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

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 (<i>Glycine soja</i>), 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 <i>Fusarium verticillioides</i> through comprehensive morphological and molecular analyses. Subsequent inoculation assays conducted on maize (<i>Zea mays</i>) 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.