Diversity and bioactive potential of culturable fungal endophytes of Dysosma versipellis; a rare medicinal plant endemic to China

The plant Dysosma versipellis is known for its antimicrobial and anticancer properties but is a rare and vulnerable perennial herb that is endemic to China. In this study, 224 isolates were isolated from various tissues of D. versipellis, and were classified into 53 different morphotypes according to culture characteristics and were identified by sequence analyses of the internal transcribed spacer (ITS) region of the rRNA gene. Although nine strains were not assignable at the phylum level, 44 belonged to at least 29 genera of 15 orders of Ascomycota (93%), Basidiomycota (6%), and Zygomycota (1%). Subsequent assays revealed antimicrobial activities of 19% of endophytic extracts against at least one pathogenic bacterium or fungus. Antimicrobial activity was also determined using the agar diffusion method and was most prominent in extracts from four isolates. Moreover, high performance liquid chromatography (HPLC) and ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry analyses (UPLC–QTOF MS) showed the presence of podophyllotoxin in two Fusarium strains, with the highest yield of 277 μg/g in Fusarium sp. (WB5121). Taken together, the present data suggest that various endophytic fungi of D. versipellis could be exploited as sources of novel natural antimicrobial or anticancer agents.

Resistance to antibiotics and drugs in pathogenic bacteria and fungi and overuse of antibiotics are the major challenges for researchers all over the world 1 . Thus, safer and novel antimicrobial drugs are eagerly awaited 2 , and natural secondary metabolites from endophytic fungi are increasingly considered due to their diverse structural classes and various bioactivities. These include antifungal 3 , antibacterial 4 , anticancer, anti-HIV 5 , and other promising bioactivities 6,7 . In addition, endophytic fungi are nontoxic and, thus, provide a promising source of novel drugs 8 .
Endophytic fungi inhabit living plant tissues without causing apparent disease or injury to the host 9 and are ubiquitous in vascular plant species 10,11 . Currently, less than 10% of the approximately one million known terrestrial endophytes have been investigated 12 . However, several rare medicinal plants produce important bioactive compounds to survive in unique environments and may host novel and diverse fungal endophytes 7,13 , and these have rarely been isolated and characterized.
Dysosma versipellis (Hance) M. Cheng ex Ying (Fig. 1a) is commonly referred to as podophyllum, hemipilia, fatsia, or octagonal lotus, and is a rare and vulnerable perennial herb of the Berberidaceae family 14 . This plant species is endemic to China and is mainly distributed in high altitudes ranging from 200-2400 m above sea level in disjunct stands of warm-temperate, deciduous, montane forests (Fig. 1b) across central and eastern China 15 . Dysosma species including D. aurantiocaulis, D. difformis, D. majorensis, D. pleiantha, D. tsayuensis, D. veitchii, and D. versipellis have been identified in previous studies and six of these are endemic to China 16 . As a traditional Chinese medicine, extracts from the rhizomes of this plant has been used as antibacterial treatments for syphilis and an antidote for snake bites 17 . In recent decades, D. versipellis has attracted increasing pharmaceutical attention due to the discovery of podophyllotoxin (PTOX), which is a pivotal lignan and is used as a natural source of various anticancer PTOX derivatives 18 . Recent studies show antiviral and anti-inflammatory properties of the flavonoids quercitrin and kaempferol from this plant 19 . However, due to overexploitation and slow growth, all Dysosma species have been  In further analyses, 49 representative morphotypes belonged to four classes of the Ascomycota phylum, including Dothideomycetes, Eurotiomycetes, Leotiomycetes, and Sordariomycetes. Most of the isolates (n = 28) from D. versipellis belonged to Sordariomycetes class in this study. This class was represented by seven orders: Glomerellales (7 isolates), Hypocreales (13 isolates), Diaporthales (2 isolates), Xylariales (4 isolates), Magnaporthales (1 isolate), Ophiostomales (1 isolate), Sordariales (1 isolate); and 13 genera: Acremonium, Arthrinium, Colletotrichum, Cylindrocarpon, Dactylonectria, Diaporthe, Fusarium, Hypoxylon, Ilyonectria, Pestalotiopsis, Pestalotiopsis, Volutella and Xenocremonium. Six isolates (WB5119, WB5133, WB5147, WB5148, WB5149 and WB5150) had no sequence similarities with any reference species from the GenBank database.
The present data show that D. versipellis roots and rhizomes contain a rich diversity of endophytic fungi, and we found that the most ubiquitous phylum of fungi is Ascomycota, which is reportedly among the most prevalent group of eukaryotes globally 24,25 . In addition, Sordariomycetes was the most prevalent class of endophytic species in the present study, followed by Dothideomycetes, Eurotiomycetes, and Leotiomycetes, as shown previously. We also found that 77.4% of endophytic fungi are present in roots and rhizomes of D. versipellis, and only 22.6% of fungal isolates were found in stems and leaves. Colletotrichum is a common fungal genus 26 and was abundant in the stems and leaves, but was absent in roots and rhizomes. Cylindrocarpon, Fusarium, Ilyonectria, and Rhizoctonia only colonized roots and rhizomes, whereas Alternaria, Arthrinium, Mucor, Pestalotiopsis, Phialophora, Phoma, Rhizoctonia were exclusively detected in roots. Another 19 isolates only colonized rhizomes, and Acremonium and Ochroconis were exclusively present in leaves. Pseudocercospora, Ramichloridium only colonized stems. Based on these varying spatial distributions of endophyte communities in D. versipellis, we suggested that these microbiotas have adapted to distinct tissue microenvironments, resulting in clear tissue specificity among endophytic fungi in D. versipellis, as indicated in a previous study of Indian medicinal plants 27,28 .
Additionally, the isolates WB5143 (Ramichloridium sp., Fig. 1c), WB5104 (Ascomycota) and WB5136 (Cadophora sp.) have darkly pigmented and septate hyphae of thick walls. These are referred to as dark septate fungi (DSE) and were isolated from roots. Jumpponen & Trappe suggested that DSE frequently colonize roots of mycorrhizal or nonmycorrhizal plants and play unique roles in terrestrial ecosystems 29 . However, in contrast with the common root tissue habitat of DSE, Ramichloridium sp. (WB5143) was isolated from stems of plants.
Antimicrobial activity of ethanolic fraction of culture supernatants of endophytic fungal species.
In this study, antimicrobial-producing fungi belonged to the genera Fusarium, Cladosporium, Ilyonectria,  Microsphaeropsis, Cadophora, Phoma, Rhizoctonia, Virgaria. In addition, the ethanolic extracts of two unidentified isolates also inhibited the microbial growth (Table 3). Endophytic strains of Fusarium are well-known producers of various metabolites screened in the host plants 30 ; the commercially important drug precursor PTOX was originally found in the endangered genus Dysosma 21 but is also produced by the endophytic F. oxysporum from Juniperus recurva plants 23 . Other natural agents include Taxol which was originally found in Taxus plants and was produced by endophytic F. proliferatum from Taxus x media 31 . Additionally, 2-methylbutyraldehyde-substituted α-pyrone, beauvericin, and subglutinol A and B are dominant antimicrobial compounds that are produced by endophytic Fusarium spp. isolated from medicinal plants [32][33][34] . Most members of the genus Cladosporium also produce antimicrobial compounds, and C. uredinicola from Tinospora cordifolia was found to possess anti-insect properties, potentially protecting plants against insect pests 35 . In the present study, Cladosporium sp. (WB5106) exhibited high antimicrobial activity against S. aureus, E. coli, B. subtilis, and C. tropicalis, but did not show any activity against A. fumigatus.
Interestingly, all of the present endophytic fungal strains that produce antimicrobial compounds were isolated from roots or rhizomes of D. versipellis. Similar studies had also showed medicinal plants with antifungal, antibacterial, anticancer, and antioxidant activities may provide more feasible opportunities to isolate and culture endophytic fungal producers 6,36 . However, further studies are required to characterize dynamic changes of endophytic communities 6 and uncultured fungi 30 and to confirm fungal tissue specificity in D. versipellis.
Screening of PTOX-producing fungi. Crude extracts of endophytic fungi were screened for fungal PTOX using HPLC and UPLC-QTOF MS analyses. In these analyses, PTOX from Fusarium sp. WB5121 and WB5122 had retention times that corresponded with the standard PTOX (Fig. 2) and corresponding yields were 277 and 1.25 µg/g (wet weight of crude extracts), respectively, after culture in 200 mL of potato dextrose broth (PDB) at 26 °C ± 2 °C with shaking at 125 rpm for 10 days. Associated MS spectra showed the same peak MH + at m/z 459.12 for standard and fungal PTOX from Fusarium sp. WB5122, and that of the fungal PTOX from Fusarium sp. WB5121 yielded a peak MH + at m/z 459.13 (Fig. 3), indicating the presence of endogenous PTOX in isolates of Fusarium sp. WB5122 and WB5121 strains.
In conclusion, D. versipellis harbors a rich and diverse range of endophytic fungi and provides a fungal resource for the study of PTOX and other unique secondary metabolites. Among the present endophytic fungi, 18.9% and 3.7% of strains produced antimicrobial and anticancer metabolites, respectively. Hence, future studies of metabolic pathways, mutual relationships, and fungal species identification are warranted.

Materials and Methods
Collection of plant material. The wild plant samples of D. versipellis were collected from Yongfu county, Guangxi province of China (109°36′E; 24°37′N). Samples were placed in polyethylene bags, labeled, transported to the laboratory, and refrigerated at 4 °C, as described previously 37 . Plant specimens were identified by Dr. Tan and were preserved in the herbarium of the Guangxi Botanical Garden of Medicinal Plants.
Fungal isolation and cultivation. Endophytic fungi were isolated from stems, leaves, and roots of plants.
Procedures for surface sterilization of plant tissues and isolation and cultivation of fungi are described by Tan et al. 38 . Briefly, stems, leaves, and roots were separated from plants, were washed thoroughly in running tap water, and were surface-sterilized in a sequence of 70% ethanol (v/v) for 30 s and sodium hypochlorite solution (2.5%, v/v) for 5 min. All tissues were then rinsed three times with sterile distilled water and were surface-dried with sterile filter paper. Subsequently, 0.5 × 0.5-cm pieces were excised using a sterile blade and were placed on PDA containing 50-µg/mL oxytetracycline and 50-µg/mL streptomycin. Nine segments were plated per Petri dish (90-mm diameter). Petri dishes were then wrapped in parafilm and were incubated at 25 °C in the dark for more than one week. Samples were checked daily and colonies were routinely isolated, purified, and maintained in PDA for DNA extraction, PCR amplification, sequencing, and molecular identification. To produce fungal mycelia, all strains were grown on PDA plates at 25 °C for 10 days. Mycelia were scraped using sterile pipette tips and were then freeze-dried, and DNA from endophytic fungi were then extracted using E.Z.N.A.TM Fungal DNA Mini Kits (Omega Bio-tek, Norcross, USA) according to the manufacturers' instructions for use as templates in polymerase chain reactions (PCR). The primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) were constructed for molecular phylogenetic studies and were used to amplify ribosomal internal transcribed spacers (ITS) 39    The sequences obtained in this study were previously submitted to the GenBank database with accession numbers from KY940469 to KY940519.
Crude extract preparation of fungal fermentation broth. Fifty-three strains were precultured on PDA (potato extract, 200 g/L; dextrose, 20 g/L) for 7 days, and five plugs (6 mm of diameter) of each fungus were then pre-inoculated into 500-mL Erlenmeyer flask containing 200-mL PDB containing 200 g/L potato extract and 20 g/L dextrose. All cultures were incubated on a rotary shaker (125 rpm) at 26 °C ± 2 °C in the dark for 10 days. Cultures were then filtered to collect fermentation broth and wet mycelia were discarded. Fermentation broth was extracted with four volumes of ethanol for one day and filtrates were further concentrated in vacuo to remove organic solvent 41 . Concentrates were then volatilized in a water bath at 60 °C and dried residues and were finally stored at −20 °C. Crude extracts were diluted with 10% dimethyl sulfoxide (DMSO) to 10 mg/mL and were sterilized by filtration using a Millipore filter (0.22 µm) prior to antimicrobial assays.
Antimicrobial activity. Five pathogens, including the fungi A. fumigatus and C. albicans and bacteria E. coli, B. subtilis, and S. aureus, were used to test antimicrobial activities of 53 crude fungal EtOH extracts, and inhibitory effects were assayed using the agar diffusion method with 10-mg/mL extracts at 100 µg/disk. Ampicillin sodium (100 µg/disk) and fluconazole (25 µg/disk) were used as positive antimicrobial controls and 10% DMSO was used as a negative control. Antimicrobial activities were determined according to diameters of inhibition zones (ZI) and experiments were repeated three times.
The MS was operated in negative ion mode and was set to total ion chromatogram mode with the following mass conditions: capillary voltage = 2500 V, cone voltage = 40 V, low collision energy = 6 V, source temperature = 100 °C, desolvation temperature = 400 °C, and desolvation gas flow = 800 L/h. Data acquisition and processing were conducted using MassLynx version 4.1 (Waters, Manchester, UK).

Statistical analyses.
Colonization rates (CR%) of fungal strains isolated from D. versipellis were calculated as follows: CR% = (Nsc/Nss) × 100, where Nsc represents the number of segments infected by fungi and the Nss represents the total number of segments investigated 43 . Isolation rates (IR%) of the strains were calculated as follows: IR% = (Ni/Nt) × 100, where Ni represents the number of segments from which fungal species were isolated and Nt is the total number of segments incubated 44 . The diversity of fungal species from D. versipellis was evaluated using the Shannon-Weiner Index (H′) with the following formulas: where ni represents the numbers of individuals and N represents the total number of individuals 45 . All statistical analyses were performed using SPSS 19.0 (SPSS Inc., Chicago, IL, USA).