in Group C, and Chloroflexi and Bacteroidetes in Group D suggests that these bacteria may play a crucial role in maintaining soil health and nutrient cycling and also associated with the breakdown of complex organic compounds thus enriching the soil (Guan et al., 2018; Ding et al., 2024; Wang et al., 2019). The higher abundance of Ascomycota in Group D is important for improving soil health (Lücking et al., 2017). In Group A, the higher abundance of Basidiomycota and Mucoromycota reflects a potential competitive advantage of these phyla (Zong et al., 2021). A lower abundance of Mucoromycota was observed among all groups except Group A, suggesting that the resilience of fungal groups varies with nematode infestation levels (Nielsen et al., 2023).

In Group A, the dominance of Solirubrobacteraceae and Cellulomonadaceae families was observed, similar to what was reported by Youssef et al., 2015, suggesting their role in cellulose degradation. Gaiellaceae was the most abundant family observed in Group B, indicating its adaptability to varied environmental conditions (Vasquez et al., 2019). In contrast, Acidimicrobiaceae was the least abundant family in both Group A and B, which may indicate competitive exclusion by more dominant families in less disturbed soils (Sang et al., 2019). In Group C, Micromonosporaceae and Acidimicrobiaceae were the most abundant families, which can play a crucial role in secondary metabolite production and mitigate stress impacts (Liu et al., 2020). Group D exhibited increased abundance with Acetobacteraceae, Streptomycetaceae, and Geodermatophilaceae families, which are generally associated with nutrient cycling (Meyer et al., 2022). The predominance of Nectriaceae and Bionectriaceae in Group A suggests their vital role in plant health, nutrient mobilization, and soil ecology (Kurtzman et al., 2018). In Group B, the Plectosphaerellaceae family was the most abundant, and Trichocomaceae was the least abundant, suggesting a potential vulnerability of specific fungal taxa even with mild RN infestation (Zhao et al., 2021). In Group D, the increased abundance of Cladosporiaceae and Chaetomiaceae suggests the stability of these fungal communities that may affect plant-microbe interactions and overall soil health (Nielsen et al., 2023).

In Group A, the prevalence of Kribbella, Cellulomonas, and Solirubacter and the absence of Illumatobacter indicates that specific genera were dominant and are potentially involved in cellulose degradation and organic matter breakdown (Fierer et al., 2007; Liu et al., 2020). In Group B, the relative abundance of Gaiella and Nocardioides reflects a shift in community dynamics, which can enhance nutrient availability and promote plant growth (Zhang et al., 2018). In Group C, a higher abundance was observed in Illumatobacter, Dactylosporangium, and Rugosimonospora, indicating their resilience and adaptability in response to biotic stress (Pester et al., 2010). A decrease in the abundance of Gaiella and Cellulomonas suggests that certain nematodes can affect root-associated microbial communities essential for maintaining plant health (Jousset et al., 2017). In Groups C and D, Solirubacter was the least abundant genus potentially sensitive to higher RN infestation levels (Zhang et al., 2020). In Group A, Fusarium is the most abundant genus that plays a dual role as a pathogen and beneficial organism (Gams et al., 2011). Lower abundance of Didymella in Groups A and B may indicate shifts in community structure linked to nematode infestation levels (Grondahl et al., 2021). In Group C, the dominance of Talaromyces and Aspergillus suggests that specific fungal taxa might thrive in moderate RN infestation, likely due to their saprophytic capabilities and ability to decompose organic matter (Jaklitsch et al., 2016). The abundance of Cladosporium, Didymella, Fusarium, and Macrophomina in Group D may support the complex interactions in soil systems. A lower abundance of Talaromyces reflects competitive exclusion caused by higher nematode loads (Zhang et al., 2022).

Among the bacterial communities, 95 shared ASVs among all groups indicate a core set of taxa that persists across varying environmental conditions (Shade et al., 2012a). Identifying 61 shared fungal ASVs underscores the potential for specific fungal taxa to adapt and thrive in diverse soil environments (Glassman et al., 2017). The high number of unique ASVs in Group A (12,758) may facilitate niches that support a wider range of bacterial diversity that may be involved in improving nutrient availability and fostering ecological interactions (Lauber et al., 2009). A lower number of unique ASVs (10,709) were observed in Group B compared to Group A, reflecting a substantial diversity that may stimulate competitive interactions under mild RN infestation (Santos et al., 2018). In Group C, 12,153 unique ASVs identified belonged to diverse bacterial communities with specific functional roles in soil and plant health (Friedman and Alm, 2012). In Group D, relatively lower bacterial communities (11,360 unique ASVs) identified were possibly due to the microbial and pest competition for the available nutrients (Wagg et al., 2014). A higher diversity of fungal ASVs was identified in Group A (663) compared to Group D (326), suggesting that specific fungal communities may be more resilient to biotic stress, highlighting the interplay between fungal diversity and plant health (Zhao et al., 2021).

By suppressing plant defenses, nematodes may inadvertently alter the composition of microbial communities in the rhizosphere. This can lead to an increase in stress-resistant bacterial taxa better adapted to the modified environment. Moreover, the weakened plant defense system can also facilitate the colonization and proliferation of secondary pathogens, including bacteria, which may further exploit the compromised plant defenses (Shade et al., 2012b; McGuire et al., 2017). The increased abundance of genera such as Streptosporangium, Labrys, Pseudonocardia, Mesorhizobium, Sphingomonas, and Arthrobacter in Group D indicates a shift in stress-resistant taxa. Various ecological interactions and environmental factors influence the shift in specific microbial populations such as Burkholderia, Micromonospora, Jatrophihabitans, Gaiella, and Mycobacterium to selective pressures from nematode infestations. The change in such bacterial genera in Group D may be attributed to the selective pressure exerted by higher levels of RN infestation. Burkholderia species, particularly B. seminalis, have shown potential as biocontrol agents against nematodes like Meloidogyne enterolobii. Studies have demonstrated that specific concentrations of B. seminalis can exhibit ovicidal activity, reducing nematode egg viability and thus controlling nematode populations (Moreira et al., 2024). Burkholderia is highly attractive to certain nematodes, such as M. incognita, which can increase nematode aggregation around these bacteria. This attraction can influence the dynamics of nematode populations and their interactions with other microbial communities (Tahseen and Clark, 2014). Fungal genera such as Gibellulopsis, Latorua, Myrothecium, Podospora, Russoella, and Bovista employ a variety of mechanisms to suppress nematode infestations. These fungi are part of a broader group known as nematophagous fungi, which are recognized for their ability to control nematode populations through diverse strategies such as mechanical trapping, endoparasitism, systemic resistance, enzymatic degradation, and toxic metabolite production (Noweer, 2020). Interestingly, a significant increase in the abundance of these fungal genera was observed in Group D. A significant decrease in the fungal genera such as Tricladium, Phialophora, Nigrospora, and Candida was observed. The presence of some nematophagous fungi can potentially reduce the prevalence of non-nematophagous genera like Tricladium and Phialophora (Mo et al., 2023).

The presence of nematodes in the soil leads to increased alkaline phosphomonoesterase (ALP) activity, directly linked to higher phosphorus availability. This enhanced nutrient cycling provides a competitive edge to bacteria like Gaiella, that can efficiently utilize the available phosphorus (Zheng et al., 2022). The core bacterial genus Gaiella emerged as a dominant genus, exhibiting a prevalence of 100% and a relative abundance of 8%, indicating the crucial role in phosphorus recycling. The specific mechanism by which Conexibacter, Bacillus, Blastococcus, and Streptomyces bacteria outcompete other microbial communities under nematode infestation involves a combination of biochemical and ecological strategies. The presence of these bacteria with a relative abundance of 0.5% suggests their possible role in survival and proliferation in the rhizosphere by competing against the nematodes. Nematode-induced nutrient cycling significantly impacts the selective advantage of bacteria such as Sphingomonas, Mycobacterium, and Actinoallomurus in mixed microbial communities, as identified in Group D, with a relative abundance of 0.1%. Through predation and feeding activities, nematodes influence the availability of nutrients like nitrogen and phosphorus, affecting bacterial community dynamics and competitive interactions. This process can enhance the growth and activity of specific bacterial taxa, providing them with a competitive edge in nutrient-limited environments (Zheng et al., 2022). In Group D, Fusarium was observed with a 100% prevalence and a relative abundance of 1%. This dominance may be due to the complex relationship between Fusarium and nematodes, including antagonistic and synergistic interactions. These interactions can vary based on environmental conditions and the specific species involved (Siddiqui and Aziz, 2024). In Group D, Aspergillus, Gibberella, Cladosporium, and Lactera were identified with a relative abundance of 0.1%. These fungi can play various roles, from being parasitic to nematodes to acting as part of a broader soil microbiome that influences nematode behavior and survival.

The presence of nematodes alters the soil environment, affecting the bacterial community structure and promoting the clustering of specific bacterial taxa that can thrive under these conditions. We observed tight clustering among bacterial nodes, particularly in Actinobacteria, Acidobacteria, and Proteobacteria in the core microbiome. This clustering is likely influenced by the competitive exclusion of less adapted bacterial clades and the selective pressures exerted by the nematodes and the altered soil environment (Yergaliyev et al., 2020). The isolation of nodes within Bacteroidetes, Firmicutes, and Planctomycetes under plant parasitic nematode infestation is driven by complex interactions between the nematodes, the plant host, and the microbial communities in the rhizosphere. These interactions are influenced by the nematode’s life cycle, the plant’s response to infestation, and the environmental conditions in the soil (Yergaliyev et al., 2020). Forming closer tier networks within Ascomycota and Basidiomycota in response to RN levels provides significant evolutionary advantages. These fungi have evolved mechanisms that enhance their survival and ecological roles by forming intricate networks optimized for resource acquisition, defense, and symbiosis with host plants. Such networks are essential in environments with higher nematode levels, as they help mitigate the damage caused by these pests (Kitagami and Matsuda, 2024). Conversely, the formation of isolated networks with Glomeromycota phylum, particularly arbuscular mycorrhizal fungi (AMF), such as species from the genus Glomus, under RN infestation can be attributed primarily to their potential role in enhancing plant resistance and growth. These fungi possibly establish symbiotic relationships with plant roots, thereby improving nutrient uptake and serve as a biological control against nematodes (Chaerani and Ginting, 2023).

The study explores the relationships between reniform nematode (RN) infestation and the rhizosphere microbiome dynamics in cotton soils. It finds that RN infestation affects the diversity and composition of microbial communities, which in turn enhances plant resistance to biotic stress. These microbial shifts also impact vital biogeochemical cycles important for soil fertility. Furthermore, the research delineates specific bacterial and fungal taxa associated with RN infestation, indicating potential approaches for biological control and soil management. Our findings underscore the importance of comprehending plant-microbe-nematode interactions to formulate integrated pest management strategies that promote sustainable cotton production.

Designing individual and integrated experiments to understand tripartite interactions among plant-nematode-soil microbiomes is critical during Reniform nematode infestation. Growing and maintaining specific bacterial or fungal pure cultures identified during RN infestation will improve our understanding of these unique microbial species’ functional roles. This knowledge will facilitate the exploration of associated plant defense mechanisms, potentially leading to the development of targeted biological control strategies. Also, investigating the interactions between nematodes, rhizosphere microbiomes, and different cotton genotypes using multi-omic approaches could enhance our understanding of metabolite degradation, nutrient availability in soil, host-parasite competition, and selective pressures exerted on microbial populations during nematode infection. To further strengthen our knowledge, pot culture studies under controlled conditions with different genotypes play a crucial role in examining microbial shifts during RN infection to comprehend the link between microbial dynamics and plant resistance. Longitudinal studies assessing the impact of various nematode management practices on microbial community composition and soil health are essential. Applying these approaches to other plant-nematode systems will support our findings. This will help us understand broader ecological effects and promote sustainable farming practices.