Introduction

Due to continuous evolution of antibiotic resistance in bacterial and fungal pathogens, there is a growing demand for antibiotics, not only for human use, but also for use in livestock (Dos Santos et al. 2015), and the need for the development of new and effective antibiotic chemicals is increasing (Dos Santos et al. 2015; Yang et al. 2015; Liu et al. 2016; Chi et al. 2019). Furthermore, growth in the use of antibiotic chemicals will increase the accumulation of these chemicals in the natural system, potentially causing a threat to human health. Consequently, new treatment methods are needed to solve these problems (Qi et al. 2015). Over the years, researchers have observed that plants harbor eukaryotic fungi of great biological diversity. It is estimated that more than one million plant fungal endophytes are distributed in approximately 300,000 terrestrial plant species on Earth (Jia et al. 2016). These fungal endophytes can produce many interesting secondary metabolites with unique structures and distinct activities, which represent a promising source of antimicrobial and other medicinal therapeutic agents (Deshmukh et al. 2014; Dos Santos et al. 2015).

Microorganisms are a rich source of bioactive compounds used in pharmaceutical, agricultural, and industrial applications (Deshmukh et al. 2014). Accumulated evidence has demonstrated that many of the chemotherapeutic agents produced by medicinal plants are also produced from their endophytes (Stierle et al. 1993). For instance, taxol is one of the most well-known plant-derived anticancer drugs, and is isolated from the bark of the Pacific yew tree Taxus brevifolia. The isolation of taxol-producing endophytic fungus Cladorhinum sp. from the Pacific yew tree suggests a simpler and more practical way to produce this compound on a large scale (Heinig et al. 2013). Endophytes are microorganisms that include bacteria and fungi living within plant tissues without visibly harming the plant (De Bary 1879). They have evolved to survive in plant tissues for all or part of their life cycle and may protect the plant hosts from pathogens, insect herbivores, and even abiotic stresses, such as drought, heat and salinity (Liu et al. 2011; Meng and He 2011; Khan et al. 2013). In addition, certain endophytic fungi can produce the same natural products as their plant hosts, as well as a broad variety of other novel bioactive secondary metabolites (Heinig et al. 2013). Soil-born microorganisms have proved to be rich sources of new microbial species and novel bioactive compounds. Since the late 1980s, endophytic fungi from plants have been studied extensively in the search for new drugs. A single drug on the market, emodepside, has resulted from these efforts, and another is now in clinical phase 3 trials, suggesting the potential for identification of new bioactive metabolites from endophytic fungi (Helaly et al. 2018). Many endophytes isolated from medicinal plants produce natural products that are strong fungicides or bactericides (de Siqueira et al. 2011).

The genus Zanthoxylum (Magnoliopsida: Sapindales: Rutaceae) includes more than 250 deciduous, evergreen trees, and shrub species, distributed globally in tropical and subtropical regions (Epifano et al. 2011). The genus is economically important owing to alimentary, industrial, and medicinal applications (Wang et al. 2014), the latter including the treatment of abdominal pain, arthritis, asthma, cough, cold, fever, diarrhea, malaria, toothache, and ascarid infections, as well as muscle cramps and spasms (He et al. 2002; Lee and Lim 2008; Wang et al. 2014; Nguyen et al. 2016; Bunalema et al. 2017; Costa et al. 2018; Ekka et al. 2020). In addition, the plants also produce bioactive phytochemical compounds that display anti-inflammatory, antiplasmodial, antiparasitic, antiviral, antibacterial, and antifungal activities (Kumar et al. 2014; Nguyen et al. 2016; Costa et al. 2018; Ekka et al. 2020). Zanthoxylum simulans (Chyau et al. 1996) is a prickly shrub native to mainland China (Western Sichuan) and Taiwan (Yang et al. 2002). The stem, branches, and branchlets have spiny thorns, and the bark is aromatic. It is a popular natural spice and has been used in traditional Chinese medicine to treat various diseases, including stomachache, toothache, intestinal worms, eczema, and pruritus, and extensive phytochemical investigations have been carried out on this plant (Wu and Chen 1993; Yang et al. 2002; Wang et al. 2014). Endophytic fungi from this plant have been reported to produce secondary metabolites that inhibit the proliferation of human rheumatoid arthritis synovial fibroblasts (HFLS-RA) (Lyu et al. 2018). However, surprisingly, fungal endophytes of this plant have not been studied in depth. Therefore, the goal of this study was to isolate endophytic fungi from the leaves and stems of Z. simulans during summer and winter seasons on Kinmen Island, screen these for antimicrobial activities against indicator pathogens, and finally assay their metabolites for anti-inflammatory activities.

Materials and methods

Collection of plant materials

Leaf and stem samples of Zanthoxylum simulans (Fig. 1) were collected from Caidian (Fig. 2) in Kinmen County, Taiwan. A total of ten twigs (each including five leaves) were cut from ten randomly-selected individual Z. simulans plants, among which the first five samples were collected in summer (August 2014) and the second five in winter (February 2015). Samples were placed into separated open plastic bags, brought to the laboratory within 30 min, maintained at 4 °C, and processed within 24 h. A voucher sample (NO. Chi 0003) was kept in the Department of Food Science, National Quemoy University, Kinmen, Taiwan.

Fig. 1
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Distribution of Zanthoxylum simulans in Caidian, Kinmen County, Taiwan, from where the samples were collected

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Fig. 2
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The natural habitat (A), leaves (B), flowers (C), and fruit (D) of Zanthoxylum simulans

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Isolation of endophytic fungi

Prior to surface sterilization, leaves and stems were thoroughly washed with running tap water to remove surface particles. They were then surface-sterilized following the protocol of Chi et al. (2019), i.e., immersed in 70% ethyl alcohol for 10 s, then soaked in 4% sodium hypochlorite for 30 s, and finally washed twice in sterile distilled water. It was crucial to ensure that the fungal isolates from the plant Zanthoxylum simulans were truly endophytes, and were not due to contamination of epiphytes. We confirmed that all the epiphytic microbes were removed, as no microbial growth occurred on control agar plates after 30 days of culture. The surface-sterilized leaf and stem segments were placed on 2% malt extract agar (MEA; BD Biosciences, Franklin Lakes, NJ, USA) containing 0.5 g/L penicillin G (Sigma-Aldrich, St. Louis, MO, USA) and 0.5 g/L streptomycin sulfate (Sigma-Aldrich). The inoculated plates were incubated at 25 °C and observed daily for one month. Fungi with different mycelial morphotypes were isolated and sub-cultured with MEA.

ITS rDNA sequencing and phylogenetic analysis

Endophytic fungal isolates that exhibited antimicrobial activity were identified based on sequencing of a fragment spanning 18S to 28S, including internal transcribed spacer 1 (ITS1), 5.8S rDNA, and ITS2. The universal primers pairs ITS1: 5′-TCCGTAGGTGAACCT GCGG-3′ (or ITS5: 5′-GGAAGTAAAAGTCGTAACAAGG-3′) and ITS4: 5′-TCCTCCGCT TATTGATATGC-3′ were used to amplify the ITS rDNA sequence (White et al. 1990). Fungal genomic DNA extraction and PCR amplification were performed following protocols described previously (Chi et al. 2019). All the obtained ITS rDNA sequences were compared against the UNITE Database (User-friendly Nordic ITS Ectomycorrhiza Database) (Nilsson et al. 2019) using the nucleotide BLAST program (Altschul et al. 1990).

Neighbor-joining analysis with Kimura’s two-parameter model was performed using MEGA software ver. 7.0.26 (Tamura et al. 2011). The robustness of the phylogenetic tree was evaluated by bootstrap analysis of 1000 randomly-resampled data sets. Leucoagaricus gongylophorus SES090115-02 (Basidiomycota) was used as the outgroup.

Preparation of crude extract

The endophytic fungi were cultured by inoculating two agar plugs (8 mm in diameter) of fungi into 250-mL flasks containing 100 ml GYP broth (1 g/L dextrose, 0.2 g/L peptone, and 0.1 g/L yeast extract). Each flask was incubated at 25 °C for 14 days on a shaking incubator at 220 rpm. The culture broth and the mycelia were separated by filtration after incubation; then, the filtered broth was extracted twice with an equal volume of ethyl acetate (EtOAc) and concentrated in a vacuum to dryness (Chi et al. 2019). The crude extracts obtained were kept at − 20 °C and used for in vitro antimicrobial, anti-inflammatory, and cell viability assays. Test solutions of endophytic fungi were obtained by dissolving crude extracts in methanol to a final concentration of 0.5 mg/mL.

Indicator microorganisms

The indicator microorganisms used for antimicrobial activity assays throughout this study were as follows: five Gram-negative bacteria, including Escherichia coli, Edwardsiella tarda, Pseudomonas anguilliseptica, Vibrio alginolyticus, and Vibrio parahaemolyticus; four Gram-positive bacteria, including Lactococcus garvieae, Staphylococcus agalactiae, Streptococcus aureus, and Streptococcus iniae; and two fungi, Candida albicans (Hazen 1995) and Cryptococcus neoformans (Kwon-Chung et al. 2017). Fungal strains C. albicans and C. neoformans were kindly provided by the Health Bureau of Kinmen County; the nine bacterial strains were supplied by the Institute of Fisheries Science, National Taiwan University.

Indicator strains were cultured as follows: E. coli was grown in Luria broth (LB) at 37 °C for 18 h and maintained on LB agar (LA). E. tarda, L. garvieae, P. anguilliseptica, S. agalactiae, S. aureus, and S. iniae were grown in tryptone soy broth (TSB) for 18 h at 37 °C and maintained on tryptone soy agar (TSA). V. alginolyticus and V. parahaemolyticus were grown in marine broth (MB) for 18 h at 37 °C and maintained on marine broth agar (MA). C. albicans and C. neoformans were grown in yeast malt broth (YM) for 48 h at 30 °C and maintained on YM agar (YA) (Chi et al. 2019).

Determination of antimicrobial activity

The antimicrobial activities of metabolites of the endophytic fungi against the indicator bacterial and fungal strains were measured using the agar well diffusion method (Rios et al. 1988). Culture broths of bacterial indicators were diluted using beef extract peptone (BEP) medium (5 g/L beef extract, 5 g/L NaCl, 10 g/L peptone, 15 g/L agar) to achieve a final concentration of 1 × 106 CFU/mL and poured into BEP plates. Culture broths of fungal indicators were spread evenly on YA and cultured at 25 °C for 3–5 days. The fungi were diluted using molten Sabouraud agar (SA) to achieve a final concentration of 1 × 105 spores/mL, and 10 mL of this diluted medium was poured into a plate containing 8 mL of solidified SA medium. A sterilized cork borer with an external diameter of 7.8 mm was used to make circular wells in the BEP and SA agar, and 40 μL of the fungal test solutions was added into the wells. The diameters of inhibition zones were measured after incubation of bacterial indicators for 24 h at 37 °C and fungal indicators for 48 h at 25 °C. No activity was observed for any of the indicator microbes in blank tests.

Determination of anti-inflammatory activity and cell viability

Inhibition of inducible nitric oxide synthase (iNOS) activity by treatment with crude extract was determined through decreasing NO release in the supernatants of lipopolysaccharide (LSP)-stimulated cells using the Griess reagent method (Wang et al. 2007) following previously-established procedures (Chi et al. 2019). Two positive controls, nitroarginine (a non-selective iNOS inhibitor) and aminoguanidine (a selective iNOS inhibitor) plus vehicle (0.1%, DMSO), were added to RAW 264.7 cells (a mouse leukemic macrophage cell line) in the presence of 200 ng/mL LSP. NO formation was measured using the Griess reagent (Wang et al. 2007).

The cell viability of RAW 264.7 cells treated with crude extract was studied using the redox indicator Alamar Blue according to the method described by Chi et al. (2019). The mouse macrophage cell line RAW 264.7 was obtained from the Bioresource Collection and Research Center (BCRC; Hsinchu, Taiwan).

Nucleotide sequence accession numbers

The nucleotide sequences obtained in the present work are available in the GenBank database (MN220648–MN220655 and MN368169–MN368183).

Results

Endophytic fungi isolation and antimicrobial activity analysis

Using the culture-dependent method, a total of 113 endophytic fungi were isolated from all the samples, among which 51 (45.13%) were obtained from the summer samples and 62 (54.87%) from the winter samples. Most of the endophytic fungi (110 isolates, 97.34%) were isolated from leaves, with only 3 isolates (2.65%) isolated from stems.

All crude extracts of the isolated fungi were then tested for antimicrobial activity against 11 indicator microorganisms. Using the agar well diffusion method, 23 isolates (20.35%) were found to possess antimicrobial activity against at least one indicator microorganism (Table 1). The percentages of fungal isolates possessing antibiotic activities obtained from the summer and winter samples were 23.53% (12) and 17.74% (11) (Table 1), respectively. Among the 23 antibiotic-producing fungal isolates, 56.52% (13) exerted activity against Gram-positive bacteria, 21.74% (5) showed activity against Gram-negative bacteria, 8.70% (2) showed activity against both Gram-positive and Gram-negative bacteria, 47.83% (11) showed activity against fungi, and 17.39% (4) showed activity against both bacteria and fungi. Most of the isolates (18 isolates, 78.26%) were able to inhibit one indicator microorganism, and only two isolates (8.70%) inhibited more than three indicator organisms (Table 1).

Table 1 Antimicrobial activities of 23 antibiotic-producing endophytic fungi isolated from Zanthoxylum simulans Hance against indicator microorganisms using the agar well diffusion method
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The percentages of susceptibility of E. coli, P. anguilliseptica, S. agalactiae, S. aureus, C. albicans, and C. neoformans to the 23 antibiotic-producing isolates were 17.39, 4.35, 21.74, 39.13, 39.13, and 8.70% (Table 1), respectively. None of the isolates exhibited activity against indicator microbes E. tarda, V. alginolyticus, V. parahaemolyticus, L. garvieae, and S. iniae.

Identification of endophytic fungi producing antibiotics

The ITS rDNA sequences of the 23 antimicrobial substance-producing fungi were sequenced and compared with the UNITE database, a curated database for fungal species identification. All 23 isolates belonged to the phylum Ascomycota (Table 2). According to the morphology and molecular identification from the ITS rDNA sequence (Table 2), Penicillium (26.09%, 6 isolates) was the most abundant genus obtained from Z. simulans, followed by Colletotrichum (21.74%, 5), Diaporthe (21.74%, 5), Daldinia (17.39%, 4), Alternaria (8.70%, 2), and Didymella (4.34%, 1) (Table 2). In addition, a neighbor-joining tree showing the affiliation of the ITS rDNA sequences from these antibiotics-producing fungi to selected reference sequences is presented in Fig. 3. The topology of the phylogenetic tree (Fig. 3) showed that 20 strains in the genera Diaporthe, Penicillium, Daldinia, and Colletotrichum formed a major clade I, under a bootstrap confidence value of 92%, while 3 strains in the genera Didymella and Alternaria, phylogenetically related to order Pleosporales, clustered to form another clade II (bootstrap value, 100%).

Table 2 Blast analysis of ITS-rDNA sequences of 23 antibiotic-producing endophytic fungi isolated from Zanthoxylum simulans Hance
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Fig. 3
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Phylogenetic analysis of fungal endophytes with antimicrobial abilities isolated from medicinal plant Zanthoxylum simulans. The phylogenetic tree was constructed based on the ITS-rDNA sequences by the neighbor-joining method, using Kimura’s two-parameter model. Bootstrap percentages greater than 70% are shown at branch points. The ITS-rDNA sequence of Leucoagaricus gongylophorus, a Basidiomycota species, was used as the outgroup. The scale bar represents 0.05 substitutions per nucleotide position

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Figure 4 compares the taxonomic compositions of fungal endophytes isolated in different seasons at the genus level. We observed that endophytic fungi of genus Diaporthe were only present in winter specimens in high abundance (45.45%), whereas endophytic fungi of genus Daldinia were only present in summer specimens in high abundance (33.33%). Alternaria and Didymella were not found in the winter plant samples. Moreover, we found that the patterns of susceptibility of pathogens to summer and winter fungal isolates of the same genus differed; for example, S. aureus, C. albicans and C. neoformans were only susceptible to Penicillium isolates collected in summer (Tables 1 and 2). All the above results suggested that endophytic fungi communities might vary in a season-dependent fashion.

Fig. 4
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Taxonomic distribution of endophytic fungi with antimicrobial activities isolated from medicinal plant Zanthoxylum simulans in different seasons at the genus level

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Anti-inflammatory analysis

The crude extracts of the 23 endophytic fungal isolates with antimicrobial activity were further evaluated for anti-inflammatory activity using the Griess assay. The cytotoxicities of the fungal crude extracts were determined using an Alamar Blue assay. Fourteen isolates (60.87%) showed low nitric oxide production (< 10%) in the Griess assay, among which 9 isolates (64.29%) did not produce any NO Fig. 5a. The level (% of vehicle) of the Alamar Blue assay ranged from 0 (Penicillium chermesinum NQU287; (Biourge 1923)) to 115.49 (Colletotrichum sp. NQU741) Fig. 5b; 13 (56.52%) of the 23 test extracts showed a high cell viability (> 80%) Fig. 5b.

Fig. 5
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Nitrite production (a), cell viability (b), and nitrite production against cell viability with a threshold value of 50 for both assay methods for identification of colonies with anti-inflammatory potential (c) of 23 endophytic fungi colonies collected in two sampling seasons based on low NO production and lower cytotoxicity

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In preliminary screening, endophytic fungi with the potential for medicinal use were required to match the criteria of low cytotoxicity (higher values in the Alamar Blue assay) and strong inhibition of iNOS (lower values in the Griess assay) Fig. 5c. Thus, we set the threshold value at 50 for both assay methods in order to identify potentially useful endophytic fungi. A total of 15 endophytic fungi (65.22%) matched the screening conditions Fig. 5c, among which 8 and 7 fungi colonies were isolated from plant samples obtained in winter and summer, respectively.

The best anti-inflammatory activity was observed for two isolates, P. chermesinum NQU307 and D. helianthi NQU760 (Muntanola-Cvetkovic et al. 1981), their extracts having both a high NO inhibition effect (< 15%) and a low cell toxicity (> 100% cell viability) Fig. 5a, b. Our results indicated that crude extracts from the fermentation broth of endophytic fungi isolated from medicinal plant Z. simulans are a potential source of natural anti-inflammatory active compounds.

Discussion

Many previous studies have indicated that endophytes of plants, especially medicinal plants, are a promising and untapped source of functional metabolites (Cui et al. 2011; Vieira et al. 2014; Dos Santos et al. 2015; Yang et al. 2015; Liu et al. 2016). However, only very small numbers of plant endophytes and their metabolites have been identified and characterized. In this study, endophytic fungi of Zanthoxylum simulans from Kinmen Island were thoroughly screened for metabolites with antimicrobial activity, and a total of 113 endophytic fungi were isolated. The crude ethyl acetate extracts of 23 isolates (20.35%) were observed to exhibit antimicrobial activity towards at least one indicator microorganism. Previous studies have demonstrated antimicrobial activities of fungal endophytes from medicinal plants Indigofera suffruticosa Miller (Dos Santos et al. 2015), Cephalotaxus hainanensis Li (Liu et al. 2016), and Melastoma malabathricum L (Mishra et al. 2016), and reported that 27.69%, 34.74%, and 26.37% of the fungal endophytes possessed antimicrobial activity, respectively; these results were consistent with our findings. In contrast, the percentage of fungal endophytes possessing antimicrobial activity in our study was lower than the percentages for Aquilaria sinensis (46.4%) and Cephalotaxus hainanensis Li (80·95%), and higher than that of Baccharis trimera (12.8%) (Cui et al. 2011; Vieira et al. 2014; Yang et al. 2015). In general, the percentage of endophytic fungi obtained from medical plants that possess antimicrobial activity varies widely from study to study (Luo et al. 2015). However, it is worth noting that the percentage can be affected by the number of indicator microbes used, the culture conditions of the endophytic fungi, and the assay method employed (Kennedy et al. 2009; Chen et al. 2012; Luo et al. 2015).

In this study, more than half (13 isolates, 56.52%) of the isolates with antimicrobial activities were specific to Gram-positive bacteria S. aureus and S. agalactiae, and only five isolates (21.74%) were found to produce inhibitors against Gram-negative bacteria E. coli and P. anguilliseptica. These results were in agreement with many previous studies in which the antibacterial activities of endophytic fungi obtained from medicinal plants were analyzed, and exhibited more potent activity against Gram-positive bacteria than Gram-negative bacteria (Phongpaichit et al. 2006; de Siqueira et al. 2011; Kaul et al. 2012). The outer layer of the cell wall of Gram-negative bacteria is composed of lipopolysaccharide, in contrast to the thick layer of peptidoglycan present in Gram-positive bacteria. Owing to differences in the cell-wall composition, antibacterial activity against Gram-positive and Gram-negative bacteria differs (Chi et al. 2019). In addition, ethyl acetate extracts are comparatively less-polar natural products than other solvents such as methanol, and these less-polar natural products might be responsible for the antimicrobial activity of the majority of the extracts against Gram-positive bacteria (Borquaye et al. 2016).

Some fungi, such as Diaporthe helianthi NQU760, and Diaporthe longicolla NQU772 (Santos et al. 2011), isolated from Z. simulans are well-known plant pathogens (Heller and Gierth 2001; Li et al. 2017). Several previous studies have shown that plants harbor diverse communities of endophytic fungi with antimicrobial activity, some of which are plant pathogens (Ashour et al. 2011). For example, Diaporthe helianthi, which has been reported to cause severe sunflower disease (Battilani et al. 2003), was isolated from the Brazilian plant Luehea divaricata, and found to produce natural products with antimicrobial activity (Specian et al. 2012), while Wagenaar and Clardy (2001) isolated antibiotic-producing Diaporthe longicolla, which can cause Phomopsis seed decay in soybean plants (Li et al. 2017). Barley pathogen Ramularia collo-cygni, which spends most of its life cycle as an endophyte and turns necrotrophic after months of symptomless growth in the host, was found to produce secondary metabolites with antimicrobial activity (Dussart et al. 2020). These pathogenic isolates were obtained from Z. simulans plants that did not exhibit any visible injury; therefore, a symbiotic relationship may be established between pathogenic fungi and host, with the fungi changing themselves into endophytes, which is in agreement with the hypothesis that endophytic fungi develop from pathogenic fungi of plants (Hormazabal and Piontelli 2009).

Zanthoxylum spp. include around 250 species, which have been used in Asia, Africa, and America to treat a number of diseases in humans and animals (Javier Patino et al. 2012). Among the Zanthoxylum genus, Zanthoxylum bungeanum is the only species in which endophytic fungi have been studied. Li et al. (2016) studied the diversity and antifungal activity of endophytic fungi in Z. bungeanum and found that Alternaria (30.85%), Fusarium (13.72%), and Phoma (12.77%) were the most abundant genera. However, this result differed from our findings that the most frequent species with antimicrobial activity found in Z. simulans were Penicillium (26.09%), Colletotrichum (21.74%), and Diaporthe (21.74%) (Table 1). In contrast, Araujo et al. (2018) reported that Colletotrichum, Penicillium, and Diaporthe were the three most frequent endophytic genera cultured from Brazilian Amazon rubber tree Hevea guianensis, which was consistent with our results.

In this study, we found that in summer and winter, the composition of fungal endophytes with antimicrobial activity (Table 1) and the patterns of susceptibility of pathogens to these fungi differed (Fig. 4). It should be noted that we only identified fungal isolates with antimicrobial activity, which therefore did not represent the diversity of endophytic communities. However, a possible explanation of our result could be that the composition and dominance of endophytic species in Z. simulans differs in different seasons. This result was in line with several previous studies of the diversity of fungal endophytes (de Souza Sebastianes et al. 2013), which indicated that the diversity and richness of fungal endophytes depend not only on the season but also on the geographical location (González and Tello 2011) and the part of the plant from which the sample was obtained (Gazis and Chaverri 2010).

As shown in Fig. 5a, b, we found that crude extracts of Penicillium chermesinum NQU307 and Diaporthe helianthi NQU760 possessed strong anti-inflammatory efficacies. A recent study showed that four compounds obtained from Diaporthe sp., an endophytic fungus from the Arjun tree Terminalia arjuna (Roxb.), showed anti-inflammatory activities (Patil et al. 2017). It has also been reported that plastatin and puteosporin, two naturally occurring PLA2 (porcine pancreatic phospholipase A2) inhibitors, from Penicillium chermesinum, isolated from a soil sample collected in Nova Scotia, Canada, exhibited anti-inflammatory activities (Singh et al. 1985). These results indicated that crude extracts from fermentation broth of endophytic fungi obtained from medicinal plant Z. simulans are a potential source of natural anti-inflammatory active compounds. In addition, it should be noted that these two fungi isolated in this study also exhibited antimicrobial activity. Therefore, they should be good candidates for further isolation and identification of their secondary metabolites, which may provide new drug leads.

Conclusions

As a renowned traditional medicine, the biology, ecology, and metabolite production of endophytic fungi associated with Zanthoxylum simulans have not been exploited. The present study confirmed the presence of inhibitors in crude extracts of endophytic fungi obtained from Z. simulans for the first time and also provided a foundation for the development and utilization of fungal resources obtained from this medicinal plant for the potential improvement of human welfare. Further studies to characterize the bioactive constituents of the extracts should be conducted in order to gain a greater understanding of the potential and mechanisms of these natural inhibitors.