Introduction

Coptis chinensis Franchet (Ranunculaceae), which is known as Weilian (one of three kinds of Huanglian) in China, is an important and well-known traditional Chinese medicinal plant. Its rhizomes, which contain alkaloids as the main bioactive components and some other types of natural products including organic acids, phenylpropanoids, lignans, flavones, volatile oils, saccharides, and steroids (Rui et al. 2020), have been frequently utilized for the treatment of various diseases including bacillary dysentery, typhoid, tuberculosis, epidemic cerebrospinal meningitis, empyrosis, pertussis, and other illnesses (Meng et al. 2018; Wang et al. 2019). Recent studies have indicated that the rhizome of C. chinensis has wide pharmacological activities, including antibacterial, antiviral, antifungal, anti-atherosclerosis, antimyocardial ischemia/reperfusion injury, anti-hyperlipidemia, antihypertension, anti-obesity, anticancer, anti-Alzheimer’s disease, anti-inflammation, antioxidation, and aging-related diseases (Xu et al. 2017; Ran et al. 2019; Wang et al. 2019, 2020a). Due to its age-old use in traditional medicine and perennial dug, the resource of wild C. chinensis is extremely rare. Furthermore, the cultivated C. chinensis requires long-term cultivation and a strict growth environment to maintain its quality. Therefore, we studied the endophytic fungi as a possible alternative to C. chinensis.

Endophytic fungi refer to groups of fungi that inter and/or intracellularly colonize healthy tissues of host plants during all or part of their lifecycle without causing obvious pathogenic symptoms (Aly et al. 2010; Xiao et al. 2021). There is a very complex relationship between endophytic fungi and their host plants (Du et al. 2020). Endophytic fungi have the ability to produce hormones and/or promote the biosynthesis of host plant secondary metabolites (Ming et al. 2013), enabling host plants to grow quickly and resist external biotic and abiotic stresses(Yan et al. 2019), and produce many products with various biological activities (Gupta et al. 2020). These bioactive compounds, which belong to various structural groups including alkaloids, peptides, steroids, terpenoids, phenols, quinones, phenols, and flavonoids, possess a variety of biological properties that include antibacterial, antiviral, antifungal, antiprotozoal, antiparasitic, antioxidant, immunosuppressant, and anticancer functions (Manganyi and Ateba 2020).

Staphylococcus aureus, the most common pathogen of food contaminants, is recognized for its virulence and its ability to produce staphylococcal enterotoxins and cause a variety of diseases, such as local suppurative infection, osteomyelitis, pneumonia, pseudomembrane, enteritis, meningitis, pericarditis, and toxic shock syndrome (Odeyemi et al. 2018; Wang et al. 2020b; Matias et al. 2021). Antibiotic therapy is an important strategy for S. aureus control. However, as a response to the selective pressure of antimicrobials, the multi-drug resistant S. aureus appeared and the treatment options for clinicians and veterinarians have been narrowed (Wang et al. 2018). It reported that methicillin-resistant S. aureus (MRSA), one kind of multi-drug resistant S. aureus, spread across the world causing a variety of nosocomial infections, community infectious diseases and contaminated exceeded 50% of cases in intensive care unit (ICU) patients since 1999 (Matias et al. 2021). Therefore, it is in high demand for developing novel antibacterial agents for MRSA infection treatment.

As a rich source of novel natural products, the endophytic fungus is a treasure house for developing novel antibacterial agents. Moreover, C. chinensis was reported to have strong antibacterial properties against MRSA (Kim et al. 2020). Therefore, this study aimed to determine the diversity and anti-MRSA activity of endophytic fungi from the medicinal plant C. chinensis. In this research, the diversity of the endophytic fungi isolated from the healthy fibrous root, rhizome, and leaf of C. chinensis was evaluated, and the antimicrobial activities against MRSA were examined. To the best of our knowledge, this work is the first report on the biodiversity, phylogeny, and assessment for the anti-MRSA activity of endophytic fungi harbored in C. chinensis.

Materials and methods

Collection of plant material

Healthy and symptomless 3-year-old C. chinensis plants were collected from Huangshui Town, Shizhu County, Chongqing, China. Selected plant samples were immediately transported to the laboratory and preserved at 4 °C. The C. chinensis plants were identified by Professor Peng Li from the College of pharmacy at the Army Medical University. For isolation of fungal endophytes, samples were processed within 24 h after harvest.

Isolation and purification of fungal endophytes

The isolation of fungal endophytes from collected plant parts was according to a standard procedure established previously (Stierle et al. 1993). The fibrous root, rhizome, and leaf of C. chinensis plants were used to isolate endophytic fungi. These three kinds of tissue were thoroughly washed in running tap water for 3–6 h followed by drying with sterile filter paper. Then, the cleared tissue was surface-sterilized by sequential immersion in 75% ethanol for 30 s, 1.3 mol/L sodium hypochlorite (3–5% available chlorine) for 2–5 min, and 75% ethanol for 30 s. All tissues were then rinsed three times in sterile water to remove any excess surface sterilants. After sterilization, the samples were dried off with sterile filter paper and cut into small pieces (about 0.5 cm) using a sterile blade. These tissue pieces were placed on potato dextrose agar (PDA) plates with 50 mg/L penicillin at 25 °C to isolate the fungal endophytes. The tissue pieces were observed every day for mycelia growth. Once the fungal mycelia grew out of the tissue pieces, the hyphal tips were transferred using a sharp sterile sharp needle to fresh PDA plates. Morphologically distinct isolates were similarly sub-cultured several times to purify and obtain pure isolates. The pure isolates were numbered and kept in a storage tube at − 20 °C for further study.

Molecular identification

Identification of fungal isolates was based on molecular and morphological analysis. The molecular identification was carried out using the Internal Transcript Spacer regions (ITS1 and ITS2) and the intervening 5.8 S rRNA region sequencing. Each fungal isolate was cultured on a PDA plate at 25 °C for up to 10 days, and the fungal mycelia were collected. The genomic DNA was extracted using the traditional cetyltrimethylammonium bromide (CTAB) method (Saghai-Maroof et al. 1984). The isolated DNA was air-dried, dissolved in 20 μL of sterile Millipore water, and stored at − 20 °C for further study. Polymerase chain reaction (PCR) was performed to amplify the ITS region of the fungal isolates using the universal ITS primers, ITS5 (5′-GGAAGTAAAAGTCG TAAGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). The PCR reaction mixture (25 μL) contained 1 μL template (50 ng/μL purified DNA sample), 15 ρmol of each primer, 12.5 μL 2 × Taq PCR Master Mix, and 10.5 μL ddH2O. PCR conditions performed were as follows: initial denaturation at 95 °C for 3 min, followed by 35 cycles of 94 °C for 40 s, 52 °C for 50 s, and 72 °C for 1 min, and a final extension at 72 °C for 10 min. The amplified PCR products were checked on 1% agarose gel and then sent to Sangon Biotech (Shanghai) Co., Ltd for sequencing. All obtained fungal ITS sequences have been deposited in GenBank and analyzed using BLAST search in the National Center of Biotechnology Information (NCBI) database to compare the sequence homology with closely related organisms. Then, the sequences from closely related organisms were downloaded to conduct the phylogenetic analysis using the neighbor-joining (NJ) method. Bootstrap analysis was carried out using 1000 replications with MEGA6.

Diversity analyses of endophytic fungi

The relative frequency (RF) was used to estimate an endophytic-specific taxon from the sampled plants (Yao et al. 2017). The colonization frequency (CF) (formula: \(CF=\frac{N\mathrm{col}}{Nt}\times 100\mathrm{\%}\), Ncol is the number of segments colonized by an individual species and Nt is the total number of incubated segments) reflect the extent of endophyte infection (Zhang et al. 2021). The diversity of fungal species from C. chinensis was evaluated using the species richness index (S), Shannon–Wiener index (H′) and Simpson’s diversity index (1-D). The species richness index (S) was obtained by counting the number of endophytic fungal species in corresponding plant tissues. The Shannon–Wiener index (H′) and Simpson’s diversity index (1-D) were evaluated using the Past ver. 3 software (Fan et al. 2020). Sorensen’s index of similarity (QS) (formula: \(QS=\frac{2a}{2a+b+c}\), a is the number of common species in both endophytic tissues, while b and c are the number of species specific to the compared tissues, respectively) was used to evaluate the similarity of endophytic fungal assemblage among different tissues (Zhang et al. 2021).

Extraction of secondary metabolites of different endophytic fungi

Submerged cultivation of the fungal endophytes was carried out to produce secondary metabolites. Five agar plugs (5 mm diameters) of grown isolates were transferred to 250 mL of the Erlenmeyer flask containing 100 mL potato dextrose broth, followed by incubation for 5–7 days at 28 °C under constant shaking (180 rpm). The final cultivation step was performed by transferring the pre-inoculum into 500 mL of the Erlenmeyer flask containing rice solid medium supplemented with 2 g peptone and incubating for 6 weeks at room temperature. After cultivation, the secondary metabolites of each endophyte were extracted by ultrasonic with methanol (analytical grade). Then, the extract solution was concentrated under reduced pressure and dissolved in fresh methanol.

Bacteria and culture methods

Three S. aureus strains (ATCC 25923, clinical strains sequence type (ST)-5 and ST-59) were provided by professor Hao Zeng from the National Engineering Research Center of Immunological Products. Bacteria were grown on Mueller–Hinton agar (MHA) plates at 37 °C overnight, followed by picking and inoculating single colonies in Mueller–Hinton broth (MHB) at 37 °C for 12 h with agitation (220 rpm). An appropriate dilution of bacteria was done with PBS, and the bacterial count was adjusted by determining the absorbance spectrophotometrically at 600 nm.

Antibacterial activity

Crude methanol extracts of each fungal endophyte were evaluated for their antibacterial activity against S. aureus ATCC 25923, ST-5, and ST-59. Five milliliter extract solution of each fungal endophyte was dried at 60 °C and re-dissolved with 50% methanol at the concentration of 50 mg/mL. Sterile filter paper discs with 5 mm diameters were soaked in the test samples and control (methanol) for 2 h followed by natural volatilization and ultraviolet sterilization. Subsequently, the filter papers were placed separately on plates inoculated with an appropriate count of different strains of S. aureus. The plates were incubated at 37 °C for 24 h. The antibacterial potential of fungal endophyte extracts was determined following the diameter of the zone of inhibition around the filter paper. Extract samples that showed the zone of inhibition around the filter papers were considered as positive, while those without the zone of inhibition around the filter papers were negative. Experiments were replicated three times and data were presented as means ± SEM.

Results

Isolation and identification of culturable endophytic fungi from C. chinensis

In this study, different healthy tissue of C. chinensis (Fig. 1a), including fibrous root (Fr), rhizome (R), and leaf (L), were used to isolate endophytic fungi. We isolated 79 endophytic fungal strains from those tissues of seven C. chinensis plants. Among these strains, nine (11.39%) were isolated from fibrous roots, 20 (25.32%) were isolated from the rhizomes, and 50 (63.29%) were isolated from leaves, respectively (Fig. 1b). These results indicated that the leaves contained the most endophytic fungi and the fibrous roots contained the least endophytic fungi.

Fig. 1
figure 1

C. chinensis and its different tissue a; isolation rate of endophytic fungi from different tissue of C. chinensis b; endophytic fungi isolated from C. chinensis c

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Based on the fungal morphological characteristics and appearance on PDA, all the endophytic fungal strains isolated from C. chinensis were assigned to 27 representative morphotypes (Fig. 1c). Subsequently, ITS rDNA sequences of each morphotype were generated and were submitted to NCBI for BLASTn comparison. Table 1 shows the identified endophytic fungal isolates, including GenBank accession number, homolog sequences, sequence identity, and closest accession number. The maximum sequence identity of the ITS rDNA sequences of endophyte compared with those available in GenBank ranged from 98.26 to 100% (Table 1). Endophytic fungi with 100% base similarity of ITS gene sequence between the reference isolates in GenBank are considered to be identified into species. The others with base similarity of ITS gene sequence between the reference isolates at the range of 98 ~ 99.9% are considered to be identified to genera. Therefore, 10 of the 27 representative morphotypes were identified to genus, while the other 17 were identified to species (Table 1; Fig. 2). Furthermore, all the endophytic fungal isolates were identified to belong to two phyla (Ascomycota and Basidiomycota), three classes (Agaricomycetes, Dothideomycetes, and Sordariomycetes), six orders, 11 families, 12 genera, and 27 species (Fig. 2).

Table 1 The ITS sequence identification and the relative frequency of the endophytic fungal strains from C. chinensis
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Fig. 2
figure 2

Neighbor-joining phylogenetic tree of 27 representative isolates from C. chinensis. The phylogenetic tree is based on ITS gene sequences. The values at each node represent the bootstrap values from 1000 replicates, and the scale bar represents 0.02 substitutions per nucleotide. Penicillium verruculosum (JQ889708) was used as an outgroup. (diamond, the maximum identity ITS rDNA sequences of the endophyte available in GenBank.)

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The relative frequency (RF) analyses of endophytic fungi from C. chinensis

The relative frequency (RF) values of the 27 representative isolates are shown in Table 1. By comparing the RF values, the most frequent species of C. chinensis belonged to C. gloeosporioides and F. avenaceum with RF both of 11.39% (Table 1). In addition, the most frequent genera of C. chinensis associated endophytic fungi belonged to Colletotrichum and Fusarium with RF of 49.37 and 26.58%, respectively (Fig. 3a). These results indicated that the dominant fungal species of C. chinensis were C. gloeosporioides and F. avenaceum, and the dominant fungal genera of C. chinensis were Colletotrichum and Fusarium, respectively.

Fig. 3
figure 3

Relative frequency (RF) of endophytic fungi from C. chinensis at the level of genus a and in different tissue (Fr, fibrous roots; R, rhizomes; L, leaves) of C. chinensis at the level of genus b

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On the basis of different tissues, the most frequent endophyte in fibrous root or rhizome belonged to F. avenaceum, and the most frequent endophyte in leaf belonged to C. gloeosporioides (Table 1). Furthermore, the most frequent genera in fibrous root or rhizome belonged to Fusarium with RF of 77.78 or 70.00% respectively, while the most frequent genera in leaf belonged to Colletotrichum with RF of 78.00% (Fig. 3b). These results indicated that the dominant endophyte in fibrous root or rhizome was F. avenaceum, and that in leaf was C. gloeosporioides. In addition, the dominant genus in fibrous root or rhizome was Fusarium and that in leaf was Colletotrichum.

Diversity of endophytic fungi from C. chinensis

As shown in Table 2, the indices of colonization frequency, species richness (S), Shannon–Wiener (H′), and Simpson’s diversity index (1-D) are listed to characterize the endophytic community from the three kinds of plant tissue. The colonization frequency of the endophytic fungi in whole plant of C. chinensis was 37.86%. Among the different tissue, colonization frequencies of the endophytic fungi were found in the following order: leaf (53.57%) > fibrous root (28.57%) > rhizome (26.79%). The colonization frequency of the endophytic fungi in leaf was significantly higher than that in fibrous root or rhizome. The difference in the colonization frequency between fibrous roots and rhizomes was not significant.

Table 2 Colonization frequency and diversity indices of endophytic fungi isolated from different tissues of C. chinensis
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The species richness (S) of endophytic fungi from C. chinensis was 27. Among the different tissue, species richness (S) of the endophytic fungi was found in the following order: leaf (15) > rhizome (9) > fibrous root (6). The Shannon–Wiener Index (H′) of endophytic fungi from C. chinensis was 2.992. Among the different tissue, the Shannon–Wiener Index (H′) of the endophytic fungi was found in the following order: leaf (2.447) > rhizome (2.013) > fibrous root (1.581). The Simpson’s diversity index (1-D) of the endophytic fungi from C. chinensis was 0.9373. Among the different tissue, Simpson’s diversity index (1-D) of the endophytic fungi was found in the following order: leaf (0.8968) > rhizome (0.8450) > fibrous root (0.7407). These results indicated that C. chinensis harbored abundant fungal resources, and the diversity of endophytic fungi from leaves was more abundant than that in the other two tissues.

Tissue specificity of endophytic fungi from C. chinensis

The Sorenson similarity analysis for the endophytes among the three kinds of tissue indicated the various species of endophytic fungi from C. chinensis with obvious tissue specificity. The Sorenson similarities for the endophytes between fibrous root and rhizome, between fibrous root and leaf, and between rhizome and leaf were 0.40, 0.00, and 0.00, respectively (Table 3). It indicated that the endophytic community in the leaf was quite distinct from that in the rhizome or fibrous root. Similar results were observed in Fig. 2. At the order classification level, endophytic fungi of Sordariales, Glomerellales, and Pleosporales in C. chinensis were almost distributed in the leaf except for Paraboeremia litseae HL-17 (Pleosporales). Furthermore, endophytic fungi of Hypocreales and Cantharellales were only distributed in rhizome or fibrous root. At the genus classification level, endophytic fungi of Colletotrichum, Calophoma, Botryosphaeria, Leptosphaeria, Cladorrhinum, and Didymella were only distributed in leaf of C. chinensis. Furthermore, endophytic fungi of Clonostachys and Diaporthe were only distributed in rhizome, while that of Paraboeremia, and Trichoderma were only distributed in fibrous root. Otherwise, endophytic fungi of Fusarium were distributed in both fibrous root and rhizome. All these results indicated that the endophytic fungi from C. chinensis had an obvious tissue specificity.

Table 3 Sorenson’s similarity for endophytic fungi from different tissues of C. chinensis
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Anti-MRSA activity screening of the methanol extracts from endophytic fungal cultures

To estimate the anti-MRSA activities of culturable endophytic fungi from C. chinensis, antimicrobial activities of methanol extracts of 27 representative isolates grown on rice solid fermentation media were evaluated by the measurement of the diameter of the zone of inhibition against the three S. aureus strains (ATCC 25923, clinical strains ST-5 and ST-59). S. aureus ATCC 25923, which is no methicillin-resistant, is used as a standard laboratory testing control strain. S. aureus ST5 is a hypertransmissible, strong biofilm-forming, methicillin-resistant, and virulent genotype that is frequently encountered in today’s operating room environments (Loftus et al. 2018). S. aureus ST 59 is an epidemic lineage of community-associated methicillin-resistant S. aureus in Asia (Feng et al. 2017).

As shown in Table 4, nine endophytic fungal methanol extracts exhibited inhibitory activities against at least one S. aureus strain and the other 18 extracts did not show antimicrobial activities (data not shown). These nine strains belonged to genera of Fusarium (4), Colletotrichum (3), Calophoma (1), and Paraboeremia (1). The zone of inhibition of Colletotrichum fungi (HL-10, HL-13, and HL-25), Calophoma fungus (HL-5), and Paraboeremia fungus (HL-17) against S. aureus ATCC 25923 were significantly larger than that of Fusarium fungi (HL-1, HL-7, HL-23, and HL-27). Furthermore, no significant difference in the diameters of the zone of inhibition found among the Colletotrichum fungi (HL-10, HL-13, and HL-25), Calophoma fungus (HL-5), and Paraboeremia fungus (HL-17). Among the nine strains, three (P. litseae HL-17, Fusarium sp. HL-23, Fusarium sp. HL-27) exhibited obvious inhibition against all the three strains of S. aureus (ATCC 25923, ST 5, and ST 59) (Fig. 4) and the other 6 just exhibited obvious inhibition against S. aureus ATCC 25923 (Table 4). It is interesting to note that only two strains (HL-23 and HL-27) of the four Fusarium fungi exhibited significant inhibition against all the three strains of S. aureus and the inhibition of HL-17 against all the three strains of S. aureus was much stronger than that of HL-23 or HL-27.

Table 4 Anti- MRSA activities of endophytic fungi obtained from C. chinensis
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Fig. 4
figure 4

Anti-MRSA activities of endophytic fungi Paraboeremia litseae HL-17, Fusarium sp. HL-23, and Fusarium sp. HL-27 were obtained from C. chinensis. The white sterile filter paper discs soaked in methanol for 2 h followed by natural volatilization and ultraviolet sterilization were regarded as negative controls

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Discussion

As an important and well-known traditional Chinese medicinal plant, C. chinensis has been used to treat various inflammatory disorders and related diseases for a thousand years. As far as we know, there is no report about the endophytic fungal diversity of C. chinensis. In our study, 79 endophytic fungi isolated from C. chinensis fibrous root, rhizome, and leaf were divided into 27 species, 12 genera, 11 families, six orders, three classes, and two phyla. Two phyla of all endophytic fungi are Ascomycetes and Basidiomycetes. Ascomycetes (74 isolates) are the most common representatives of endophytic fungal communities, while Basidiomycetes constitutes the rest five isolates. This result is consistent with the reports of endophytic fungal contents of most plants (Du et al. 2020). Among the 12 genera, Colletotrichum and Fusarium were the dominant ones. Thus, the host has a strong affinity towards establishing symbiotic associations with the fungi belonging to the genera Colletotrichum and Fusarium. Among the 27 species, C. gloeosporioides and F. avenaceum were the dominant ones. However, F. avenaceum has been previously reported as a pathogen confirmed by re-inoculated back on C. chinensis (Mei et al. 2020). Several studies indicate that the endophytes isolated from asymptomatic plant tissues express either mutualistic, commensal, or parasitic lifestyles when re-inoculated back on the original host species (Rodriguez and Redman 2008). Therefore, there is a balance between the fungi and the host plants. When the fungus was inoculated to the host plant, it would be an endophytic fungus if a balanced antagonism is established. Otherwise, it would be a pathogen resulting in disease.

In our study, the analysis of colonization frequency, species richness (S), Shannon–Wiener (H′), and Simpson’s diversity index (1-D) indices indicated that C. chinensis harbored abundant fungal resources. The diversity of endophytic fungi in the above-ground tissues (leaf) of C. chinensis was more than that in the under-ground parts (rhizome and/or fibrous root). The result is similar to fungal endophytes from Glycyrrhiza glabra (Arora et al. 2019). In the research of Dillenia indica, the percent frequency of endophytic fungi was also highest in leaves (Kumar and Prasher 2022b). Furthermore, obvious tissue specificity of endophytic fungi from C. chinensis was observed through the Sorenson similarity analysis. The endophytic fungal community structure of leaf was utterly distant from that of rhizome or fibrous root. The difference of micro-environments in the different tissue would be responsible for the different microbiota.

MASA has been considered as the prototype of multiresistant nosocomial pathogens. Methicillin belongs to the β-lactam class of compounds that are hydrolyzed by β-lactamase. β-Lactams are the most widely used broad-spectrum antibiotics, and resistance to β-lactams is a serious threat for infectious disease management, surgery, and organ transplantation. The emergence of MRSA indicates an urgent need for the control of the use of antibiotics and the development of novel therapeutic agents. The main active constituents of C. chinensis are protoberberine alkaloids, which can affect the accumulation of amyloid fibers in MRSA biofilm phenol-soluble modulins (PSMs), thus inhibiting the formation of MRSA biofilm and increasing the bactericidal activity of antibiotics. However, the low bioavailability and weak effect of berberine limit its clinical application. Therefore, structural modification and new isomers of berberine are required. Endophytic fungi are a rich source for developing novel host-like biomolecules. Some endophytic fungi isolated from the antimicrobial medicinal plants had shown significant antimicrobial activity (Al Mousa et al. 2021; Deshmukh et al. 2022; Kumar and Prasher 2022a). In our research, methanol extracts of nine endophytic fungi (33% of total screened) showed inhibitory activities against S. aureus ATCC 25923. Furthermore, three (P. litseae HL-17, F. sp. HL-23, F. sp. HL-27) of the nine endophytic fungi exhibited obvious inhibition against the two methicillin-resistant S. aureus strains (ST-5 and ST-59). Genus of Fusarium was previously reported to show antibacterial activity against S. aureus (Ratnaweera et al. 2015; Wen et al. 2015). However, new antibacterial compounds were constantly being discovered from the fungus of Fusarium (Uz Zaman et al. 2021). Moreover, there is no report about P. litseae producing antimicrobial compounds. Thus, three endophytic fungi obtained in this study with antimicrobial activity against the three S. aureus strains have the potential to produce natural products of novel structures. Furthermore, three new polyketides and three new pyrrole alkaloids, which had potential anti-inflammatory activity, were isolated from three endophytic fungi of C. chinensis (Wei et al. 2022; Yin et al. 2022a, b) and one endophytic fungus isolated from C. chinensis was reported to produce berberine (Zhang et al. 2016). Therefore, it is noteworthy to further investigate the secondary metabolites of the three endophytic fungi (P. litseae HL-17, Fusarium sp. HL-23, and Fusarium sp. HL-27) from C. chinensis.

In this study, we report for the first time the diversity and anti-MRSA activity of fungal endophytes from C. chinensis. This research extended our knowledge on the distribution of endophytic fungi in C. chinensis and endophytic fungi from C. chinensis could be considered as a potential source for anti-MRSA agent in future biotechnology applications.