Identification and Transcriptomic Analyses of Two Endophytic Fungi WDR2 and WDR5 from Wild Soybean Elucidates Mechanistic Aspects of Alkali Stress Tolerance

TL;DR

This study identifies two fungi from wild soybean that help plants tolerate alkaline soil stress, offering insights into how these fungi adapt and improve crop resilience.

Contribution

The study identifies two novel alkali-tolerant endophytic fungi and reveals their distinct molecular adaptation strategies to alkaline stress.

Findings

WDR2 and WDR5 are alkali-tolerant endophytic fungi that promote plant growth under stress.

Transcriptomic analysis reveals strain-specific and shared mechanisms for alkali stress adaptation.

The fungi show potential as bio-inoculants to improve crop productivity in alkaline soils.

Abstract

Soil alkalinization constitutes a significant abiotic stress factor that severely constrains global agricultural productivity. The application of alkali-tolerant endophytes represents a promising strategy for enhancing crop resilience. This study focused on the isolation and characterization of alkali-resistant endophytic fungi derived from wild soybean (Glycine soja), aiming to elucidate their potential in promoting host plant growth and to investigate their molecular responses to alkali stress. From an initial collection of 39 wild soybean endophytic fungal isolates, 12 strains demonstrated significant alkali tolerance, as evidenced by increased mycelial dry weight under both mild and intense alkali stress. Among these, two strains, designated WDR2 and WDR5, demonstrated particularly pronounced biomass enhancement and were taxonomically identified as Fusarium verticillioides through comprehensive morphological and molecular analyses. Subsequent inoculation assays conducted on maize (Zea mays) revealed that both fungal strains significantly alleviated the inhibitory effects of alkali stress on root system architecture. Comparative evaluations in soybean indicated that the growth-promoting effects of these endophytes were host-specific and varied according to fungal strain, stress intensity, and inoculation timing. Transcriptomic profiling via RNA-Seq under mild alkali stress identified 589 and 182 differentially expressed genes (DEGs) in WDR2 and WDR5, respectively, with only 43 DEGs shared between the two strains, indicating largely strain-specific transcriptional adaptations. Functional enrichment analysis revealed several shared mechanisms underlying alkaline adaptation in both fungi species, including the maintenance of ion homeostasis, remodeling of the cell wall, and regulation of protein complex assembly and oxidative stress responses. Moreover, distinct metabolic adaptations were identified: WDR2 exhibited significant enrichment in cellular integrity and modulation of membrane-related processes, as well as amino sugar and nucleotide sugar metabolism pathways. In contrast, WDR5 was enriched in phosphate ion transport and related transporter functions, glycerol kinase activity, and glycerolipid and glutathione metabolism. In summary, this study successfully identified two novel alkali-tolerant wild soybean endophytic fungi, WDR2 and WDR5. The results provide valuable insights into their divergent molecular adaptation strategies and highlight their potential application as bio-inoculants to enhance crop productivity in alkaline soils.

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1. Introduction

As sessile organisms, plants are consistently exposed to a range of abiotic stresses, including extreme temperature fluctuations, drought, flooding, and variations in soil salinity or alkalinity [1,2]. Among these stressors, soil alkalinity—characterized by elevated pH levels due to the accumulation of soda salts such as NaHCO_3_ and Na_2_CO_3_—significantly constrains plant growth and markedly reduces crop yields worldwide. Soil alkalinity adversely affects plants primarily through four mechanisms: elevated soil pH, osmotic stress, ionic imbalance, and secondary oxidative stress [3,4]. Consequently, soil alkalinity represents a critical challenge to agricultural productivity and land-use efficiency, underscoring the urgent need for effective reclamation strategies for alkalized soils.

Plants respond to alkali stress through a range of morphological, physiological, and molecular adaptations; however, these intrinsic defense mechanisms are frequently insufficient to cope with high soil alkalinity. In recent years, utilizing the symbiotic relationship between plants and microbes has emerged as a promising strategy to enhance plant resistance to abiotic stresses [5,6]. Among these beneficial microorganisms, endophytic fungi have attracted particular interest due to their unique functional traits and potential applications [7]. Endophytic fungi are symbiotic organisms that inhabit the internal tissues of living plants without causing apparent disease symptoms. These fungi establish mutualistic relationships with their host plants during at least part of their life cycle, residing within plant cells without inducing destructive infections or producing phytotoxic compounds. Through this unique symbiosis, endophytic fungi enhance the host plant’s ability to tolerate abiotic stresses while minimizing damage via multiple mechanisms, including the regulation of root system architecture [8], production of protective metabolites and osmolytes [9,10], maintenance of ionic homeostasis [11], enhancement of antioxidant systems [12], modulation of phytohormone levels [13], and improved nutrient acquisition [14]. In fact, abiotic stress itself may alter root morphology and stimulate plants to recruit beneficial endophytes that help mitigate these conditions [15]. For instance, Khan et al. [16] demonstrated that inoculation with Fusarium proliferatum SL3 and Aspergillus terreus MGRF2 reduced metal accumulation in plants by modifying root architecture.

As a close relative of cultivated soybean (Glycine max), wild soybean (Glycine soja) exhibits valuable inherent agronomic traits, including enhanced tolerance to abiotic stresses such as salinity, alkalinity, drought, and cold, as well as increased resistance to diseases [17,18]. Furthermore, G. soja is reported to harbor a more diverse microbial community compared to its cultivated counterpart [17]. We therefore propose that wild soybean serves as an ideal reservoir for the isolation of diverse endophytic fungi, which may contribute to its observed resilience. For instance, Gao et al. [19] isolated the endophytic fungus Alternaria angustifolia (strain YD09) from wild soybean using tissue culture methods, demonstrating its potential in promoting plant growth and alleviating salt stress in soybean seedlings. Further supporting this potential, a large-scale screening in the cold regions of China identified 302 endophytic fungal strains from soybean plants, of which 215 were specifically derived from wild soybean. Notably, the majority of endophytic fungi isolated from root tissues were identified as belonging to the genus Fusarium [17]. Despite these findings, research on endophytic fungi from wild soybean in natural alkaline environments remains limited. Moreover, there is an absence of comprehensive functional analysis that integrates plant phenotypic response with the elucidation of fungal tolerance mechanisms under alkali stress. Therefore, the screening and identification of novel alkali-tolerant endophytic fungi, along with the elucidation of their tolerance mechanisms, are of significant scientific and agricultural importance.

This study involved the isolation of endophytic fungi from wild soybean plants growing in saline-alkaline soils in northeastern China. Two alkali-resistant strains (WDR2 and WDR5) were identified and assessed for their effects on root systems of maize and soybean under mild and intense alkali stresses. Furthermore, transcriptomic analyses were conducted to elucidate the molecular responses of WDR2 and WDR5 to mild alkali stress. These findings contribute to the discovery of novel endophytic fungal strains from wild soybean for agricultural applications and offer new insights into developing microbial strategies for improving crop alkali tolerance.

2. Materials and Methods

2.1. Screening of Alkali-Tolerant Endophytic Fungi Strains

The wild soybean (Glycine soja) seeds were collected in October 2018 from the Acheng district (45°32′00.00′′ N, 126°59′00.00′′ E) in Harbin, China, and subsequently propagated in the experimental field of Northeast Agricultural University. From the rhizomes and leaves of the resulting seedlings, a total of 39 endophytic fungal strains were isolated. These strains were inoculated onto potato dextrose agar (PDA) medium (containing 200 g/L potato extract, 20 g/L glucose, 15 g/L agar, pH 7.0) and incubated at 26 °C for five days to achieve activation. To screen for alkali-tolerant endophytic fungi, agar plugs (8 mm in diameter) bearing activated mycelium were excised using a cork borer and transferred into 50 mL aliquots of potato dextrose broth (PDB) medium, which was adjusted to pH 7.0 (control), pH 9.0 (mild alkali stress), and pH 11.0 (intense alkali stress). The cultures were incubated on a rotary shaker at 120 rpm and 25 ± 2 °C for durations of 3, 7, and 10 days. Following incubation, mycelium biomass was separated from the culture broth by centrifugation at 12,000 rpm. The harvested mycelium was then dried at 60 °C to constant weight, and the dry weight was subsequently recorded.

2.2. Taxonomic Identification of Alkali-Tolerant Endophytic Fungi

Fungal strains were inoculated onto PDA plates. Once colonies covered the plates, microstructure observations were conducted using an Axioskop 2 plus FL upright microscope (Zeiss, Jena, Germany). Image capture and subsequent analysis were performed with Axioplan 2 imaging MOT software (version 4.8).

For molecular identification, the internal transcribed spacer (ITS) regions were sequenced. Genomic DNA was extracted from the endophytic fungi using the cetyltrimethylammonium bromide (CTAB) method [20]. The ITS regions were amplified via polymerase chain reaction (PCR) with the universal primers ITS1 (5′-CTTGGTCATTTAGACGAAGTAA-3′) and ITS4 (5′-GCATATCAATAAGCGGAGGA-3′) [17]. PCR products were verified by 1% agarose gel electrophoresis and subsequently sequenced by Shanghai Bioengineering Company (Shanghai, China). The obtained sequences were analyzed using the Basic Local Alignment Search Tool (BLAST, version 2.13.0, https://blast.ncbi.nlm.nih.gov, accessed on 15 October 2024). Phylogenetic trees were constructed from the ITS sequences employing the neighbor-joining method implemented in MEGA software (v11.0).

2.3. Characterization of Alkali Tolerance Induced by Endophytic Fungal Inoculation

The alkali tolerance conferred by endophytic fungal inoculation was assessed in seedlings of maize (Zea mays) and soybean (Glycine max). The plants were subjected to alkali stress through irrigation with NaHCO_3_ solutions at concentrations of 50 mM (mild stress) and 80 mM (intense stress). At the seedling emergence stage, each of the two NaHCO_3_ concentration groups was further divided into four subgroups based on the timing of endophytic fungi inoculation yielding a total of eight distinct experimental treatments (2 concentrations × 4 inoculation timings). The four inoculation subgroups with the same NaHCO_3_ concentration were defined as follows: C (Control): Irrigation with NaHCO_3_ solution only (no endophytic fungi inoculation); IP1 (Inoculation protocol 1): Inoculation with endophytic fungi three days post NaHCO_3_ exposure; IP2: Inoculation three days prior to NaHCO_3_ exposure; IP3: Simultaneous inoculation and NaHCO_3_ exposure. For each combination of NaHCO_3_ concentration and endophyte fungal strain, a minimum of three seedlings were analyzed. Following a 20-day treatment period, root morphological parameters were quantified using a root scanning system (ScanMaker i800 Plus, Microtek, Shanghai, China).

2.4. RNA Extraction and Illumina RNA-Seq

The endophytic fungi strains WDR2 and WDR5 were cultivated under mild alkali stress conditions (pH = 9.0) and control conditions (pH = 7.0), respectively. Following a 7-day culture period at 160 rpm, mycelia samples were harvested for RNA-Seq analysis, with the pH 7.0 samples serving as the controls. Total RNA was extracted from the mycelial samples using the SPARKeasy Total RNA Rapid Extraction Kit (Sikejie Biotechnology, Binzhou, China). RNA concentration and purity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA), and RNA integrity was verified by agarose gel electrophoresis through the clear visualization of sharp, distinct ribosomal RNA (18S and 28S) bands on the agarose gel. Subsequently, a transcriptome library was constructed from 5 μg of total RNA using the Illumina Truseq^TM^ RNA sample Prep Kit (Illumina, San Diego, CA, USA). This library was then sequenced on the Illumina Novaseq 6000 sequencing platform ** **(Illumina, San Diego, CA, USA), generating 150 bp paired-end reads.

2.5. Raw Data Processing, Reads Mapping, and Differentially Expressed Gene Analysis

Raw sequencing reads in FASTQ format were generated through base calling using bcl2fastq (v1.8.4). RNA-Seq reads were aligned to the reference genome employing TopHat (v2.1.1) [21]. Differential gene expression analysis was performed using DESeq2 (version 1.38.3) [22], with genes meeting the criteria of |log_2_ Fold change| ≥ 2 and an adjusted p-value < 0.05 classified as significantly differentially expressed genes (DEGs). Functional enrichment analyses of the identified DEGs were conducted utilizing Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, with significantly enriched terms and pathways (significance threshold: p < 0.05).

2.6. Quantitative Real-Time PCR Validation

RT-qPCR was performed to validate the RNA-Seq results. Total RNA was extracted from mycelia samples as described in Section 2.4, and cDNA was synthesized using the RevertAid Master Mix Kit (Thermo Fisher Scientific, Waltham, MA, USA). The RT-qPCR amplifications were conducted using the 2× SYBR Green qPCR Master Mix II (Universal) (Seven Innovation Biotechnology, Beijing, China) with gene-specific primers for ten DEGs, listed in Table S1. The thermal cycling protocol consisted of an initial denaturation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 30 s. For each DEG, three biological replicates and three technical replicates were analyzed to obtain cycle threshold (CT) values. 18S rRNA gene was used as the reference. Relative gene expression levels were calculated using the 2^−ΔΔCt^ method [23]. For direct comparison with the RNA-Seq data, the results are presented as log_2_ (fold changes).

2.7. Statistical Analysis

Statistical analyses were conducted using GraphPad Prism software (v8.0.2) (GraphPad Software, San Diego, CA, USA). Error bars presented in the figures correspond to standard deviations (SD). Significant differences between mean values were determined using Student’s t-test for pairwise comparisons or one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons. Statistical significance was defined as p < 0.05.

3. Results

3.1. Screening of Alkali-Resistant Endophytic Fungi

A total of thirty-nine wild soybean endophytic fungi were screened by measuring mycelia dry weight under mild (pH 9.0) and intense (pH 11.0) alkali stress conditions. Significant inter-strain variability was observed in mycelial dry weight following ten days of exposure to both stress levels. From this screening, twelve alkali-tolerant strains—WDS-6, Y2S4, Y6L-14, ZX-WDS4, WDR2, Z1L-18, Y6R1, Y6R15, WDR5, Z1R1, Y2S1, and Y6S-26—were identified based on their increased mycelia dry weight under alkaline conditions (Figure 1). Notably, strains WDR2 and WDR5 demonstrated notable alkali tolerance under both pH treatments. Specifically, after ten days of mild alkali stress, the mycelial dry weight of WDR2 and WDR5 increased by 24.8% and 24.3%, respectively, compared to the control. Under intense alkali stress, their biomass increased by 25.8% and 25.6%, respectively. The hyphal dry weight data for the remaining 27 alkali-sensitive endophytic fungi from wild soybean subjected to mild and intense alkali stresses for durations of 3, 7, and 10 days are presented in Figure S1. Based on their superior alkali tolerance observed in these initial screenings, the subsequent in-depth alkali resistance assessment and transcriptomic analyses were focused on the two most promising isolates, WDR2 and WDR5.

3.2. Identification of Alkali-Resistant Endophytic Fungi

Morphological characterization of the two alkali-tolerant endophytic strains, WDR2 and WDR5, was performed. Strain WDR2 displayed a flat, milky white colony with a smooth surface, scarce mycelial growth, and colorless exudates. In contrast, strain WDR5 formed flat, light-yellow colonies with a rough surface, limited mycelium, and distinctive white, shiny spots. Microscopic examination of both strains revealed the presence of septate hyphae and elliptical spores (Figure 2a,b). The microscopic features of the ten additional alkali-resistant endophytic strains isolated from wild soybean are provided in Figure S2.

To elucidate the taxonomic classification and evolutionary relationships of the isolated alkali-tolerant strains WDR2 and WDR5, a phylogenetic analysis was conducted using the internal transcribed spacer (ITS) region, a widely accepted genetic marker for fungal identification. The resulting cladogram clearly delineates the genetic affiliations of both endophytic fungal strains within the Fusarium genus (Figure 2c).

3.3. Effects of the WDR2 and WDR5 Strains Inoculations on Plant Root Systems Under Alkali Stress

To evaluate the impact of endophytic fungi on root system architecture under alkali stress conditions, maize plants were inoculated with fungal strains WDR2 and WDR5 subjected to both mild (50 mM NaHCO_3_) and intense (80 mM NaHCO_3_) alkali stress conditions. Key parameters related to root architecture—including total root length, root dry weight, root average diameter, root surface area, root volume, and root-shoot ratio—were measured across different inoculation timings (T1–T3). Under mild alkali stress, inoculation with either fungal strain significantly mitigated the inhibitory effects on root growth compared to the uninoculated control (T0). Specifically, WDR2 treatment elicited a marked increase in all root-related physiological parameters, except root average diameter, across all inoculation timings (T1, T2, T3) (Figure 3a). A comparable positive effect was observed for strain WDR5 under mild stress conditions (Figure 3b), with at least one inoculated group (T1–T3) demonstrating improvements in all measured root parameters, excluding root average diameter, compared to the T0 control.

In contrast, under intense alkali stress, the beneficial effects of fungal inoculation exhibited greater variability and appeared to be dependent on the timing of treatment (Figure 3c,d). Notably, the T1 inoculation treatment yielded the most pronounced improvements, achieving the highest values for the majority of root parameters for both WDR2 and WDR5, indicating the efficacy of post-stress inoculation. Collectively, these findings indicate that endophytic fungal strains WDR2 and WDR5 enhance maize tolerance to alkali stress by promoting root system development, with post-stress inoculation (T1) representing the most effective strategy under intense alkali stress conditions.

To evaluate host-specific responses to these endophytic fungi, the same experimental design was applied to soybean plants, and outcomes were compared with those observed in maize. As illustrated in Figure S3, inoculation with either fungal strain generally alleviated alkali stress in soybean; however, the effectiveness was highly dependent on the strain, stress intensity, and inoculation timing.

3.4. Transcriptomic Profiles of WDR2 and WDR5 in Response to Alkali Stress

To investigate the potential molecular mechanisms responsible for the differential microbial alkali tolerance observed in microbial species, high-throughput transcriptional profiling was conducted on the endophytic fungi WDR2 and WDR5 under a seven-day treatment of mild alkali stress (pH 9.0) using the Illumina Novaseq 6000 platform. Following the removal of adaptor sequences and low-quality reads, an average of over 4.36 × 10^7^ clean reads per sample were obtained. The total length of these clean reads reached 5.12 × 10^10^ nucleotides, with an average GC content of 52.72%. Base-calling accuracy was notably high, with over 97.72% of bases achieving a Q20 score and 93.62% reaching Q30. Furthermore, approximately 89.48% of the clean reads from each sample were successfully aligned to the reference genome of Fusarium verticillioides (Figure 4a). Among these aligned reads, over 88.38% of the mapped reads were uniquely aligned, with 82.55% specifically mapped to the coding sequence (CDS) regions (Figure 4b,c). Subsequently, a total of 16,867 unigenes were annotated against one or more public databases. Of these, 10,589 unigenes, representing 62.78% of the total assembled unigenes, matched entries in the GO database. Additionally, 4548 unigenes (26.96%), 14,405 unigenes (85.40%), 16,243 unigenes (96.30%), 9246 unigenes (54.82%), and 10,952 unigenes (64.93%) demonstrated significant sequence similarity to entries in the KEGG, COG, NR, Swiss-Prot, and Pfam databases, respectively (Figure 4d). Notably, 3812 unigenes, accounting for 22.60% of the total, showed high similarity across all six public databases.

Applying the thresholds of |log_2_ Fold change| ≥ 2 and an adjusted p-value < 0.05, a total of 727 genes were identified as differentially expressed in response to mild alkali stress. Specifically, in the endophytic fungus WDR2, 589 differentially expressed genes (DEGs) were detected, including 119 upregulated and 470 downregulated genes. In contrast, the endophytic fungus WDR5 exhibited 182 DEGs, comprising 42 upregulated and 140 downregulated genes under identical stress conditions (Figure 4e and Tables S2 and S3). Additionally, 43 DEGs were common to both strains, suggesting a conserved transcriptional response to alkali stress conditions (Figure 4f). Conversely, under both neutral and alkaline conditions, WDR2 and WDR5 exhibited distinct gene expression patterns, indicating strain-specific responses potentially mediated by divergent genes or molecular pathways.

3.5. Functional Enrichment Analysis of Alkali Stress Induced DEGs in Endophytic Fungi WDR2 and WDR5

To understand the biological roles of these DEGs in response to mild alkali stress in the endophytic fungi strains WDR2 and WDR5, we annotated the identified DEGs by using GO function terms. Only one GO term—"integral component of membrane” (GO: 0016021) within the cellular component (CC) category—was significantly enriched in the WDR2 strain at a false discovery rate (FDR) threshold of less than 0.05 (Table S4). Conversely, in the WDR5 strain, one biological process (BP) term, “phosphate ion transport” (GO:0006817), along with two molecular function (MF) terms, “transmembrane transporter activity” (GO:0022857) and “glycerol kinase activity” (GO:0004370), reached significance at this threshold (Table S5). When applying a less stringent significance criterion (p < 0.05), five GO terms were commonly enriched across both strains. These included the BP term “phosphate ion transport” (GO:0006817), the CC term “fungal-type cell wall” (GO:0009277), and the MF terms “protein dimerization activity” (GO:0046983), “heme oxygenase (decyclizing) activity” (GO:0004392), and “urate oxidase activity” (GO:0004846) (Figure 5a,b). Moreover, to pinpoint specific genetic players, we extracted the common DEGs associated with these shared GO terms (Table S6). Among these, three DEGs (FVEG_12069, FVEG_08445 and FVEG_03134) were annotated as high-affinity phosphate transporters, one DEG (FVEG_06538) was characterized as a structural constituent of the cell wall, one DEG (FVEG_10312) was identified as uricase, one DEG (FVEG_04784) was annotated as heme oxygenase, and one DEG (FVEG_13127) was associated with protein dimerization function and identified as a homolog of sterol regulatory element-binding proteins. These findings indicate that the DEGs from both endophytic fungi, including these specific candidate genes, participate in a range of shared cellular and physiological processes, mediating responses to mild alkali stress.

To further explore the functional implications of the DEGs identified under mild alkali stress, KEGG pathway enrichment analysis was performed for both WDR2 and WDR5 strains. The results revealed that the majority of alkali-responsive DEGs in both fungi were associated with metabolic pathways (Figure 5c,d; Tables S7 and S8). Specifically, no KEGG terms were significantly enriched among the DEGs of both WDR2 and WDR5 strains at an FDR threshold of less than 0.05. However, when a less stringent criterion (p < 0.05) was employed, nine and seven KEGG pathways were found to be significantly enriched in the WDR2 and WDR5 strains, respectively. In the WDR2 strain, the most significantly enriched pathway was “Amino sugar and nucleotide sugar metabolism”, followed by “Mannose type O-glycan biosynthesis” and “Galactose metabolism”. Conversely, in the WDR5 strain, the top enriched pathways included “Glycerolipid metabolism”, “Glutathione metabolism”, and “Thiamine metabolism”. Collectively, these results reveal distinct metabolic and regulatory adaptations to mild alkali stress between the two strains, suggesting species-specific mechanisms underlying alkali tolerance. These insights contribute to a deeper understanding of the molecular strategies employed by endophytic fungi to adapt to environmental stressors.

3.6. Validation of RNA-Seq Results by RT-qPCR

To validate the RNA sequencing results, ten DEGs were randomly selected for RT-qPCR analysis. The selected DEGs represented various functional categories, including stress and signaling (e.g., SOD1, SOD2, fhkC, and pzh1), carbohydrate and amino acid metabolism (e.g., LEU2, leu2A, fer4, and BGL1B), and protein turnover (e.g., srpA and SPAC3C7.07c). The selection included both up-regulated and down-regulated DEGs to objectively verify the transcriptomic expression trends of candidates related to alkali tolerance. The RT-qPCR results corroborated the RNA-Seq findings, thereby confirming the reliability of the sequencing analysis. Specifically, under alkali stress, RT-qPCR verified the upregulation of two genes (fer4 and LEU2) and the downregulation of six genes (fhkC, sprA, SPAC3C7.07c, leu2A, pzh1, and BGL1B) in the WDR2 strain (Figure 6a). Similarly, in the WDR5 strain, RT-qPCR validated the upregulation of two genes (SOD2 and pzh1) alongside the downregulation of six genes (fhkC, sprA, SPAC3C7.07c, LEU2, SOD1 and BGL1B) (Figure 6b).

4. Discussion

Soil alkalinity poses a major challenge to global agriculture productivity, necessitating the development of sustainable solutions to improve crop resilience. In this study, twelve alkali-tolerant endophytic fungal strains were isolated and characterized from wild soybean. Notably, strains WDR2 and WDR5 exhibit pronounced tolerance, as demonstrated by notable increases in mycelial dry weight under both mild (pH 9.0) and intense (pH 11.0) alkaline conditions. Subsequent inoculation experiments revealed that these strains help alleviate alkali stress in maize and soybean by promoting root system development. Additionally, transcriptomic analyses were conducted to uncover the molecular mechanisms underlying the alkali stress adaptation of WDR2 and WDR5. The findings of this study hold significant potential for developing novel bio-inoculants derived from endophytic fungi to enhance crop tolerance to alkali stress.

In the present study, morphological and molecular analyses identified the WDR2 and WDR5 strains as F. verticillioides. This finding presents a paradox, given that F. verticillioides is predominantly recognized as a highly destructive fungal pathogen affecting maize and other cereal crops. Nonetheless, the finding is consistent with our previous reports of stress-resistant endophytic fungi Fusarium sp. isolated from wild soybeans in cold regions of China [17]. Similarly, another study identified a Fusarium sp. as the dominant endophyte in the roots of Dendrobium moniliforme [24]. Although the majority of Fusarium species are pathogenic, some are capable of existing as symptomless endophytes. Under the experimental conditions of this study, both strains promoted plant growth without inducing disease symptoms, indicating a mutualistic rather than pathogenic interaction. This shift in lifestyle from pathogenicity to mutualism is likely influenced by host genotype, environmental conditions, and fungal genetic makeup, which highlights the context-dependent nature of plant-fungal interactions. Moreover, the identification of both morphologically distinct strains (e.g., differing in hyphae septation) as the same species underscores the considerable genetic and phenotypic plasticity inherent within F. verticillioides.

The present study reveals that inoculation with the fungal strains WDR2 and WDR5 enhances alkali tolerance in maize, primarily through improvements in root architecture. Under mild stress, the consistent positive effects observed across all inoculation timings suggest the presence of a robust plant-growth-promoting mechanism. Conversely, under intense alkali stress, the effectiveness of inoculation was contingent upon timing; the IP1 treatment (post-stress inoculation) proved most effective, implying that these fungi function more effectively as mitigators of stress-induced damage rather than as agents of pre-emptive priming under extreme conditions. This timing-dependent effect has important practical implications for application strategies. Additionally, the positive, albeit variable, effects observed in soybean indicate that the plant-growth-promoting traits of WDR2 and WDR5 are not strictly host-specific, highlighting their potential utility as broad-spectrum bio-inoculants for improving crop resilience in alkaline soils.

Beyond modulating root architecture, endophytic fungi employ additional strategies to enhance host plant tolerance to alkali stress. For example, under alkaline conditions, Epichloë endophytes upregulated the expression of the HbNHX1 gene in the host plant Hordeum bogdanii, facilitating the sequestration of Na^+^ ions within vacuoles. This sequestration mitigated sodium toxicity and contributed to an increase in K^+^ and related anions, thereby alleviating the effects of alkaline stress [11]. In a separate study, the mycobiont Endocarpon pusillum was observed to secrete D-mannitol—a polyol—into the hyphal networks of its host Diplosphaera chodatii. This transfer enhanced cellular viability to 78.3% under severe saline–alkali conditions, indicating an osmoprotective mechanism [10]. To further elucidate the molecular regulatory mechanisms underlying alkali tolerance, we conducted transcriptomic analyses on two endophytic fungi, WDR2 and WDR5. The identification of five GO terms exhibiting significance at a threshold of p-value less than 0.05 reveals a conserved set of functional responses shared by both strains. The mutual enrichment of terms such as “phosphate ion transport” and the “fungal-type cell wall” highlights the critical roles of ion homeostasis and cell wall remodeling in alkaline adaptation in these fungi. Furthermore, the common overrepresentation of activities associated with protein dimerization, heme oxygenase, and urate oxidase implies that both strains utilize shared pathways involving protein complex assembly and oxidative stress regulation, potentially mediated through the catabolism of heme and purine compounds.

Additionally, our transcriptomic analysis revealed a distinct divergence in the gene expression responses of the endophytic fungi WDR2 and WDR5 under alkali stress. WDR2 exhibited a substantially greater number of DEGs, suggesting a broader transcriptomic reprogramming that may underlie its enhanced tolerance to alkali conditions. Although a limited subset of 43 DEGs was shared by both strains, indicating the presence of shared fundamental stress response mechanisms, the majority of DEGs were strain-specific. This finding highlights that different genetic pathways predominantly contribute to the differential resilience observed between the two strains. This distinction is further corroborated by functional enrichment analysis. Under a stringent FDR threshold for GO analysis, WDR2 demonstrates significant enrichment exclusively for the term “integral component of membrane”, suggesting a potential role in preserving cellular integrity and regulating membrane-associated processes, which may serve as an initial defense mechanism against ionic and osmotic disturbances. In contrast, WDR5 shows significant enrichment for “phosphate ion transport” and related transporter activities, indicating a possible specialization in phosphate homeostasis, a crucial factor for pH buffering, energy metabolism, and signal transduction under alkaline conditions. Moreover, WDR2 exhibits significant enrichment in pathways related to amino sugar and galactose metabolism in KEGG analysis. This enrichment likely facilitates cell wall reinforcement, as amino sugars (e.g., N-acetylglucosamine) and nucleotide sugars (e.g., UDP-glucose) serve as essential precursors for synthesizing key fungal cell wall components such as chitin and glucans [25,26]. Under high pH stress, alkaline ions disrupt hydrogen bonding and electrostatic interactions within the cell wall polysaccharide network, leading to structural loosening and increased permeability. The observed upregulation of genes within this pathway is therefore posited to enhance chitin and glucan biosynthesis and cross-linking, thereby increasing cell wall thickness and rigidity [27]. This structural reinforcement strengthens the cell wall, effectively resisting alkaline-induced damage to its integrity and ultimately promoting fungal survival under extreme alkaline conditions. Conversely, WDR5 shows enrichment in glycerolipid and glutathione metabolism pathways, suggesting a strategy focused on mitigating oxidative stress and preserving membrane integrity. High pH disrupts electrostatic interactions between membrane lipids and proteins, increasing membrane fluidity and permeability [28]. An expanded glutathione pool potentially fuels the ascorbate-glutathione cycle for reactive oxygen species (ROS) scavenging and provides substrate for glutathione S-transferases (GSTs) to detoxify stress-induced lipid peroxidation products [29]. Concurrently, alterations in glycerolipid metabolism suggest membrane remodeling to maintain optimal fluidity and integrity. This may involve increased incorporation of saturated fatty acids or a regulated balance between bilayer-stabilizing phospholipids and non-bilayer-forming lipids to minimize leakiness under alkaline stress, a mechanism documented in response to other abiotic stressors [30]. Consequently, the differential alkali tolerance between these two strains appears to arise from their reliance on different defense strategies—structural reinforcement in WDR2 versus metabolic and redox adjustments in WDR5. Both strategies were observed in an early microarray analysis of the maize pathogenic fungus Ustilago maydis, which revealed that its response to mild alkaline stress (pH 9.0) involves modifications to both the cell wall and plasma membrane through altered polysaccharide, lipid, and protein metabolism. This adaptive response is likely mediated by the Pal/Rim signal transduction pathway [31].

5. Conclusions

In conclusion, this study successfully isolated and characterized alkali-tolerant endophytic fungi from wild soybean. Among the initial isolates, two strains, designated WDR2 and WDR5, identified as Fusarium verticillioides, were selected as the most promising candidates due to their robust biomass accumulation under alkali stress. These endophytes significantly enhanced plant tolerance to alkali stress, as demonstrated by inoculation assays on maize, which showed a marked alleviation of stress-induced inhibition of root system development. Furthermore, the observed effects exhibited host specificity, varying according to plant species, fungal strain, stress intensity, and inoculation timing. Transcriptomic analyses provided fundamental insights into the molecular mechanisms underlying this tolerance, revealing predominantly strain-specific adaptive responses. Both strains demonstrated multiple common mechanisms associated with alkaline adaptation, encompassing the preservation of ion homeostasis, remodeling of the cell wall, and regulation of protein complex assembly alongside oxidative stress responses. Additionally, the WDR2 strain showed notable enrichment in the biological process related to cellular integrity and membrane modulation and the metabolism of amino sugar and nucleotide sugars. Conversely, the WDR5 strain exhibited enrichment in functions associated with phosphate ion transport and related transporter activities, glycerol kinase activity, and glycerolipid and glutathione metabolic pathways. Collectively, these findings not only highlight the potential of wild soybean-derived endophytes like WDR2 and WDR5 as novel bio-inoculants to enhance crop resilience in saline-alkaline soils but also delineate their unique molecular adaptations, thereby paving the way for future mechanistic investigations and applied agricultural research.