Ascomycetous Endophytic Fungi Drive Root Fungal Community Assembly in Wheat Under Moderate Drought

Abstract

Drought stress severely limits wheat growth, development and yield. Endophytic fungi play a crucial role in plant growth and drought resistance. In agricultural production, they hold significant application potential as biocontrol agents capable of mitigating drought-induced damage. However, the mechanisms underlying changes in endophytic fungal community structure under drought stress remain unclear. Our study employed amplicon sequencing to investigate the structure of endophytic fungal communities in wheat roots under different water treatments, comparing structural and functional changes between different treatments. Results revealed that drought stress led to the greatest accumulation of relative abundance in the phylum Ascomycota (86.4%). At the genus level, Stachybotrys (increase 994.2%), Fusarium (increase 94.6%) and Aspergillus (increase 295.6%) showed the most significant increases in relative abundance. Co-occurrence network and Sankey diagram analysis revealed that wheat roots formed a drought-specific endophytic fungal community centered around Stachybotrys, Fusarium and Aspergillus, which indirectly enhanced crop drought tolerance. Our findings provide a theoretical foundation for future agricultural strategies to improve crop drought resistance through precise regulation of microbial communities.

1. Introduction

As a critical staple crop, wheat (Triticum aestivum L.) feeds over one-third of the world’s inhabitants, and its stable production is vital for global food security and social stability [1]. The compounding pressures of global population growth and climate change are subjecting agricultural production to increasingly severe abiotic stresses, such as drought, salinity, heat, and cold [2,3,4]. The stress factors constrain crop growth and development, causing significant declines in yield and quality, thereby jeopardizing global food security [5]. Current responses to the challenges primarily rely on genetic modification and intensive inputs of water and fertilizers, but the measures are often costly and may impose environmental pressures. Against the backdrop, seeking a green and sustainable solution has become a focal point of modern agricultural research [6].

In recent years, research on drought stress mitigation has shifted from long-term breeding of drought-tolerant varieties toward leveraging plant–microbe interactions to alleviate drought-induced damage and enhance plant drought resistance. The approach capitalizes on the function of plant-associated microbial communities [7], including plant-growth-promoting bacteria and fungi, in plant growth and responses to environmental changes such as drought [8,9]. Plant microbial communities, particularly endophytic fungi, demonstrate immense application potential as powerful “biological tools” [10,11]. In combating abiotic stresses, endophytic fungi establish a robust “internal defense line” for crops through multiple sophisticated physiological and molecular mechanisms. When facing drought stress, certain endophytes can regulate stomatal opening and closing in host crops, promote root development, and induce the production of Osmoprotectants (proline and soluble sugars), thereby effectively helping crops retain water and maintain physiological activity [12,13]. On the other hand, fungal infection following drought stress can promote drought recovery [14]. Among these endophytic fungi, phosphate-solubilizing fungi play a crucial role in helping plants withstand drought stress through their ability to solubilize phosphorus. They convert phosphorus in the soil into forms that plants can absorb, thereby enhancing the efficiency of plant uptake of essential nutrient [15]. Phosphate-solubilizing fungi not only promote root growth, increasing root depth and density, but also help plants access water from deeper soil layers, finally strengthening their drought resistance.

The structure of endophytic fungal communities within roots is not static but rather a dynamic structure that adapts to changes in the living environment and the plant’s own regulatory mechanisms [16]. Soil fungi enter the roots through root deposition, forming root fungal communities. Some fungi enter the plant roots under the host’s regulation, forming an endophytic fungal community within the roots alongside fungi originally colonizing plant roots [17,18]. The dynamic process involves plants modulating microbial communities to adapt to environmental changes. Previous studies show that plant genotype, growth stage, physiological state, geographic location and environmental factors like climate, temperature and rainfall all influence endophytic fungal composition and biodiversity. Gehring suggests that environmental factors exert a greater influence on endophytic fungi than host genetics [19]. Under drought conditions, changes in root architecture alter root exudates [16,20]. The modifications may influence the diversity of root-associated endophytes, regulating and reorganizing the endophytic fungal community structure. The reshapes between plants and their endophytic communities mitigate drought-induced damage, enhance plant drought tolerance, and significantly impact growth and development. Endogenous microorganisms have been proven to be key biological factors enabling crops to enhance their stress resistance [21] and achieve sustainable high yields [22].

Plant–fungal symbiosis is crucial for host phosphorus acquisition. Classical research centers on arbuscular mycorrhizal fungi (Glomeromycota), obligating mutualists that extend root phosphorus uptake via extensive extraradical mycelium. However, the symbiosis, dependent on stable soil conditions, is often impaired under drought [23]. In contrast, the role of Ascomycota endophytes in nutrient mobilization under stress remains less explored. The fungi colonize roots endosymbiotically without forming mycorrhizal structures, engaging in more flexible host relationships. Previous studies show that many ascomycete endophytes can activate insoluble phosphorus in soil by secreting organic acids, protons, and phosphatases, demonstrating independent phosphorus-solubilizing capabilities [24]. Therefore, we hypothesize that under drought stress, ascomycete endophytes (such as Aspergillus and Fusarium) with greater metabolic plasticity and environmental tolerance may play a crucial substitute or complementary role in rhizosphere phosphorus cycling and host phosphorus nutrition supply, compared to inhibited AMF.

Therefore, in-depth exploration of the relationship between plants and their endophytic fungi not only deepens our understanding of “plant-microbe” interaction mechanisms but also provides valuable microbial resources and theoretical foundations for developing novel, efficient microbial inoculants (biofertilizers/biostimulants). By strategically regulating the microbial communities within crops to enhance their adaptability to climate change, we are charting a promising path toward transforming agriculture into a resource-efficient, environmentally friendly and sustainable development model [25].

We investigated the impact of drought stress on the wheat root microbiome, with a focus on structural changes in its endophytic fungal communities. It identified important fungi following the changes and examined their relational networks within the community. Further analysis explored the implications of such community shifts for plant health and predicted their functional roles. We found that while diversity of endophytic fungal communities in wheat roots under drought stress showed no significant alteration, the proportion of unique fungi decreased by 23.17%. Under drought conditions, Stachybotrys, Aspergillus, and Fusarium became the dominant genus within the endophytic fungal community. Aspergillus and Fusarium were not only abundant in the root system but also demonstrated phosphate-solubilizing capabilities. Oure findings suggest a theoretical basis for the future application of such microorganisms to alleviate drought stress and enhance crop yield.

2. Materials and Methods

2.1. Test Materials and Culture Conditions

The wheat variety Yangmai 25 which our study utilized is a spring wheat variety bred by the Jiangsu Lixiahe Agricultural Science Research Institute, exhibiting balanced quality characteristics across all aspects. The wheat variety Yangmai 25 was provided by Jiangsu Lixiahe Agricultural Science Research Institute. Soil was collected from the Yangzhou Crop Variety Regional Trial Station (119°53′ E, 32°31′ N). Soil texture was clay (organic matter 20.48 g·kg⁻¹, total nitrogen 1.15 g·kg⁻¹, alkaline-hydrolyzable nitrogen 228.3 mg·kg⁻¹, available phosphorus 42.5 mg·kg⁻¹, available potassium 109 mg·kg⁻¹, pH 6.21).

Prior to the experiment, plump and uniformly sized wheat seeds were placed in 50 mL centrifuge tubes for surface disinfection. The seeds were treated with 10% NaClO for 15 min and subsequently rinsed five times with sterile distilled water. The surface-disinfected wheat seeds were evenly placed on Petri dishes and moistened with an adequate amount of sterile distilled water. The dish was placed in a 28 °C constant-temperature incubator for dark germination. After approximately 36 h of growth (with roots reaching about 2 cm in length), the seedlings were set aside for later use. We collected topsoil (0–20 cm), air-dried it, and sieved it through an 18-mesh sieve. After weighing, we added sterile distilled water to adjust the moisture to 35% field capacity. The moist soil was then thoroughly mixed and re-sieved to ensure homogeneity. We prepared 700 mL opaque cups by punching nine uniform drainage holes in the bottom of each. Then, we added 500 g of the soil mixture into each cup. After that, we transplanted uniformly sized wheat seedlings (after 36 h of germination induction) into the cups and placed the cups on trays with varying amounts of distilled water added to the tray bottom: 80% soil moisture (Well-Watered, WW) and 50% soil moisture (Moderate Drought, MD) [26,27,28]. Finally, they were placed in a 24 m² indoor smart climate chamber for 8 days. The greenhouse environment was set to a 14 h light/10 h dark cycle, with a temperature cycle of 26 °C/22 °C. The relative humidity in the greenhouse was 40%, and the light intensity was 100 μmol m⁻²s⁻¹.

2.2. Collection and Sequencing of Root-Interior Fungal Samples

Wheat plants were gently removed from the soil under well-watered (WW) and moderate drought (MD) treatments. The root surface was rinsed to remove loose soil using 1× PBS solution; this was repeated three times. Subsequently, the wheat roots were rinsed with sterile distilled water; this was repeated three times. The washed roots were placed in 1.5% NaClO (v/v) for 15 min disinfection and rinsed 3–5 times with sterile distilled water. Root tissue was incised with a sterile scalpel to release microbial cells then transferred to a 50 mL centrifuge tube containing sterile glass beads and 0.9% sterile saline. This was then shaken at 30 °C for 4 h to release endophytes, filtered through a sterilized 5 µm membrane, then centrifuged at 12,000 rpm and 4 °C for 10 min to collect the pellet. The pellet was immediately frozen in liquid nitrogen for storage. Total DNA was extracted from collected wheat root samples using the FastDNA SPIN Kit. Fungal primers are listed in the table (Table S1). Purified PCR products were ligated with sequencing adapters to construct a sequencing library, which was subsequently subjected to Illumina MiSeq high-throughput sequencing.

2.3. Amplifier Sequencing Analysis

The analysis workflow proceeds sequentially through species annotation, α-diversity analysis, β-diversity analysis, and community functional prediction. After obtaining raw reads from sequencing, low-quality reads are first filtered. Assembly follows, where paired-end reads are assembled into tags. These tags undergo further filtering, yielding clean tags. Clustering is then performed on the clean tags, removing chimeric tags detected during the clustering alignment process. The final output is Effective tags. The representative sequence (the most abundant tag for OTUs or the consensus sequence for ASVs) was determined for each feature; subsequently, these sequences were classified using the Naïve Bayesian algorithm of the RDP Classifier (v2.13) against the UNITE (v10.0) database. A confidence threshold of 0.85 was applied for all taxonomic assignments. Our study employed OTUs as the phylum level and utilized Bray–Curtis distance based on principal coordinates analysis (PCoA) to investigate endophytic fungi community structure differences within wheat roots under varying moisture treatments. For principal coordinates analysis (PCoA), permutational multivariate analysis of variance (PERMANOVA) was performed using the Adonis function with 999 permutations based on Bray–Curtis distances. To predict the functional potential of the fungal community, we performed phylogenetic investigation of communities by Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2). Briefly, the representative sequences of amplicon sequence variants (ASVs) were placed into a fungal reference phylogenetic tree. The copy numbers of gene families (KEGG Orthologs) were then inferred using hidden state prediction (HSP). Subsequently, the ASV abundance was normalized by these predicted copy numbers to estimate the community-level abundances of metabolic pathways. The analysis utilized the built-in fungal ITS reference dataset. To ensure the reliability of predictions, quality control was applied based on the weighted Nearest Sequenced Taxon Index (NSTI), whereby predictions derived from ASVs with an NSTI value greater than 2 were filtered out. Finally, a functional abundance table annotated with the KEGG databases was generated.

2.4. Statistical Analyses

All experimental data were statistically analyzed using SPSS version 17.0. Each group was replicated at least three times. Differences between two groups were analyzed using Student’s t-test. A p value < 0.05 was considered indicative of a significant difference between data sets. Data collection and basic processing were performed using Microsoft Excel 2025, while graphical analysis was conducted using GraphPad Prism 9.

3. Results

3.1. Analysis of Endophytic Microbial Community Diversity in Wheat Roots Under Different Moisture Conditions

To investigate changes in fungal communities within wheat roots and identify beneficial fungi promoting wheat growth under drought stress, our study performed ITS rDNA sequencing on two root samples from wheat plants subjected to WW and MD treatments. The ITS sequencing yielded 721 operational taxonomic units (OTUs), with an average of 317 OTUs (

Table 1. The number of tags and OTUs in sequencing samples determined by ITS rDNA.

Sample ID	Total Tags	Taxon Tags	Unclassified Tags	Singleton Tags	OTUs
MD-1	107,066	106,544	46	476	330
MD-2	124,140	123,712	11	417	287
MD-3	96,596	96,278	34	284	349
WW-1	35,542	35,082	309	151	257
WW-2	109,349	106,910	2103	336	370
WW-3	52,594	52,020	298	276	308
Avg	87,548	86,758	467	323	317

).

To assess the adequacy of our sequencing depth, Shannon diversity index-based rarefaction curves were generated (Figure 1). All curves reached a plateau rapidly with increasing sequence reads, indicating that the sequencing depth was sufficient to reliably capture fungal diversity. The saturation curves of the well-watered (WW) control group were consistently higher than those of the moderate drought (MD) group, suggesting a potential reduction in root endophytic fungal diversity under drought stress.

To analyze the impact of different water conditions on the overall structure of the endophytic fungal community, we analyzed the diversity of the endogenous fungal community via employing alpha-diversity and beta-diversity analysis (Figure 2). We found that the alpha-diversity (based on the Chao1 index and Shannon index) of the endophytic fungal community showed no significant change (p > 0.05) under drought stress (Figure 2a,b). However, the beta diversity of the endogenous fungal community changed. Principal coordinate analysis (PCoA) was employed based on Bray–Curtis distances. The PCoA plot showed that the first two principal coordinates (PCo1 and PCo2) accounted for 86.97% and 10.97% of the total variation, respectively (Figure 2c). A clear separation between the WW and MD samples along the PCo1 axis was observed, forming distinct clusters, which suggests fundamental differences in endophytic fungal community structure. To quantify this divergence, PERMANOVA was performed. The analysis showed that the drought treatment explained 61.96% of the total variance in community structure (R² = 0.6196), showing a trend towards significance (F = 6.515, p = 0.100) (Figure S1), though the statistical power was limited by sample size. Furthermore, a test for multivariate homogeneity of groups dispersions (Betadisper) confirmed no significant difference in within-group dispersion between the two treatments (F = 0.900, p = 0.601) (Table S2), validating the PERMANOVA assumption and supporting that different water conditions were the primary driver of community differentiation. The results show that the moderate drought condition changed the endophytic fungal community.

3.2. Stachybotrys, Fusarium and Aspergillus Form the Important Fungal Genera Under Moderate Drought Condition

The Venn diagram illustrates the taxonomic categories under WW and MD treatments (Figure 3a). A total of 63 OTUs were uniquely present in the MD treatment, showing the change in the community under drought stress. A comparison between the WW and MD treatments revealed that the number of root-specific fungal OTUs decreased by 19, and their proportion declined by 23.17% under MD conditions (Figure 3a).

ITS analysis revealed that the endophytic fungal community in wheat roots comprised 9 phyla: Blastocladiomycota, Zoopagomycota, Monoblepharomycota, Kickxellomycota, Mucoromycota, Chytridiomycota, Mortierellomycota, Basidiomycota and Ascomycota. Analysis results show that Ascomycota accounted for the highest proportions in both WW and MD treatments. Compared to WW, relative abundances of all groups decreased in MD except for Ascomycota and Kickxellomycota, with Ascomycota showing the largest increase (9.05%) (Figure 3b). Our result suggests that Ascomycota is not only the dominant phylum but also the most positively responsive phylum to moderate drought conditions in the wheat root.

Further analysis of the endogenous fungal community structure at the genus level revealed the top ten fungal genera by relative abundance: Mortierella, Aspergillus, Chaetomium, Penicillium, Cladosporium, Bipolaris, Stachybotrys, Malassezia, Fusarium and Alternaria (Figure 4a). There was a notable difference in the composition of the endogenous fungal community between moderate drought and well-watered conditions. Aspergillus, Fusarium, Stachybotrys, Bipolaris, and Chaetomium exhibited higher abundance in MD (Figure 4b). Among these, Stachybotrys, Fusarium and Aspergillus showed the most significant abundance disparity between MD and WW (Figure 4c,d). Our results show that Stachybotrys, Fusarium and Aspergillus are the important fungal genera under MD treatment, and their interaction with the host plant may be key to enhancing wheat’s drought tolerance.

3.3. Ascomycota Are Biomarkers in Wheat Roots Under Moderate Drought Condition

LefSe (LDA Effect Size) is a tool for biomarker analysis in high-dimensional data, capable of identifying biomarkers with statistical significance and biological relevance across multiple groups. It is widely applied in microbial diversity research, particularly suited for comparing differences between groups. LefSe analysis determines whether the microorganisms identified through the above steps constitute biomarkers significantly distinguishing one treatment or sample from others (Figure 5). The analysis threshold was set to display only significantly differential species, from domain to genus, with LDA scores ≥ 3.8. Analysis of wheat root fungi under different treatments revealed that the number of biomarkers in MD roots was comparable to that in WW roots. WW biomarkers comprised Basidiomycota. MD biomarkers included Sordariomycetes, Hypocreales, Ascomycota and Cystofilobasidiales, which are endophytic fungi (Figure 5).

3.4. Prediction of Fungal Function in Wheat Roots Under Different Water Conditions

We characterized the functional potential of wheat root endophytic fungi under moderate drought by employing PICRUSt2 on fungal ITS sequence data, and compared the sequencing results with its database. Figure 6 displays the top ten functional pathway predictions by abundance: fatty acid β-oxidation I, fatty acid elongation—saturated, aerobic respiration I, aromatic biogenic amine degradation, glyoxylate cycle, adenosine ribonucleotides de novo biosynthesis, tRNA charging, pyruvate fermentation to isobutanol, GDP-mannose biosynthesis, and superpathway of adenosine nucleotides de novo biosynthesis I. According to previous research, the results suggest a multi-level adaptation strategy. We suggest that drought stress reshapes the endogenous fungal community structure and may weaken the plant’s basal defenses, thereby creating a potential ecological niche for fungi [29]. Furthermore, the changes in the endogenous fungal community may enhance their metabolic activity and provide them with a competitive advantage in resource acquisition under moderate drought conditions [30,31,32].

3.5. Phosphate-Solubilizing Fungi Accumulate Inside Wheat Roots Under Drought Stress

As an essential mineral element, phosphorus is vital for plant growth and development. Yet, its availability is limited because plants cannot directly absorb it from its fixed states in the soil. Phosphorus-solubilizing fungi are therefore critical agents that mobilize this nutrient. Our study performed a comparative analysis by integrating amplicon sequencing data with taxa identified as key phosphorus-solubilizing microorganisms in prior research [33]. Drought stress increased the relative abundance of Fusarium and Aspergillus but decreased that of Penicillium (Figure 7). Based on previous studies [23,24] and data from our research, Fusarium and Aspergillus may assist wheat in coping with drought through phosphorus solubilization.

3.6. Stachybotrys, Aspergillus and Fusarium Emerge as Core Endophytic Fungi Under Moderate Drought Condition

To further analyze changes in wheat endogenous fungal community under drought stress, we employed co-occurrence network analysis on fungal communities. By utilizing pooled data from fungal OTUs, we analyzed fungal community abundance and co-occurrence networks. As shown in Figure 8, we found that drought triggers a shift in the fungal community, which converges around Stachybotrys, Aspergillus, and Fusarium as important species in response to stress. The results are consistent with the analysis of endogenous fungal community structure, further demonstrating that endophytic fungi within wheat roots may alter their community structure to adapt to drought stress.

4. Discussion

Over the course of their long evolutionary journey, plant–microbe interactions under abiotic stress represent a key frontier in plant science and sustainable agriculture. Elucidating the underlying mechanisms of the interactions is imperative for formulating effective mitigation strategies and exploring sustainable solutions [34]. Previous studies have shown that certain endophytic fungi, acting as natural “probiotics” for plants, demonstrate significant application potential [35]. Endophytic fungi live endophytically within plant tissues without causing apparent disease, establishing a mutualistic symbiosis [36]. Previous studies have revealed that the symbiotic relationship enhances plants’ survival capacity under adverse conditions [37].

Endophytic fungi, as a vital component of plant microbiomes, exhibits dynamic plasticity and adaptive regulation in their community structure when confronting environmental stress. Our study found no significant shift in the diversity of wheat root endophytic fungi in response to drought (Figure 2a–c). The result is consistent with the findings of María Julia Carbone [38], which showed that fungal community diversity in grape roots did not change significantly under drought stress. When plants undergo drought stress, endophytic fungal communities undergo a series of changes in composition, structure, and function. The alterations are not random but follow specific ecological patterns [39]. Endophytic fungi may respond to plant drought signals by restructuring their communities to adapt to aridity. Through plant–microbe interaction, they enhance the host’s drought resistance [40]. Afsoun Kamyab’s study on the drought resistance of Pinus Picea revealed that exogenous addition of the endophytic fungus Fusarium sp. significantly increased the number of leaves, root width, and fresh leaf weight in Pinus Picea under drought stress [41]. Our study observed a significant increase in Fusarium abundance in drought-stressed wheat roots (Figure 4c). The result may represent a targeted adjustment of root microbiota in response to drought stress. Additionally, our study observed significant increases in the relative abundances of Aspergillus (Figure 4d), which may contribute to enhanced plant drought resistance.

From the perspective of community composition, abiotic stress induces significant alterations in the species richness and diversity of endophytic fungal communities. For instance, under extreme drought conditions, OTU richness of endophytic fungi in desert plants is markedly higher than in other plant species [42,43]. In contrast, our study found that the number of unique endophytic fungal species in wheat roots under drought stress decreased by 23.17% compared to well-watered conditions (Figure 3a), showing slight divergence from previous research. The difference is likely due to our use of moderate drought treatment, in contrast to extreme drought treatment employed in previous studies.

From an ecological perspective, plants respond to drought stress by altering the structure of their endophytic fungal communities, involving complex interspecific interactions and niche allocation. Sulaimon Basiru revealed that within arbuscular mycorrhizal fungal (AMF) communities, specialized taxa such as Septoglomus and Funneliformis function as pivotal hubs within root fungal networks, while generalist taxa like Rhizophagus and Entrophospora drive cross-sector connections, forming close interactions with bacteria including rhizobia and Sphingomonas. Cross-sector interactions are crucial for plant adaptation under arid conditions [44].

When plants encounter stress, their endophytic fungal communities undergo not random fluctuations but a highly directed ecological succession process. The process is driven by both “environmental selection” and “active regulation by the plant host.” For example, under drought stress, dark septate endophytes (DSE) with thick-walled spores, well-developed mycelium systems or the ability to produce large amounts of polysaccharides become enriched due to their strong water retention and transport capabilities, with their relative abundance increasing by over 30% [45]. Research on the fungal community at the roots of Chinese juniper revealed [46] that as soil moisture decreased from 40% to 10%, the abundance of ectomycorrhizal fungi (EMF) significantly increased, while the prevalence of plant pathogens relatively decreased. In our study, drought stress altered the structure of the wheat root endophytic fungi community. The relative abundances of Fusarium, Aspergillus, and Chaetomium significantly increased (Figure 4c–e and Figure 8). The change suggests a multi-layered adaptive response. Based on functional predictions from PICRUSt2 (Figure 6) and previous studies, we infer that under moderate drought condition, the weakening of wheat root defenses may provide an opportunity for colonization by fungi, whose metabolic potential could be enhanced [29]. The changes in the function of endogenous fungal communities may drive a transition of the root endophytic fungal community from a stable symbiotic state toward a “high-alert,” metabolically active state with heightened stress-responsive capacity [47]. Their increased abundance may reflect a coping strategy of wheat to drought stress. Additionally, functional prediction must be treated with caution. The PICRUSt2 carries inherent limitations when applied to fungal ITS data. The results serve as preliminary predictions and require further validation.

Phosphorus, as one of the three essential macronutrients for plants, serves not only as a core component of nucleic acids and membrane lipids—fundamental building blocks of life—but also as the “universal currency” driving all cellular energy metabolism [48,49]. Under increasingly frequent abiotic stresses such as drought, salinity, and low temperatures, phosphorus emerges as a key regulator in plant survival strategies by modulating root architecture, enhancing stress resistance [49]. Recent advances in microbiology and biotechnology have revitalized interest in phosphorus-solubilizing microorganisms as agents to improve soil phosphorus availability and enhance plant stress tolerance [50,51]. Among diverse phosphate-solubilizing microorganisms, bacteria dominate, with fungi constituting a smaller proportion [52]. However, studies show that phosphate-solubilizing fungi often demonstrate greater phosphorus solubilization capacity and stability compared to their bacterial counterparts [53]. Amira Susana Nieva observed that Fusarium solani effectively alleviated adverse responses in lotus plants caused by phosphorus deficiency [54]. Ramalingam Radhakrishnan found that the soybean endophytic fungus Fusarium verticillioides RK01 enhanced superoxide dismutase (SOD) activity, significantly reducing oxidative stress [55]. Our study found that under moderate drought conditions, the phosphorus-solubilizing fungi Fusarium and Aspergillus significantly accumulated in wheat root systems (Figure 7). The results may represent a plant response to drought stress by adjusting the structure of its endophytic fungal community to reduce stress-induced damage, consistent with previous research findings.

Future agricultural drought-resistance strategies should fully leverage the power of plant–microbe interactions. By precisely regulating microbial communities, crop drought tolerance can be enhanced, reducing reliance on chemical pesticides and fertilizers, and steering agricultural production toward more ecologically friendly and sustainable practices.