Fungal endophytes diversity and influencing factors in Liquidambar orientalis Mill. in Türkiye

Beram, R. C., and Akyol, S. (2025). "Fungal endophytes diversity and influencing factors in Liquidambar orientalis Mill. in Türkiye," BioResources 20(1), 1251–1272.

Abstract

Fungal endophytes were isolated from the leaves and petioles of Liquidambar orientalis Mill., an endangered species in Türkiye. Plant material was collected from 10 sites in September 2023, yielding 499 fungal isolates, classified into 38 morphological groups. DNA extraction and polymerase chain reaction (PCR) amplification of ITS and Beta-tubulin regions were conducted on representative isolates. All fungi belonged to the Ascomycota phylum, comprising 11 genera and 26 species across 15 families, with one group unidentified. The most prevalent families were Diaporthaceae (34.9%), Pleosporaceae (23.4%), and Botryosphaeriaceae (22.2%), with Diaporthe eres (15.0%) and Phomopsis sp. (12.4%) being dominant species. Fungal diversity was assessed using Shannon, Simpson, and Chao1 indices, revealing tissue type as the strongest factor influencing species diversity, followed by media and spatial factors. The presence of pathogenic families, such as Botryosphaeriaceae, highlights potential threats to the species. This is the first study to report fungal endophytes in L. orientalis, as well as the first records in Türkiye for several species, including Alternaria destruens, Alternaria alstroemeriae, Stemphylium majusculum, Diaporthe cynaroidis, Pseudopithomyces rosae, Nothophoma variabilis, Cladosporium endophyticum, Cladosporium colombiae, Muyocopron sp., Sphaerulina rhododendricola, Constantinomyces macerans, and Aequabiliella effusa.

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**Fungal Endophytes Diversity and Influencing Factors in Liquidambar orientalis Mill. in Türkiye**

Refika Ceyda Beram ,* and Sultan Akyol

DOI: 10.15376/biores.20.1.1251-1272

Keywords: Anatolian sweetgum; Endophytic fungi; Fungal pathogen; Mycobiota; Türkiye

Contact information: Pamukkale University, Faculty of Science, Department of Biology, 20160, Denizli, Türkiye; *Corresponding author: rberam@pau.edu.tr

INTRODUCTION

Endophyte research dates back to 1866, when German botanist Anton de Bary defined microorganisms residing within plant tissues as “endophytes” (de Bary 1866). The discovery of taxol present in an endophytic fungus associated with Taxus species (Stierle et al. 1993) drew significant attention to the species diversity and bioactive potential of these microorganisms (Reis et al. 2022). Endophytes, which include both fungi and bacteria, colonize the internal tissues of plants without causing visible harm, although certain species may become pathogenic under specific stress conditions (Wilson 1995; Tejesvi et al. 2007; Rodriguez and Redman 2008). Some fungi can transition from being asymptomatic endophytes to latent or opportunistic pathogens, particularly when triggered by environmental stressors, such as drought, hail, extreme temperatures, or mechanical injury (Swart and Wingfield 1991; Diekmann et al. 2002; Blumenstein et al. 2021). These stressors can lead to sudden outbreaks of diseases, and cryptic or latent pathogens are now recognized as major contributors to emerging fungal diseases in forests (Ghelardini et al. 2016).

Endophytes have garnered substantial scientific interest due to their multifaceted roles in plant health, development, and defense mechanisms (Arnold et al. 2003; Hartley and Gange 2009). They can colonize various plant organs, such as leaves, stems, and roots, forming complex associations with their hosts. Beyond their ecological roles, endophytes are prolific sources of bioactive compounds with applications in medicine, agriculture, and biotechnology industry (Trejo-Estrada et al. 1998; Guo et al. 2008; Priti et al. 2009). Exploring the diversity and biochemical capabilities of endophytes continues to unlock new opportunities for benefiting both plant and human health (Kusari and Spiteller 2012).

Extensive research on fungal endophytes has shown that they inhabit a wide range of taxonomic groups, vegetation types, and ecological settings (Arnold et al. 2000; Porras-Alfaro and Bayman 2011). The production of bioactive secondary metabolites can vary based on factors such as environmental conditions, geography, host species, and tissue type (Bacon and White 2000). Understanding endophyte communities in different plant tissues and species is essential to fully harness the potential of these valuable resources (Singh et al. 2017b). Many fungal genera are host-specific, with colonization influenced by the phytochemistry and nutrient content of plant tissues (Arnold et al. 2003). Furthermore, the richness and diversity of endophytes within the same plant species can be highly dynamic, affected by various biotic and abiotic factors (Özdemir et al., 2017; Reis 2022; Özdemir 2024).

Liquidambar orientalis, known as the “sweetgum tree” in Türkiye, is a paleoendemic species that first appeared around 60 million years ago (Davis 1982). It is part of the genus Liquidambar, the subfamily Buclanoidae, and the family Hamamelidaceae. Historically widespread, this relict species is now found only in limited regions, specifically in southwestern Türkiye and on the island of Rhodes (Kurt and Ketenoğlu 2008; Güner 2012). The name “Liquidambar” derives from the Latin “Liquidus,” meaning liquid, and the Arabic “Amber,” which refers to a resinous substance, describing the balsam found in the tree’s trunk. L. orientalis is classified as “Endangered” by the IUCN, with its last assessment in 2017 listing it under criterion A2c due to habitat loss and other environmental threats (Kavak and Wilson 2018). Significant threats include agricultural activities, fires, pollution, contaminated water, tourism, and overgrazing. Changes in water availability due to rainfall variability and climate change-induced droughts are also critical threats to the species’ habitat (IUCN 2024). Its conservation status is crucial for regional ecosystems and biodiversity preservation.

The decline of L. orientalis habitats in Türkiye is well documented and reflects a broader trend of habitat loss. In 1949, sweetgum forests covered 6,312 hectares, but by 2016, this had decreased to 1,416 hectares (GDF 2021). Efforts to conserve this protected species continue, given its ecological, cultural, and economic significance. L. orientalis is not only important for biodiversity but is also used in various contexts. Its pleasant aroma has made it valuable in products ranging from cosmetics and perfumes to parasiticides and medicines. Additionally, it is used for its calming effects in therapy forests and as incense in religious and cultural ceremonies (Lee et al. 2009; Selim and Sönmez 2015).

The restricted distribution and historical context of L. orientalis highlight its importance in understanding environmental changes and conservation needs. Due to ongoing and emerging threats, continuous monitoring and protection of this species are essential for maintaining biodiversity and ecosystem health. Moreover, many studies globally have emphasized the role of such plant species in providing habitats for endophytic fungi. These plants often thrive in unique ecosystems that offer specific conditions, making them suitable for hosting microorganisms such as endophytes. Research on these plant species serves as a crucial resource for discovering new fungal species, thereby enriching the scientific understanding of global fungal diversity. Such efforts not only aid in developing effective conservation approaches but also improve predictions related to global fungal biodiversity.

The diversity of endophyte communities in trees is influenced by numerous factors, making it challenging to identify and understand the specific contributions of individual elements to these communities. However, studies with adequate sample sizes that account for various environmental and methodological variables can help elucidate these interactions and their individual contributions. Understanding these factors is essential for advancing knowledge on the ecology and dynamics of endophytic communities.

Despite the importance of L. orientalis and its potential as a host for diverse endophytes, no previous research has specifically investigated its endophyte associations. This study is the first comprehensive report on the diversity of fungal endophytes within L. orientalis, exploring both the Turkish and global contexts. In Muğla province, various natural populations of L. orientalis were identified, and 10 sampling sites were selected based on distinct geographical features. The selection aimed to include representative sites with diverse habitat characteristics to capture ecological variability. Samples from these sites were collected and analyzed to identify endophytic fungi using both morphological and molecular approaches. The research investigated variations in endophyte communities concerning spatial, individual, directional, tissue type, tissue region, and nutrient media factors.

EXPERIMENTAL

Determination of Sample Sites

In Muğla province, various natural populations of Liquidambar orientalis were identified, resulting in the selection of 10 different sampling sites based on their distinct geographical features. The identification of these sites utilized management plans, which allowed for the mapping of areas where L. orientalis occurs in a digital format. Topographic variables, such as elevation and aspect, as well as climate variables, including annual average temperature and total annual precipitation, were defined for these areas.

Elevation data were downloaded from the EarthData database with a resolution of 30 m. The data were used in ArcMap (Environmental Systems Research Institute, Inc., version 10.2, Redlands, CA, USA) to create an aspect map (EarthData 2024). Climate data were obtained from the WorldClim database, using maps with a resolution of 30 arc seconds (~1 km). These four variables were transferred to ArcMap, where the distribution areas of L. orientalis were overlaid on the maps.

Finally, the most representative sampling sites with diverse habitat characteristics were selected for field sampling. Geographic Information Systems (GIS) were employed throughout the process of site selection and sample collection. Using ArcGIS, the study area in Muğla was divided into a grid system, creating sub-sampling areas. Random points were generated within these grids, and trees located at these points were selected for sampling (Oruç et al. 2017).

Sampling

The sampling was conducted in September 2023 at 10 different sites located in the Muğla district in southwestern Türkiye (Fig. 1). Beram and Akyol collected 40 leaf and petiole samples from 20 trees. In each sub-sampling area, two sample trees were selected, spaced 50 meters apart to represent different microhabitats.

Fig. 1. Location of the sampling sites in Muğla province, south-western Türkiye (Sampling Sites: K1, Köyceğiz; K2, Köyceğiz; KZ, Kızılkaya; D1, Dalaman; M, Marmaris; F, Fethiye; F1, Fethiye; F2, Fethiye; F3, Fethiye; F4, Fethiye; A1, Tree1; A2 Tree2)

The host characteristics of the stands, including age (Haglöf Sweden increment borer), diameter (Haglöf Mantax black 1020 mm diameter gauge), and height (measured with a Blume Barl Leiss height meter), along with current stand type (Table 1), were recorded in the field notebook.

Samples of healthy leaves and petioles from branches 4 to 8 m above the ground were collected using pruning shears (Meşem, Türkiye) from one-year-old growth on the selected trees (Oono et al. 2015) (Fig. 2). This height range was chosen to standardize sampling from similar canopy levels across all trees. Leaves were taken from the leaf just below the terminal shoot leaf, following a consistent sampling method for each tree (Gamboa and Bayman 2001). A total of four leaves (two from the north and two from the south) showing no signs of disease were collected (Dos Reis et al. 2022).

The collected leaves were transported in labeled sealed bags with the corresponding sampling site number, sample number, and sampling date, using ice packs to maintain suitable conditions in the laboratory. The samples were stored at 4 °C ± 1 °C until analyzed.

Table 1. Characteristics of Sampling Sites and Sampling Trees in Muğla District, South-Western Türkiye

Fig. 2. Sampling and shredding method: (a) Selection of the area for leaf sampling on the tree, (b) Leaf selection for sampling, and (c) Shredding method for leaves and petioles

Fungal Culture Isolation

Isolation is a critical step in the identification of fungi. The isolates obtained during this process, representing pure fungal colonies, were employed in both morphological and molecular diagnostic studies. During the isolation process, media-dependent variations were analyzed to enhance the understanding of how methodological factors influence fungal diversity.

Surface Sterilization of Plant Material

The sterilization procedure was performed to eliminate external contamination and epiphytic fungi, ensuring the accurate isolation of endophytic fungi for analysis. The leaves and petioles underwent surface sterilization procedures. For surface sterilization, each sample was initially thoroughly rinsed in distilled water. Subsequently, the samples were subjected to a surface sterilization process, wherein each sample was immersed in 75% ethanol for 1 min, followed by a 5-min immersion in 5% sodium hypochlorite (NaOCl), and then dipped in 75% ethanol for 30 s. Finally, the samples underwent a sequential rinse of 1 min in sterile distilled water, and this was repeated five times (Gamboa et al. 2003; Ibrahim et al. 2021). Post-sterilization, the leaves and stems were left to air-dry within a under sterile conditions in a biological safety cabinet.

Cultivation of Fungi from Leaf and Petiole Samples

Each leaf was dissected into four approximately 10 x 5 mm² fragments using a sterile scalpel: one near the tip along the midrib, one near the base along the midrib, one from the area closest to the right edge, and another from the area closest to the left edge. Subsequently, each of these fragments was further subdivided into four equal sub-samples. Each sub-sample was individually placed onto Potato Dextrose Agar (PDA), Potato Carrot Agar (PCA), Water Agar (WA), and Malt Extract Agar supplemented with plant material (PL-MEA), allowing the cultivation of leaf samples from all four fragments on each growth medium (Fig. 2) (Cannon and Simmons 2002).

The same procedure was applied to petioles. Each stem was initially divided into four equal vertically along the length segments, and then each of these segments was further divided into four equal transverse sections. Petri dishes were incubated in the dark at 25 ± 2 °C for 21 days. Regular microscopic examinations were conducted daily using a stereomicroscope. Fungal hyphae actively developing in plant tissues were transferred to the same type of culture media for purification, and the obtained cultures were incubated again at 25 ± 2 °C (Khalil et al. 2020).

Morphological and Molecular Identification of Fungal Isolates

Fungal isolates grown on plates were initially grouped based on their morphological characteristics, including colony shape, size, color, texture, growth pattern, and reproductive structures, to tentatively identify them at the genus level. Distinct morphotypes were selected for molecular identification. Genomic DNA was extracted from fresh mycelium using the High Pure Polymerase Chain Reaction (PCR) Template Preparation Kit (Roche, Basel, Switzerland), following the manufacturer’s protocol. Molecular identification involved PCR amplification and sequencing of the ITS1-5.8S-ITS2 region (ITS1-2) using the universal primer pairs ITS1 and ITS4 (White et al. 1990), along with the beta-tubulin (tub2) gene using primers Bt2a and Bt2b (Glass and Donaldson 1995). The PCR was performed using Xpert Fast Hotstart Mastermix (Grisp, Portugal) in a 25 μL reaction mixture.

The PCR products were sequenced at BMLabosis (Ankara, Türkiye), and the DNA sequences were analyzed and edited using MEGA 11 software (MEGA Development Team, MEGA 11 version, Philadelphia, PA, USA). The sequences were compared against the NCBI GenBank database using BLAST (Altschul et al. 1990), with species-level identification requiring a minimum of 98% coverage and 98.5% identity. Genus-level identification was set between 94% and 97% similarity (Singh et al. 2017a). For ambiguous species assignments, published studies were used for confirmation (Hernández et al. 2023).

Evolutionary relationships were analyzed using the Neighbor-Joining method (Tamura et al. 2004), with bootstrap values calculated from 500 replicates (Felsenstein 1985). Evolutionary distances were measured using the Maximum Composite Likelihood method, and ambiguous positions were removed via pairwise deletion. Only isolates identified with both markers were considered accurately identified at the species level.

Finally, selected isolates underwent further morphological characterization using an Olympus compound microscope (Olympus Corporation, Tokyo, Japan) and the Olympus DP-Soft program. Representative isolates were preserved and stored in triplicate at the Fungal Biotechnology Laboratory, Department of Biology, Pamukkale University, Denizli, Türkiye.

Statistical Analysis of Fungal Endophyte Diversity

To evaluate the diversity of the fungal endophyte community across sampling sites and tissue types, statistical analyses were conducted. The statistical analysis was performed using R (R Core Team, R 4.3.1 Version, Auckland, New Zealand). Shannon diversity indices and the Bray-Curtis similarity index (Shannon 1948; Bray and Curtis 1957) were used to characterize the diversity and composition of fungal communities, while the Simpson index was employed to estimate dominance (Simpson 1949). Beta diversity has been employed to identify the dissimilarities among the sampling sites (Bray and Curtis 1957; Jost 2007; Legendre and De Cáceres 2013; Ricotta et al. 2021). In this study, a universal beta diversity calculation based on Shannon entropy was preferred. Additionally, the Chao1 diversity index was calculated for parameters with one or two OTUs (Operational Taxonomic Unit).

RESULTS

This study includes findings related to the prevalence of fungi obtained from the leaves and petioles of L. orientalis collected from 10 different sampling sites within Muğla province. A total of 40 leaf and petiole samples were collected from 20 trees, and these samples were inoculated onto 1280 Petri dishes.

From the collected samples, 499 isolates were obtained. All isolated fungi were identified as members of the Ascomycota. Morphological and molecular analyses resulted in the identification of a total of 37 fungi from 15 different families, including 26 species at the species level and 11 genera at the genus level. Additionally, one unidentified species was obtained (Table 2).

Upon reviewing the data, the most common families identified were as follows: Diaporthaceae with 174 isolates (34.9%), Pleosporaceae with 117 isolates (23.4%), and Botryosphaeriaceae with 111 isolates (22.24%). These three families accounted for 80.5% of all isolates, indicating their dominance within the fungal community. Other families contributed less significantly, such as Didymosphaeriaceae (7.4%), Aspergillaceae (6.8%), and Hypocreaceae (3.4%). At the species level, D. eres (75 isolates – 15.0%) was the most commonly isolated and dominant species across all sites, followed by Phomopsis sp. (62 isolates – 12.4%) and Alternaria sp. (56 isolates – 11.2%).

Fungal diversity was assessed using Shannon and Simpson alpha diversity indices, as well as the Chao1 index. The analyses considered various factors, including spatial, individual, orientational, tissue type, tissue region, and media-dependent variations. These findings indicate that specific environmental and methodological factors influence fungal diversity. Among the isolates obtained, 71.0% were recovered from all tissue types. While these endophytes were widespread, some were specific to particular environmental and methodological conditions.

Notably, tissue type emerged as the strongest factor affecting species diversity within the endophytic community, followed closely by media-dependent and spatial factors, with only minor differences between them. Collectively, these factors shaped the structure of the endophytic mycobiota community in L. orientalis. Additionally, this research provides the first records of the following species in Türkiye: Alternaria destruens, Alternaria alstroemeriae, Stemphylium majusculum, Diaporthe cynaroidis, Pseudopithomyces rosae, Nothophoma variabilis, Cladosporium endophyticum, Cladosporium colombiae, Muyocopron sp., Sphaerulina rhododendricola, Constantinomyces macerans, and Aequabiliella effusa.

Spatial Alpha Diversity Analysis

Upon examining the data collected from the sampling sites, the most dominant species were FBL7, FBL23, and FBL56, each with a frequency value of 10. These were followed by FBL6, FBL11, and FBL32, with a frequency value of 9. Shannon and Simpson alpha diversity indices have been calculated. The sites with the highest diversity for both Shannon and Simpson indices were F and F2, while the sites with the lowest diversity were F3 and K1 (Table 3).

Table 2. Classification of Isolated Fungi, Morphological and Molecular (Based on ITS Region) Identification of Different OTUs, Their Closest Match from NCBI Database with Their Accession Number, Query Coverage (%QC) and Similarity (%ID)

Table 3. Spatial Diversity Analysis Data