Taxonomic Diversity, Functional Guilds, and Spatial Distribution of Endophytic Fungi in Healthy Spinach via Amplicon Sequencing

Endophytic fungi, residing within plant tissues without causing disease, play critical roles in plant health and ecosystem dynamics. This study characterized the taxonomic composition, diversity, functional guilds, and spatial distribution of endophytic fungal communities in healthy spinach (Spinacia oleracea L.) leaves using ITS1 amplicon sequencing. Surface-sterilized leaf samples were processed for DNA extraction, followed by Illumina MiSeq sequencing. Sequences were clustered into 24 operational taxonomic units (OTUs) using QIIME2 and the UNITE database. Fusarium spp. dominated (77.23% of reads), followed by Thanatephorus cucumeris (19.90%) and Plectosphaerella spp. (2.73%). Alpha diversity analysis revealed moderate richness (24 OTUs) but low evenness (Shannon index: 2.097, Simpson index: 0.702). FUNGuild annotation assigned ecological roles to 21 OTUs, with saprotrophs comprising 94.82% of abundance, symbiotrophs 0.04%, and other guilds 5.14%. Multifunctional taxa, such as Fusarium (saprotroph-pathotroph-endophyte), were prevalent. Dispersion analysis revealed a highly aggregated community (VMR: 53,647.54, Morisita’s Index: 6.76), characterized by the dominance of specific genera. Rank-abundance curves and guild distribution visualizations, generated using R (ggplot2), highlighted Fusarium dominance and saprotrophic prevalence. Most taxa exhibited filamentous morphology, aligning with saprophytic strategies. These findings suggest a low-diversity, highly aggregated fungal community with saprotrophic dominance, which may potentially influence spinach health. This study provides a foundation for understanding endophytic fungal ecology in agricultural systems, with implications for crop management.

High-throughput sequencing revealed that Fusarium spp. and Thanatephorus cucumeris dominate the endophytic fungal community in healthy spinach leaves, comprising over 97% of the total abundance. The overwhelming presence of Fusarium spp. and T. cucumeris in the spinach endosphere suggests these fungi may be functionally significant in maintaining plant health or enhancing resilience, despite Fusarium's common association with pathogenicity. Their prevalence in asymptomatic tissues implies a potential shift from a pathogenic to an endophytic or mutualistic lifestyle under certain environmental or physiological conditions.

Comparable patterns of Fusarium dominance have been observed in endophytic communities of other crops. Imazaki and Kadota (2015) reported that the majority of endophytic Fusarium isolates from tomato stems belonged to the F. oxysporum, F. fujikuroi, and F. solani species complexes, which were also prevalent in surrounding soils. Similarly, Mamaghani et al. (2024) and Osman et al. (2025) identified Fusarium spp. as one of the most abundant endophytes in healthy potato and medicinal plants, respectively, across different agro-climatic regions, with several strains demonstrating mycotoxin production potential. These findings collectively indicate that Fusarium spp. are widespread and adaptable fungal endophytes in various plant hosts (Asim et al., 2022; Seema et al., 2023; Ravoori et al., 2024).

The coexistence of Fusarium spp. and T. cucumeris as dominant fungi in healthy spinach leaves suggests they may participate in complex plant–fungal interactions. Their mutualistic roles could include modulating host immune responses, facilitating nutrient acquisition, or competing with pathogenic microbes. Such interactions may suppress visible disease symptoms, thereby maintaining an asymptomatic but colonized state.

Thanatephorus cucumeris, the teleomorph of Rhizoctonia solani, is a well-documented soil-borne phytopathogenic basidiomycete responsible for considerable yield losses in numerous economically important crops worldwide (Fernandes et al., 2022; Akber et al., 2023; Shaheen et al., 2023; Jash & Sarkar, 2025). In foliar vegetables, R. solani causes a wide range of symptoms including sheath blight, foliar blight, leaf blight, and leaf spot. Particularly in lettuce, an economically important raw-consumed leafy vegetable, R. solani is known to cause severe diseases such as late sugar beet rot, damping-off, and bottom rot, with potential yield losses of up to 70% (Ohkura et al., 2009; Maneea et al., 2024; Benitez-Andrade et al., 2025). Based on hyphal anastomosis reactions, Rhizoctonia isolates are divided into 13 anastomosis groups (AGs), each having distinct host specificity (Nandeesha et al., 2021; AlHussain et al., 2022; Malcangi et al., 2023; Naqvi et al., 2024). A comprehensive study in Greece revealed multiple Rhizoctonia solani AGs and Pythium spp. associated with damping-off in baby leafy vegetables, showing varying pathogenicity and broad host range (Tziros & Karaoglanidis, 2022; Bulusu & Cleary, 2023; Bolay et al., 2024). Interestingly, despite this known pathogenicity, the dominant presence of T. cucumeris in healthy spinach tissues may reflect a latent, ecologically balanced role that differs from its aggressive behavior in other crops. Its endophytic colonization might therefore represent a shift in its ecological function—from virulence to conditional mutualism—within the spinach microbiome.

Furthermore, Scherwinski et al. (2008) demonstrated that bacterial antagonists can actively suppress R. solani in lettuce with negligible short-term effects on non-target microbial communities, indicating that pathogen behavior is strongly influenced by microbial interactions. This supports the possibility that T. cucumeris, though closely related to a known pathogen, could assume a benign or even beneficial role within a balanced endophytic community.

These findings underscore the need to understand better the dual roles of endophytic fungi, which are traditionally viewed as pathogens. Unravelling the ecological functions of their components within the plant microbiome could aid in developing microbiome-based strategies for crop protection and yield enhancement. Furthermore, assessing the environmental or host-driven triggers that facilitate a transition from endophytism to pathogenicity is critical for managing food safety risks associated with latent toxin producers and ensuring sustainable crop production. 

Alpha diversity discussion

The alpha diversity analysis of the spinach endophytic fungal community revealed moderate diversity, with 24 observed operational taxonomic units (OTUs), a Shannon index of 2.097 and a Simpson index of 0.702, indicating community has several species present, but a few may be more abundant than others, alongside complete sampling coverage (Good’s coverage = 1.000). These metrics suggest a community characterized by limited evenness, dominated by a small number of abundant taxa, notably Fusarium and Thanatephorus. The high Simpson index (approaching 1) indicates low evenness, a pattern consistent with endophytic fungal communities in other crops, such as the Olive plant (Olea europaea L.), where dominant genera similarly drive community structure despite moderate OTU richness (Costa et al., 2021; Pisano et al., 2023). The near-perfect sampling coverage further underscores that this dominance is not an artifact of under sampling but reflects true ecological dynamics.

Fungal endophytes, which asymptomatically colonize plant tissues, occupy a dynamic niche along the mutualist-pathogen continuum, often conferring increased fitness to their hosts through pathogen suppression, stress tolerance, or nutrient acquisition (Dipalma et al., 2022; Grabka et al., 2022). In this study, the high prevalence of Fusarium within the spinach endophytic community aligns with growing evidence that certain Fusarium species function as beneficial endophytes rather than pathogens. For instance, Fusarium proliferatum isolated from Cissus quadrangularis produces phenolics, terpenoids, and unsaturated alkenes that inhibit fungal pathogens like Rhizoctonia solani and Fusarium oxysporum at concentrations as low as 0.2 mg/mL, demonstrating its potential as a biocontrol agent (Singh et al., 2021b; Marian et al., 2024). Similarly, Fusarium oxysporum can act as a root endophyte, providing biocontrol through endophyte-mediated resistance (De Lamo & Takken, 2020; Fiodorova et al., 2022). These antifungal mechanisms underscore Fusarium’s dual ecological role: while often perceived as pathogens, their endophytic strains can enhance host health by suppressing competing pathogens or priming plant immune responses (Singh et al., 2021a; Dongmo & Tamesse, 2022).

The dominance of Fusarium in the spinach endosphere may thus reflect a mutualistic relationship where host plants selectively enrich microbial partners offering protective benefits. Such associations are increasingly recognized in agricultural contexts, where endophyte-mediated pathogen inhibition reduces reliance on synthetic fungicides (Grabka et al., 2022). This parallels findings in medicinal and crop plants, where Fusarium endophytes contribute to host fitness through secondary metabolite production, niche exclusion, or induced systemic resistance—a paradigm shift in understanding this genus’ ecological versatility (Singh et al., 2021a, 2021b).

Trophic mode discussion

Trophic structure through FUNGuild analysis of the spinach endophytic fungal community revealed that saprotrophic fungi, especially Fusarium spp. and Thanatephorus cucumeris, unitedly dominated the community—comprising 97.75% of classified sequence reads—while pathotrophic and symbiotrophic guilds contributed minimally (0.65% and 1.47%, respectively). This striking dominance suggests that decomposition processes within the spinach endosphere are primarily mediated by these saprotrophic taxa, potentially playing a significant role in nutrient cycling despite their occasional secondary pathogenic roles. Similar patterns have been documented in rice roots, where Fusarium spp. were also prevalent and likely instrumental in nutrient recycling, as demonstrated by Pili et al. (2016), who identified Fusarium oxysporum and Gibberella fujikuroi species complexes as major components of the rice root endophytic community; notably, Fusarium oxysporum isolates showed clear divergence between irrigated and upland ecosystems, underscoring the influence of environmental conditions on endophyte community structure. Mechanistically, the saprotrophic predominance of Fusarium and Thanatephorus is likely to facilitate organic matter breakdown in the spinach endosphere, thereby enhancing nutrient availability. At the same time, their pathogenic traits remain suppressed in healthy plants. This mechanism is further supported by Pili et al. (2016), who observed the widespread adaptability and functional importance of Fusarium spp. across diverse rice agroecosystems. The predominance of saprotrophic fungi in the spinach endosphere underscores the potential for harnessing these taxa to enhance nutrient cycling, which could inform sustainable agricultural practices and biocontrol strategies. Moreover, as emphasized by Pili et al. (2016), endophytic fungi represent a valuable resource for future searches for biological control and growth-promoting agents, reinforcing their ecological and agronomic relevance.

High-throughput sequencing (HTS)

HTS has enabled a detailed and accurate assessment of the endophytic fungal community structure, overcame the limitations of culture-dependent methods and revealed the true ecological diversity within plant tissues. In the current analysis of spinach, the functional guild composition of endophytic fungi was overwhelmingly dominated by mixotrophic modes—specifically, pathotroph-saprotroph-symbiotroph (141,622 reads, 12 OTUs) and pathotroph-saprotroph combinations (35,418 reads, 6 OTUs)—which together accounted for 177,040 reads (99.99%) across 18 OTUs. In contrast, only 19 reads (2 OTUs) were assigned exclusively to the saprotroph guild (0.01%). The key taxa contributing to this dominance were Fusarium spp. (136,749 reads, 77.23%) and Thanatephorus cucumeris (35,227 reads, 19.89%), reflecting their ecological versatility and adaptability to complex plant microenvironments. In contrast, fungi exhibiting a single trophic mode, such as saprotrophs, were exceedingly rare, contributing only 19 reads (0.01%) from Leptospora sp. and Nigrospora vesicularifera. Additionally, a small fraction of OTUs (~0.011%) remained unclassified due to insufficient taxonomic resolution, highlighting the current limitations of functional annotation databases like FUNGuild. This mixotrophic dominance aligns with findings of Wang et al. (2022) in case  Sophora alopecuroides, where saprotrophs and mixotrophs were also prevalent, and supports the notion that endophytic fungi often possess flexible ecological strategies to thrive in diverse and fluctuating internal plant environments The presence of mixotrophs suggests that these fungi can shift between nutritional modes—acting as decomposers, pathogens, or mutualists—depending on the physiological state of the host tissue and environmental conditions. Such versatility may be crucial for their survival and ecological function, enabling them to contribute to nutrient cycling during plant senescence or stress, while remaining benign or even beneficial during healthy plant growth. This dynamic functional structure underscores the importance of considering both community composition and ecological guilds when evaluating the roles of endophytic fungi in plant health, stress resilience, and potential biotechnological applications (Wang et al., 2022).

Aggregation patterns

The high values across all aggregation indices confirm that the endophytic fungal community in spinach is strongly clustered, with dominant taxa such as Fusarium and Thanatephorus forming localized aggregations. These fungi, known for their multi-trophic versatility, can function as saprotrophs, pathogens, or mutualists, depending on the plant's physiological conditions. This aligns with ecological theories suggesting that trophic plasticity supports niche dominance in competitive environments such as the plant endosphere.

Such functional and spatial clustering likely enhances resource capture, colonization efficiency, and resilience to environmental fluctuations. For instance, Morisita's Index (6.76) and an exceptionally high VMR (53,647.54), along with Lloyd’s Index of Patchiness (62,499.49), indicate that these fungi do not distribute randomly but instead occupy specific ecological niches, possibly as a survival or competitive strategy. Similarly, the remaining three indices reflect the same trend.

This interpretation is further validated by Griffin et al. (2001), who used Lloyd’s Index of Patchiness to assess the spatial distribution of Aspergillus flavus and A. niger in peanut fields with a history of aflatoxin contamination. Their values ranged from 2.32 to 6.55, representing a moderate to high aggregation of these fungi. Importantly, they linked such aggregation to pathogen persistence and environmental risk zones within agricultural fields. Compared to Griffin et al. (2001) the orders-of-magnitude higher Lloyd’s value in the present study reflects much stronger patchiness, likely due to the compartmentalized niche structure of the endosphere and the active colonization strategies of endophytes.

This finding is also consistent with earlier studies in wheat (Gdanetz & Trail, 2017), where Fusarium exhibited multitrophic behavior and clustered colonization patterns. Their combined saprotrophic and pathogenic capacities enable them to persist during both healthy and senescent phases of plant growth, thereby participating in nutrient cycling while maintaining their ecological presence.

CONCLUSION

This study analysed endophytic fungal communities in healthy spinach leaves. It revealed a low-diversity, highly aggregated community. Fusarium spp. dominated at 77.23%, followed by Thanatephorus cucumeris at 19.90%. ITS1 amplicon sequencing confirmed these results. The community showed moderate species richness with 24 OTUs. Low evenness was indicated by Shannon (2.097) and Simpson (0.702) indices. These taxa exhibited mixotrophic behaviors, primarily saprotrophic. Secondary pathotrophic and symbiotrophic roles were also observed. FUNGuild analysis determined saprotrophs accounted for 94.82% of abundance. This suggested a critical role in nutrient cycling. It potentially enhanced plant resilience. The study identified a highly aggregated spatial distribution (VMR: 53,647.54, Morisita’s Index: 6.76). This indicated niche-specific colonization. The ecological versatility of the dominant genera likely drove this pattern. The findings challenged the view of Fusarium and Thanatephorus as solely pathogenic, and proposed potential mutualistic roles in asymptomatic spinach. The study established a foundation for understanding trophic plasticity in mixotrophic endophytic fungi. It highlighted their role in nutrient cycling and functional shifts. It also provided a basis for exploring spatial colonization and cross-kingdom microbial interactions, specifically with bacteria present in the endosphere. These will inform advanced microbiome management and food safety strategies for leafy greens. Future studies will investigate environmental and host factors that facilitate the transition of trophic structure and optimize biotechnological applications.

ACKNOWLEDGMENTS: The authors sincerely thank the Principal of Jagannath Kishore College, Purulia, India, for providing laboratory facilities for this research. We want to express our sincere gratitude to the Department of Computer Science of the same institution for providing access to the computer laboratory. Special thanks are extended to Mr. Sandip Sao and Mr. Ranjan Kumar Misra for their technical support and assistance throughout the study.

CONFLICT OF INTEREST: None

FINANCIAL SUPPORT: None

ETHICS STATEMENT: All procedures involving the collection and processing of plant material adhered to institutional, national, and international ethical guidelines. Spinach seeds were procured from a local seed shop, commonly used for field cultivation, and were grown in pots under controlled conditions. No endangered or protected plant species were involved in this study. Furthermore, no experiments involving humans or animals were conducted. Endophytic fungal species were identified through DNA extracted using a commercially available and internationally recognized DNA isolation kit.

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