Exploration and Comparison of High-Throughput Sequencing Analysis of Endophytic Fungal Communities in Morinda tinctoria and Pithecellobium dulce

Abstract

Fungal endophytes can be identified in a wide range of plant species which help to protect from both abiotic and biotic stressors. This research focused on using high-throughput sequencing (HTS) analysis to gain insight into the foliar endophytic fungal diversity between Morinda tinctoria and Pithecellobium dulce. The study obtained a total of 118,547 sequencing reads, which were grouped into 266 Operational Taxonomic Units (OTUs) with a 97% similarity threshold. M. tinctoria had more OTUs than P. dulce. Alpha diversity results show that both plant species support varied microbial communities with similar but distinct biodiversity profiles. The Shannon index revealed that M. tinctoria had considerably more fungal diversity than P. dulce. The correlation matrix and PCoA depicts the pairwise correlations between several soil metrics such as the total nitrogen level, entire phosphorus, overall potassium, and the electrical conductivity, total carbon from organic matter, pH levels, manganese, iron, zinc, copper, and boron. The OTUs were classified into 5 phyla, 18 classes, 40 orders, 70 families, and 36 genera, where the phylum Ascomycota has a relative abundance of (50–55%), followed by Basidiomycota at (55–60%). The most abundant genera were Wallemia (30–35%), Saitozyma (30–40%), and Talaromyces (20–25%), with average relative abundances. Unassigned genera show a significant proportion of fungal taxa that are still taxonomically unclear. A comparative analysis has been performed between the two plants, M. tinctoria has a higher fungal diversity, which is frequently associated with increased ecological stability, disease resistance, and better functional relationships with the host plant.

1. Introduction

Plants encompass a vast range of microorganisms, including fungi, which have both positive and detrimental consequences. The mycobiome is constituted of the microbial populations that exist within a living thing’s microhabitat and contribute to its metagenome [1,2]. Endophytes are microorganisms which invade the tissues of plants without apparent detrimental effects [3]. During their life cycle, they dwell within the plant tissue of host trees without causing significant harm. Furthermore, endophyte-produced bioactive substances contribute significantly to host plant productivity [4]. Endophytes, as one together, can boost plant nutrient intake while also protecting host plants from biotic and abiotic stressors. The endophytes of fungi have been utilized to extract antibacterial substances and generate antibiotics. Endophytic fungal species have been identified in a wide range of plant varieties [5,6].

Using omics data to analyze endophyte communities in plant hosts offers fresh insights into the environmental and microbial factors affecting plant health and genetic differentiation. It has become widely recognized as a next-generation sequencing (NGS) technology, assisting scientists in discovering and understanding the variance, function, as well as emergence of uncultivated microbes in different environments or ecosystems [7]. High-throughput sequencing is recognized as an excellent approach to identifying the nucleic acids among uncultivated fungi in various habitats. HTS reanalysis of plant-associated DNA sequences can reveal the patterns of microbe co-occurrence inside plant hosts, aid in infection pathogen tracking, and anticipate the ecological functions of endophytes. These findings could lead to the development of new biological control goods for plant disease executive management [8].

Morinda tinctoria, frequently referred to as Indian mulberry, is a variety of plant whose flowers belong to the Rubiaceae category and is native to South Asia. The various M. tinctoria leaf extracts include a wide range of secondary metabolites and have significant antibacterial activity against microorganisms. M. tinctoria plants may be employed to observe interesting natural compounds, which could lead towards the creation of new medications. These chemicals have strong bioactivities and may be beneficial in drug discovery [9]. Pithecellobium dulce (Roxb) is an ancient medicinal plant whose medicinal properties have yet to be thoroughly examined. Pithecellobium species are widespread across the temperate regions of Asia [10]. The herb is thought to be utilized as a conventional treatment for toothaches, gastric ulcers, leprosy, and earaches [11]. Its antibacterial, antidiabetic, anticancer [12] and anti-inflammatory properties have been investigated. The medicinal benefits of P. dulce have prompted various researchers to do in-depth investigations on it. There have been no reports on the identification of endophytic fungal communities present in M. tinctoria and P. dulce. We evaluated and compared the importance of fungal diversity from M. tinctoria and P. dulce, which generated beneficial findings for all of the therapeutic characteristics revealed. Nevertheless, the application of HTS to examine endophytic mycobial communities remains limited. There have been no reports on the identification of fungal communities present in M. tinctoria plant and P. dulce.

This study centers on next-generation sequencing (NGS) platforms for exploring microbial diversity across various environments. Specifically, the fungal endophytic communities associated with the leaves of M. tinctoria and P. dulce were investigated using Illumina MiSeq sequencing, revealing distinct microbial assemblages between the two host species.

As a result, the current study’s objective is to compare the diversity and community composition of endophytic fungi associated with M. tinctoria and P. dulce and analyze the differences in fungal taxonomic profiles between the two host plants using high-throughput sequencing.

2. Materials and Methods

2.1. Sample Collection

The two plant species, Pithecellobium dulce (Roxb.) Benth. (Family: Fabaceae) and Morinda tinctoria Buch. -Ham. (Family: Rubiaceae), were authenticated by the Botanical Survey of India, Southern Regional Centre, Coimbatore (Certificate Nos. BSI/SRC/5/23/2022/Tech/1574 and 1575, dated 21 March 2022).

Both plant samples were collected from Nagamalai Hills, Madurai, at the same developmental stage (fourth-true-leaf stage; about 10–15 leaf bits) to minimize growth-dependent variation in microbial community structure (Figure 1). Each plant species was represented by a single pooled sample of leaves and the corresponding rhizosphere soil collected under identical environmental conditions. The research site is located at an altitude of approximately 150 yards, at 9°54′ N latitude and 78°00′ E longitude, characterized by diverse native flora spread across ~4 km of terrain reaching 1500 ft elevation. Immediately after collection, leaves were placed in sterile polypropylene bags within portable coolers and transported to the laboratory. The samples were gently rinsed with tap water to remove debris, followed by surface disinfection using 2.0% NaCl for 3 min and 70% ethanol for 1 min, with repeated rinsing in sterile distilled water (approximately 15 s per rinse). After surface sterilization, samples were stored at −80 °C until further microbiome analysis. To minimize contamination and technical bias, all sample processing steps were performed under sterile conditions. These ensure methodological transparency and improve reproducibility by eliminating potential sampling and handling bias.

2.2. Soil Analysis

Following leaf collection, soil samples were collected from the same locations and prepared for analysis [13]. Soil pH was measured with a glass electrode [14], and electrical conductivity (EC) was measured by the solubridge method [15]. Organic carbon was estimated using the Walkley and Black wet digestion procedure [16]. Macronutrient analysis included available nitrogen by the alkaline potassium permanganate procedure [17], available phosphorus by extraction with 0.5 M sodium bicarbonate [18], and available potassium using a neutral ammonium acetate extractant [19]. Finally, plant-available micronutrients iron, manganese, copper, and zinc were quantified through the DTPA extraction process [20].

2.3. DNA Extraction and Sequencing

Genomic DNA was isolated from the homogenized leaf material using a QIAGEN soil extraction kit. The extracted DNA’s concentration and purity were initially checked. To target the nuclear ribosomal Internal Transcribed Spacer (ITS) region, we performed PCR using the ITS1 (5′-TTGGTCATTTAGAGGAAGTAA-3′) and ITS2 (5′-GCTGCGTTCTTCATCGATGC-3′) primers, each with a unique barcode. Positive and negative controls were included in all PCR runs to validate the amplification specificity and monitor contamination. PCR amplification was performed with a TAQ Master Mix, running for 30 cycles at 95 °C (45 s), 56 °C (45 s), and 72 °C (60 s), and finalized with a 10 min extension at 72 °C. Amplicons of roughly 400–450 bp were selected for further steps. The quantity and quality of these PCR products were then verified using Nano Drop spectrophotometry (via the 260/280 ratio). Ampure beads were used to purify the amplicons and remove residual primers. The libraries were prepared by performing an additional 8 cycles of PCR, this time incorporating Illumina-specific barcoded adapters. After purification, library concentrations were measured using the Qubit dsDNA High Sensitivity assay. Sequencing was conducted on the Illumina MiSeq platform (2 × 250 bp paired-end reads) at Biokart Pvt. Ltd., Bangalore, India, following their established culture-independent protocols.

2.4. Sequencing Data Analysis and Statistical Modeling

Paired-end reads were assigned to individual samples using unique barcode sequences, after which both barcodes and primer regions were trimmed. The resulting reads were merged to form contiguous sequences. To ensure taxonomic accuracy, chimeric sequences were identified and removed by aligning the tags against a reference database [6,13]. Clean, high-quality sequences were processed through the QIIME2 pipeline—a comprehensive tool for microbial community analysis [21].

Sequences with ≥97% similarity were clustered into Operational Taxonomic Units (OTUs), and representative sequences from each OTU underwent detailed annotation. Data points flagged as outliers or inconsistencies were removed from the dataset resulting in a standardized dataset for all subsequent analyses. OTUs were taxonomically assigned using the VSEARCH consensus classifier in QIIME2, referencing the UNITE ITS v8.2 fungal database. Relative abundances of OTUs across multiple taxonomic levels—phylum, class, genus—were visualized using the R programming environment. Additionally, functional traits associated with fungal taxa were examined using the FungalTraits database to provide ecological insights into community composition and potential metabolic roles [22].

Alpha diversity metrics were calculated in R Studio using R v4.2.2 [23]. Rarefaction curves were generated using the iNEXT package, which provides the interpolation and extrapolation estimates of biodiversity. For other diversity indices, including observed richness, Shannon, Simpson (1-D), and Evenness, calculations were performed using functions from the vegan package. These metrics were combined into a unified data frame and visualized using the ggplot2 package. Beta diversity was assessed using Bray–Curtis dissimilarity, computed via the vegdist function in vegan. Principal Coordinate Analysis (PCoA) was performed using the cmdscale function to explore fungal community structure. Environmental parameters were standardized using the scale function, and their correlation with PCoA ordination was evaluated using the envfit function (vegan, perm = 999), allowing the statistical testing of environmental influence on community composition. Spearman correlation analysis between fungal communities and soil physicochemical parameters was conducted using the cor function with the Spearman method. Venn diagrams were constructed using the Venn diagram function to identify shared OTUs across samples. Statistical comparisons of diversity indices between plant species were conducted using standard parametric tests (ANOVA and t-test) as preliminary approaches to evaluate group differences. The multivariate analyses such as Principal Coordinates Analysis (PCoA) and correlation matrix visualization were incorporated to assess relationships between fungal diversity and soil physicochemical parameters. These analyses provided an ecological framework for interpreting community-level differences between M. tinctoria and P. dulce.

3. Results

3.1. Soil Nutrient and Physicochemical Profile Across Plant Samples

The soil nutrients and physicochemical characteristics of two plant species, P. dulce and M. tinctoria, were investigated. Morinda’s rhizosphere has a slightly higher total nitrogen concentration (125 mg/kg) than P. dulce’s (116 mg/kg). Similarly, Morinda has a higher total phosphorus (6.4 mg/kg) and potassium (71 mg/kg) content than P. dulce. The electrical conductivity was similar at 0.74 dS/m in both samples. There was a substantial difference in total organic carbon between P. dulce (7.95%) and Morinda (2.43%). Both species had soil pH values close to neutral, with P. dulce at 7.81 and Morinda at 7.89.

The micronutrients analysis found that P. dulce contained a little more iron (2.47 mg/kg) and manganese (1.39 mg/kg), whereas Morinda contained more zinc (1.65 mg/kg) and copper (1.69 mg/kg). The element boron levels were practically comparable across the two samples (0.37 mg/kg in P. dulce and 0.36 mg/kg in Morinda (

Table 1. Summary of soil nutrients and physicochemical profile between the two plants.

Soil Nutrients	M. tinctoria	P. dulce
Nitrogen (mg kg−1)	125	116
Phosphorus (mg kg−1)	6.4	5.01
Potassium (mg kg−1)	71	62
Electrical conductivity (dS/m)	0.74	0.74
Organic carbon (mg L−1 C)	2.43	7.93
pH	7.89	7.81
Iron (mg kg−1)	2.42	2.47
Magnesium (mg kg−1)	1.39	1.69
Zinc (mg kg−1)	1.65	1.42
Copper (mg kg−1)	1.69	1.61
Boron (mg kg−1)	0.36	0.37

). These findings suggest that the two plant species differ in terms of nutrient intake and soil interactions, which could be due to changes in root exudation or ecological adaptation.

The comparative analysis of soil parameters associated with P. dulce and M. tinctoria reveals distinct nutrient and soil quality profiles. M. tinctoria exhibited higher concentrations of essential macronutrients—total nitrogen (125 mg/kg), Total Phosphorus (6.4 mg/kg), and Total Potassium (71 mg/kg)—as well as higher levels of Zinc (1.65 mg/kg) and Copper (1.69 mg/kg), suggesting its potential to enhance immediate plant growth and development. In contrast, P. dulce demonstrated significantly higher total organic carbon (7.95%), along with slightly greater iron (2.47 mg/kg) and manganese (1.69 mg/kg) levels, which are critical for long-term soil health, microbial activity, and carbon stabilization (Figure 2). Both plant-associated soils maintained a neutral pH and similar electrical conductivity, indicating comparable general soil conditions.

Overall, M. tinctoria appears more suitable for rapid nutrient supply and plant productivity, while P. dulce offers greater benefits for sustaining soil structure, organic matter content, and long-term fertility.

3.2. Taxonomical Diversity

A total reads of 69,545 for P. dulce and 49,002 reads for M. tinctoria generated by the Illumina sequencing. The GC concentration varied from 52.54 to 54.5%, with bases with Q20 at over 99.0% and bases with Q30 at over 95.0%. The Illumina Sequencer data collection exhibited good quality, as confirmed by those findings. The sequences clustered into 266 OTUs at 97% similarity level. The taxonomic classification of fungal communities associated with P. dulce and M. tinctoria organized based on relative abundance). Five fungal phyla were found in both plant species. Basidiomycota dominated the P. dulce community, accounting for roughly 55% of the total, followed by Ascomycota at around 45%. In M. tinctoria, Ascomycota was slightly more prominent, accounting for around 52%, while Basidiomycota contributed roughly 48%. Minor phyla, including Mortierellomycota (~1%), Glomeromycota (<0.5%), and Unassigned fungus (<0.5%), were found in both samples, but only made up a small percentage of the diversity. Figure 3A depicts the contrast between the fungal community structure of the two host plants, indicating a comparable fungal phylum-level composition dominated by Ascomycota and Basidiomycota, with minimal contributions from other phyla (Supplementary Table S1).

At the order level, the fungal community composition diversified even more. Tremellales represented the most widespread order in P. dulce, accounting for approximately 53%, then followed by unassigned orders at around 26%. Saccharomycetales (~5%), Sordariales (~3.5%), and Eurotiales (~3%) were among the most common orders. Tremellales accounted for around 48% of M. tinctoria, with the unassigned group accounting for approximately 30%, indicating slightly greater taxonomic ambiguity in this species. Additional orders, such as Hypocreales, Dothideales, Agaricales, Microascales, and Mortierellales, were found in lesser amounts (usually less than 3%). Rare orders such as Fungales, Glomerellales, and Xylariales made small contributions, often less than 1% (Supplementary Figure S1).

The relative abundance of fungal families associated with P. dulce and M. tinctoria provides information about the fungal community composition at the family taxonomic level. The Debaryomycetaceae family dominated the fungal community in both plant species, accounting for roughly 60–65% of the total fungal population in P. dulce and 55–60% in M. tinctoria. The Wallemiaceae family came in second, accounting for roughly 30% of M. tinctoria and 25% of P. dulce, showing an inadequate rise in M. tinctoria abundance. Several more families were found in significantly lower abundances (usually less than 2% each), accounting for the remaining percentage of the population. These included Cystobasidiaceae, Cladosporiaceae, Aspergillaceae, Chaetomiaceae, Nectriaceae, and Didymellaceae, among others. A significant number of the fungal readings were also classified as unassigned, accounting for approximately 2–3% in both host species, indicating the existence of novel or taxonomically uncertain mycobial species.

Despite sharing dominant families, the two plants differed in their relative representation of smaller families. M. tinctoria exhibited slightly higher representation from families such as Nectriaceae and Chaetomiaceae, but P. dulce had marginally higher proportions of Aspergillaceae and Cladosporiaceae. The fungal family-level profiles reveal Debaryomycetaceae and Wallemiaceae as the dominant families in both species, but it also reflects host-specific variability in the presence and proportions of lesser-known families, which contributes to the endophytic fungal community’s overall richness and diversity (Supplementary Figure S1).

In both plant species, a few genera dominated the fungal communities. Notably, the genus Wallemia was highly abundant in both hosts, accounting for roughly 30% of the total fungal diversity in P. dulce and slightly higher, at 35%, in M. tinctoria. Saitozyma was predominant in P. dulce (~40%) but less so in M. tinctoria (~30%). Talaromyces is a major genus, accounting for roughly 25% of M. tinctoria but only about 20% of P. dulce. The remaining fungal communities in both plants were composed of a broad group of low-abundance species. The fungal genus included Coprinellus, Colletotrichum, Clonostachys, Penicillium, Nigrospora, Xylaria, and others, each accounting for a modest proportion (usually less than 5%) of the total fungal population. Unassigned genera made up a sizable component of the community in both samples, showing a significant proportion of fungal taxa that are still taxonomically unclear.

Interestingly, several genera such as Coprinellus, Talaromyces, and Wallemia were consistently found in both host species, indicating that the two plants share a common fungal microbiome. However, differences in their relative abundances, as well as the presence of separate minor taxa, suggest a degree of host-specificity in fungal endophyte composition.

M. tinctoria has a more diversified, functionally rich, and ecologically advantageous endophytic fungal population than P. dulce. This finding is reinforced by Ascomycota’s increased abundance, higher representation of plant-beneficial classes and orders, and the inclusion of major genera and families with well-documented functions in plant growth and resilience. As a result, M. tinctoria is distinguished among the plant with the most robust and advantageous endophytic fungus community.

Overall, the mycobial communities of Pithecellobium dulce and Morinda tinctoria had been dominated by Basidiomycota and Ascomycota, particularly the Tremellomycetes class and Tremellales order. A significant percentage of the community at large remains unclassified at the class and order levels, indicating the presence of novel or unexplored fungal taxa. Despite minor differences between the two hosts, the fungal profiles are highly taxonomically similar, with a core group of dominating species and a lengthy tail of diverse, low-abundance groupings.

The taxonomy of fungal endophytes at the genus and family levels in P. dulce and M. tinctoria reveals distinct patterns of diversity and dominance. At the genus level (Figure 3B), both plant species were shown to be dominated by a few genera, with Wallemia being the most prevalent in Pithecellobium dulce, accounting for 30–35% of the whole fungal population. This was followed by Coprinellus, which contributed between 20 and 25%, and a significant number of unassigned genera, which accounted for about 15 to 18%. Additional taxa, including as Penicillium, Cladosporium, Colletotrichum, and Phaeophleospora, were present in smaller abundances, accounting for approximately 1–5%.

The functional trait composition associated with P. dulce and M. tinctoria reveals a pronounced dominance of saprotrophic fungi, particularly soil saprotrophs and unidentified saprotrophs, which together constitute more than 80% of the total community composition (Figure 4). This trend underscores the ecological role of these plants as reservoirs for decomposer fungi, facilitating nutrient turnover in their respective environments. The presence of minor functional groups such as mutualists, endophytes, pathogens, and parasites indicates a degree of fungal diversity, but their reduced proportions suggest limited symbiotic or pathogenic interactions relative to decomposition-driven activity.

Although both plant species share a similar fungal structure, subtle differences in trait distributions imply the influence of host-specific and environmental factors. Discrepancies may be attributed to variations in root exudate chemistry, physiological traits, or differing recruitment strategies that favor fungal taxa. Environmental variables such as soil composition, humidity, or canopy structure could further modulate fungal colonization patterns. Together, these results highlight the dynamic interplay between host identity and microbial ecology, offering a nuanced understanding of how tropical flora contributes to shaping underground fungal networks.

3.3. Fungal Diversity Analysis

Alpha diversity analyses revealed clear differences in fungal communities associated with P. dulce and M. tinctoria. A rarefaction along with extrapolation curves were developed to examine the species diversity of fungal communities corresponding to P. dulce and M. tinctoria. The x axis denotes sample size (e.g., number of genomic reads), while the y axis represents estimated species richness. Solid curves correspond to rarefaction based on observed data, whereas dotted curves illustrate extrapolation from predictive models. Shaded regions reflect the 95% confidence intervals. The rarefaction curves for both species have plateaued, indicating adequate sampling coverage. P. dulce had slightly higher microbial richness than M. tinctoria. The results obtained show that both plant species support varied microbial communities with similar but distinct biodiversity profiles (Figure 5).

Figure 6 shows an extensive analysis of mycobial alpha diversity between two plant species, M. tinctoria and P. dulce, employing four major diversity indicators: diversity, Shannon index, Simpson index, and evenness. Each bar in the chart shows the mean value for the measure for the endophytic mycobial communities linked to every plant species, with error bars reflecting standard deviation or standard error, which indicate sample variability.

The richness index shows that M. tinctoria supports a greater number of mycobial species than P. dulce. Richness is simply the total number of different species, implying that M. tinctoria supports a wider variety of microorganisms. This could be due to variations in surface leaf composition, chemical discharge, or microhabitat diversity.

In the bottom-left panel, M. tinctoria has a larger Simpson Index, which provides greater weight to dominating species within the community. A higher Simpson value indicates less predominance along with greater evenness, corroborating the hypothesis that M. tinctoria supports an additional evenly organized fungal ecosystem. This might prove biologically beneficial, as more evenly distributed groups are redundant in nature and immune to disruptions.

Finally, the evenness index in the bottom-right panel validates these findings. Evenness indicates how similarly each species is distributed in a diversity. M. tinctoria has a greater evenness value than P. dulce, signifying that the mycobial community linked to it is more homogeneous in composition, with organisms present in similar amounts.

The interaction between the soil factors with the fungal OTUs of P. dulce and M. tinctoria was investigated using a correlation matrix and a Principal Component Analysis (PCoA) biplot (Figure 7 and Figure S2). The correlation matrix shows relationships among soil parameters. Total organic carbon and total nitrogen have a strong positive correlation, while iron and boron show negative associations with other parameters. This matrix effectively reveals the interconnected nature of soil chemical properties. Spearman correlation analysis revealed weak but moderate relationships between soil and fungal OTU abundance. These results indicate that soil chemistry may play a contributory role in shaping fungal community size, supporting the importance of soil nutrient availability. On the right, the PCoA biplot diagram illustrates the way that these soil characteristics influence the division of the two plant varieties based on principal components. The arrows that follow denote the direction and magnitude of each soil variable’s influence, while the vibrant spots represent the arrangement of each plant species. M. tinctoria, displayed in green, clusters on the right side of the plot and is strongly related to characteristics such as total nitrogen, phosphorus, potassium, pH, and electrical conductivity. P. dulce, seen in red on the left, is more impacted by elements such as iron and boron. The PCoA shows considerable differences in soil parameter connections between the two species. Collectively, these illustrations offer a thorough understanding of how the chemical composition of the environment impacts the ecological niches and developmental preference of P. dulce species M. tinctoria.

The Venn diagram depicts a comparison of the elements connected with two plant species: M. tinctoria and P. dulce. M. tinctoria included 169 OTUs, 99 of which were considered unique towards the species and not present in P. dulce. P. dulce, on the other hand, included 167 OTUs, 97 of which were unique to its profile (Figure 8). Notably, 70 components were shared by both plant species, indicating a 26% high degree of similarity or comparable features among their respective ecological communities or molecular composition.

4. Discussion

4.1. Soil Parameters

Research on M. tinctoria have shown that agroecological management approaches improve soil fertility by the raising levels of essential macronutrients such as nitrogen (N), phosphorous (P), potassium (K), and soil organic carbon (SOC). Such quality of soil enhancements has been proven to be associated with improved plant physiological characteristics and fruit quality, demonstrating Morinda’s ability to promote robust plant development [24,25].

In addition, studies conducted on similar systems of agriculture reveal that the implementation of organic amendments, especially compost and biochar, might enhance soil structure by increasing water absorption, ventilation, as well as available nutrients [26,27]. This consequently improves plant absorbance of nutrients and increases overall production. Morinda’s naturally increased amounts of N, P, K, zinc (Zn), and copper (Cu) assist in establishing a nutrient-rich soil ecosystem that promotes strong and sustained plant growth [27].

Since there have been few studies on P. dulce, general research suggests that soils enhanced with organic matter increase optimized aggregation, porousness, and microbial ecosystem development—all of which are important components of the long-term fertility of the soil. Furthermore, iron (Fe) and manganese (Mn) oxides found in soil establish strong mineral–organic complexes which protect the organic matter from degradation by microbial organisms, increasing its ability to persist in the environment [28,29]. In particular, interactions with Fe hydroxides were demonstrated to inhibit glucose breakdown by more than 99.5% when compared to free glucose, suggesting a considerable stabilizing impact [29]. These results demonstrate that the higher Fe and Mn levels originally found in P. dulce could play an important role in a long-term carbon storage, enhanced soil structure, and a more conducive environment for microbial populations [30].

4.2. Functional Traits

Soil saprotrophs play an important role in biological material decomposition and nutrient mineralization, implying that those fungal communities are significantly impacted by the soil ecology and litter composition [31]. This development coincides with earlier studies that emphasize the dominance of saprophytic organisms in the rhizosphere along with leaf-associated fungal assemblages throughout tropical and subtropical plant ecosystems. Interestingly, M. tinctoria has a comparatively greater percentage of unidentified saprotrophs, whereas P. dulce has a higher prevalence of soil saprotrophs. This could be due to microhabitat-specific selection or changes in root exudate profiles that promote distinct saprotrophic species, as shown in research on host-specific fungal recruitment [32]. Minor but ecologically important guilds include plant diseases, foliar endophytes, and epiphytes. Despite the small quantities, their existence suggests possible interactions including host defense, stress tolerance, or latent infection phases—all of which are frequently described in endophytic fungal research [33,34]. Some functional categories, such as mycoparasites, dung saprotrophs, wood saprotrophs, and arbuscular mycorrhizal fungi (AMF), have low representation. Because of their importance in nutrient uptake and plant growth, AMF’s relative scarcity is noteworthy. The authors in [35] found that AMF are commonly underrepresented in datasets prepared using general ITS primers due to divergent rDNA sequences. Furthermore, the existence of unassigned functional categories indicates limits in current functional annotation methods like fungal traits, particularly when used for non-model or underexplored fungal taxa [36].

4.3. Alpha Diversity and Microbial Richness

Altogether, alpha diversity indicators frequently show that M. tinctoria has an increasingly diversified and equally distributed mycobial population compared P. dulce. This could be regulated by changes in soil nutrient concentration, leaf structure, or secondary metabolites of plants, every one of these modify the microenvironment along with selected among various microbial communities [37]. These alterations in microbial richness can have a significant impact on plant health, nutrient cycle, and ecological interactions, underlining the two botanical species’ ecological distinctness [38]. M. tinctoria had a considerably higher Shannon index for endophytic fungi compared to P. dulce, the fungal communities corresponding to M. tinctoria, especially those pertaining to the phyla Ascomycota and Basidiomycota, and families such as Debaryomycetaceae, Wallemiaceae, Chaetomiaceae, Cladosporiaceae, and Nectriaceae, are highly compatible with several well-established probiotic fungi. Although these fungal species are not probiotics, they frequently co-occur or interact with beneficial fungi in plant microbiomes [39]. Ascomycota members, particularly Chaetomium, Cladosporium, and Fusarium species, have been shown to interact positively with Bacillus subtilis, Bacillus velezensis, and Pseudomonas fluorescens, improving biocontrol efficacy and root colonization [40]. Similarly, Debaryomycetaceae, which includes Debaryomyces species, is frequently found alongside probiotic bacteria such as Lactobacillus plantarum and Enterococcus faecium in fermented environments and medicinal plant tissues, contributing to enhanced antioxidant and antibacterial activity [40,41]. Basidiomycetous families, such as Wallemiaceae and Cystobasidiaceae, are ecologically compatible with Lactobacillus spp., especially on composted and fermented substrates. The synergistic relationships demonstrate that M. tinctoria not only supports beneficial fungal endophytes, but also provides an ideal environment for the application of probiotic microbes such as Bacillus subtilis, Pseudomonas fluorescens, Lactobacillus plantarum, and Azospirillum brasilense, thereby improving plant health, secondary metabolism production, and resistance to environmental stress [40,41,42]. The use of advanced bioinformatics and ordination techniques helped establish clear correlations between fungal communities and soil physicochemical parameters, reinforcing the idea that environmental factors shape microbiome architecture [43]. It is important to note that the functional and ecological mechanisms discussed in this study—such as enhanced resistance, improved plant health, or metabolic regulation—are inferred from the previously reported roles of similar fungal taxa rather than from direct experimental validation. These interpretations are intended to provide a conceptual framework linking observed fungal diversity to potential ecological functions. Future studies integrating metabolomic, transcriptomic, or controlled inoculation experiments are required to confirm these hypothesized interactions. While these analyses demonstrate strong correlations between soil properties and fungal community composition, they do not establish direct causal relationships. The observed associations should therefore be interpreted as indicative rather than definitive, pending further experimental validation. Similarly, the functional and ecological mechanisms discussed in this study—such as enhanced resistance, improved plant health, or metabolic regulation—are inferred from the previously reported roles of related fungal taxa rather than from direct experimental evidence. These interpretations are intended to provide a conceptual framework linking fungal diversity to potential ecological functions. Future studies incorporating controlled inoculation, metabolomic, and transcriptomic analyses will be necessary to confirm these hypothesized causal linkages and validate their functional relevance. The study design was intended as an initial comparative survey of endophytic fungal diversity between two host species. As such, experimental controls for functional validation were not included. Future studies incorporating appropriate positive and negative controls will be essential to verify the causal effects of specific microbial taxa or biochemical activities. Subsequently, it is of the utmost importance to conduct controlled inoculation studies to validate their plant-growth-promoting and protecting qualities [44]. When combined, metabolomic and transcriptome investigations have the potential to provide light on the specific molecular pathways that regulate the interactions between microbes and plants that are beneficial [44,45]. As this study was comparative in nature and did not include biological replicates, the results represent the preliminary patterns of fungal community diversity. Future studies incorporating replicated sampling will be essential to confirm the observed trends statistically. Although this study employed a culture-independent metagenomic approach, the isolation and characterization of individual fungal and bacterial strains were not performed. Culturing representative endophytes from M. tinctoria and P. dulce in future studies would enable detailed functional assays and the validation of their ecological or plant-beneficial roles.

This study underscores the power of high-throughput sequencing to reveal intricate patterns of microbial diversity and composition within plant tissues. By focusing on the endophytic fungal communities of M. tinctoria and P. dulce, we demonstrate not only species-specific differences but also the ecological implications of fungal diversity within host plants. In addition to identifying 266 OTUs spanning multiple taxonomic levels—5 phyla, 18 classes, 40 orders, 70 families, and 36 genera—the predominance of Ascomycota and Basidiomycota highlights the core fungal lineages contributing to the symbiotic landscape. With M. tinctoria supporting a more taxonomically diverse and functionally promising community, particularly rich in biocontrol and plant growth–promoting families like Chaetomiaceae, Nectriaceae, and Cladosporiaceae, its endophytic microbiome may confer enhanced ecological stability and resilience against stressors. Importantly, the presence of a few unidentified fungal OTUs suggests a wealth of unexplored diversity that warrants further functional and genomic investigation. Overall, our findings suggest that M. tinctoria may serve as a valuable reservoir for beneficial endophytes with potential agricultural and ecological applications. Future studies integrating transcriptomic, metabolomics, or culture-based validation could provide deeper insight into the functional roles of these fungi and their impact on host plant health.