Endophytic Microorganism From the Endangered Plant Nervilia Fordii (Hance) Schltr. In the Southwest Karst Area of China: Isolation, Genetic Diversity and Potential Functional Discovery

The plant Nervilia fordii (Hance) Schltr. is known for its antimicrobial and antitumor properties. It is a rare and vulnerable perennial herb of the Orchidaceae family. In this study, 984 isolates were isolated from various tissues of N. fordii. and were identied through the sequence analyses of the internal transcribed spacer region of the rRNA gene. Except for 12 unidentied fungi, all others were aliated to at least 39 genera of 14 orders of Ascomycota (72.66%) and Basidiomycota (19.00%). Antimicrobial activity was determined by using the agar diffusion method. Subsequent assays revealed 20 strains of fungal endophytes exhibited antibacterial activity against at least one pathogenic bacterium or fungus. Moreover, the capability of promoting seed germination was evaluated on the basis of the interaction of Bletilla striata seeds with the isolates. Results revealed that the three isolates could promote B. striata seed germination. After 21 days, the germination rate under treatment with the best strain was 97.89%, which was higher than that under the control treatment (12.68%). Taken together, the present data suggested that various endophytic fungi of N. fordii could be exploited as sources of novel natural antimicrobial agents or used for the articial breeding of rare orchids. days, ve PDA agar plugs (diameter of 2 mm) with active mycelial growth from the colony margin were inoculated into a Petri dish (diameter 9 mm) containing 15 mL of sterile oatmeal agar (2.5 g·L −1 oatmeal, 12 g·L −1 agar, and pH of 5.2 prior to autoclaving) with ve pieces of nylon cloth (1 × 1 cm), and grown at 25 °C in dark 61,62 . The seeds of the terrestrial orchid plant B. striata were selected as the experimental subject for seasonal reasons. The seeds were surface sterilized with sodium hypochlorite solution for 10 min to remove fungal contamination. After 1 week, approximately 150 axenic seeds of B. striata (Fig. 3 a) were sown on the surface of each piece of cloth. Each treatment was replicated on ve plates. The treatment without fungi was used as the control. All treatments were placed in a tissue culture chamber under 12 h of light exposure at 25 °C for 70 days. A stereomicroscope (LEICA TL3000 Ergo) was used to assess and record seed germination and protocorm development. Stewart et al. 63 had divided the seed germination and protocorm development of orchids into six stages (0–5). Three seedling growth stages were added in this study as shown in Table 3 showing as a referencefor assessing the percentage of seed germination and protocorm development of B. striata. Daldinia, Diaporthe, Phomopsis, Leptostroma, and an unknown Magnaporthaceae genus. 181 isolates were assigned to class Dothideomycetes, comprising the ve orders of Pleosporales, Cladosporiales, Muyocopronales, Venturiales and Botryosphaeriales and 15 genera (Acrocalymma, Letendraea, Periconia, Ascochyta, Epicoccum, Phoma, Alternaria, Bipolaris, Exserohilum, Sclerostagonospora, Torula, Dictyosporium, Cladosporium, Mycoleptodiscus and Phyllosticta,). 34 isolates were assigned to fordii. And those that were mainly distributed in the rhizome. However, further studies are required to characterize the dynamic changes in endophytic communities 28 and uncultured fungi 29 and to conrm fungal tissue specicity 30 in N. fordii. The results suggested that endophytic fungi from N. fordii. are potential sources of natural antimicrobial products. Seed germination trials. This study aimed to obtain isolates with high seed germination activities from N. fordii plants. The seeds of the orchid plant Bletilla striata were selected as the experimental subject for seasonal reasons.


Introduction
Endophytic fungi have been detected in all plant species investigated thus far 1 . They inhabit living plant tissues for at least parts of their life cycles without causing any apparent disease or injury to their host 2 and are ubiquitous in vascular plant species 3 . All orchids require endophytic fungi. They play a key role in supporting and enhancing plant health and growth 4,5 , such as produce different plant hormones to enhancing plant growth 6 , protecting the host plant against phytopathogenic microorganisms or pests 7,8 and so on. In addition, they can in uence plant ecology; tness; the evolution of plant community structures; and the diversity of interacting organisms, including nematodes and insects 9,10 . Endophytes represent a relatively underexplored and attractive source of natural products that are suitable as novel sources of bioactive metabolites, including metabolites with anticancer, antimicrobial, antimalarial, and other activities, and have thus elicited considerable attention from many researchers 11,12 . Currently, although many endophytic fungi from various terrestrial plants are gradually being described and explored, they account for less than 10% of the approximately one million known terrestrial endophytes have been investigated 13 .
Nervilia fordii (Hance) Schltr. (Fig. 1 a) is a rare and vulnerable perennial herb of the Orchidaceae family; the whole plant (including rhizome, Fig. 1 b) or the aerial part is used as a traditional Chinese medicine called Qing Tian Kui 14,15 . This plant species is endemic to the southwest karst area of China ( Fig. 1 c) and is mainly distributed at altitudes ranging from 220-1,000 m above sea level in sheltered valley or hillside areas 16,17 . N. aragoana, N. cumberlegii, N. fordii, N. lanyuensis, N. mackinnonii, N. plicata (Andr.) Schltr. var. plicata, N. taiwaniana, and N. plicata (Andr.) Schltr. var. purpurea (Hayata) S. S. Ying have been identi ed as endemic to China in previous studies 18 . N. fordii is a traditional Chinese medicine that has long been used in Chinese folk medicine for the treatment of various respiratory diseases, such as bronchitis, stomatitis, acute pneumonia, and acute pharyngitis 19,20 . N. fordii has received considerable attention in modern pharmacology research because of its biological behaviors, which include antimicrobial 21 , antitumor 22 , antiviral 23 , and antiin ammatory 24 behaviors. Therefore, the present work aimed to investigate the species diversity of the culturable endophytic fungi in N. fordii collected from Guangxi Provinces, China, via rDNA internal transcribed spacer (ITS) sequences analysis. The endophytic fungi were screened for antimicrobial activities, and their bene t to seed germination was determined. The results of this report are helpful for exploring the potential sources of novel natural antimicrobial and the actual propagation and conservation of orchids.

Materials And Methods
Ethics Committee approval was obtained from the Institutional Ethics Committee of Guangxi University of Chinese Medicine to the commencement of the study. We con rm that all methods were performed in accordance with the relevant guidelines and regulations.
Collection of plant material. In 2020, he samples of N. fordii wild plants were collected from Sanhaung Townlet, Guangxi Province, China (109°66′E; 24°94′N). Healthy N. fordii plants were selected. Whole plants were dug out and placed in pots with rhizosphere soil and environmental soil, labeled, and transported to the laboratory within 12 h. The plant specimens were identi ed by Professor Tan and were preserved in the herbarium of the the Guangxi Botanical Garden of Medicinal Plants (voucher ID: SHNF20200618).
Fungal isolation and cultivation. Endophytic fungi were isolated from the corms, rhizomes, and leaves of the plant specimens. Procedures for the surface sterilization of plant tissues and the isolation and cultivation of fungi have been previously described by Tan et al. 3,10 . Brie y, roots, rhizomes, and leaves were separated from the plants; washed thoroughly in running tap water to remove dirt; and surface-sterilized sequentially in 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 lter paper. Subsequently, 0.5 cm × 0.5 cm pieces were excised by using a sterile blade and placed on PDA containing 50 µg·mL −1 oxytetracycline and 50 µg·mL −1 streptomycin. Seven segments were plated per Petri dish (90 mm diameter). The Petri dishes were then wrapped in para lm and incubated at 25 °C in the dark.
They were observed for the growth of fungi from the segments every 2 days for more than 1 week. The colonies were routinely isolated, puri ed, and maintained in PDA for identi cation and antimicrobial assays. Pure endophytic fungi were nally photographed and preserved in the Scienti c Laboratory Center, Guangxi University of Chinese Medicine.
DNA extraction, PCR ampli cation, sequencing, and molecular identi cation. For the production of fungal mycelia, all strains were grown on PDA plates at 25°C for 1-2 weeks (the diameter of fungal colony was approximately 4 cm, which could meet the requirement of DNA extraction). Mycelia were scraped by using sterile pipette tips and were then freeze-dried, DNA from endophytic fungi was then extracted using by E.Z.N.A.TM Fungal DNA Mini Kits (Omega Bio-tek, Norcross, USA) were utilized in accordance with 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) 56 The PCR mixture (50 µL) contained 25 µL of 2× SanTaq PCR Mix (Sangon Biotech, Shanghai), 2 µL of each primer (5 µM), and 2-10µL of genomic DNA (20-50 ng·µL −1 ) and brought to a volume of 50 µL with ddH 2 O. PCR was performed on a thermal cycler (BioRAD) as follows: incubation at 94 °C for 3 min; followed by 35 cycles of 94 °C for 30 s, 55 °C for 25 s, 72 °C for 30 s; and then a nal extension step at 72 °C for 7 min. Subsequently, PCR products were puri ed and sequenced at the Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. The sequences were then BLASTed the sequences of known isolates in the NCBI database (http://www.ncbi.nlm.nih.gov) 57 . Only those matching previously published sequences with high similarity were used. All identi ed isolates were categorized at the genus or family levels in accordance with the ownership criterion as follows: species of the same genera had sequence similarity (SS) ≥ 95% and those of the same families had SS ≤ 95% 58,59 .
Antimicrobial activity. Three pathogens, including the fungi C. tropicalis and bacteria E. coli and S. aureus, were used to test the antimicrobial activities of all strains. Inhibitory effects were assayed by using the fungus cake method 60 . Streptomycin (20 μL, 5 mg·mL −1 ) and tetracycline (20 μL, 5 mg·mL −1 ) were used as positive antimicrobial controls, and PDA agar plugs were used as the negative control. Antimicrobial activities were determined in accordance with the diameters of ZI. Experiments were repeated three times.
Effects of fungal strains on seed germination. All strains were grown on PDA at 25 °C in the dark. After 10 days, ve PDA agar plugs (diameter of 2 mm) with active mycelial growth from the colony margin were inoculated into a Petri dish (diameter 9 mm) containing 15 mL of sterile oatmeal agar (2.5 g·L −1 oatmeal, 12 g·L −1 agar, and pH of 5.2 prior to autoclaving) with ve pieces of nylon cloth (1 × 1 cm), and grown at 25 °C in dark 61,62 . The seeds of the terrestrial orchid plant B. striata were selected as the experimental subject for seasonal reasons. The seeds were surface sterilized with sodium hypochlorite solution for 10 min to remove fungal contamination. After 1 week, approximately 150 axenic seeds of B. striata (Fig. 3 a) were sown on the surface of each piece of cloth. Each treatment was replicated on ve plates. The treatment without fungi was used as the control. All treatments were placed in a tissue culture chamber under 12 h of light exposure at 25 °C for 70 days. A stereomicroscope (LEICA TL3000 Ergo) was used to assess and record seed germination and protocorm development. Stewart et al. 63 had divided the seed germination and protocorm development of orchids into six stages (0)(1)(2)(3)(4)(5). Three seedling growth stages were added in this study as shown in Table 3 showing as a referencefor assessing the percentage of seed germination and protocorm development of B. striata.
Statistical analyses. The IR% of the strains were calculated as follows: IR% = (Ni/Nt) × 100, where Ni represents the number of segments from which the fungal species was isolated, and Nt is the total number of segments incubated 64 . The percentages of seed germination for per stage were calculated by using the following formula 61 :

Percentage of seeds germination
The diversity of fungal species from N. fordii was evaluated by using the H′ and J with the following formulas: where S represents the total species numbers of endophytic fungi in the community, ni represents the numbers of individuals, and N represents the total number of individuals 65,66 . All statistical analyses were performed using by SPSS 19.0 (SPSS Inc., Chicago, IL, USA).

Results
Isolation, sequencing data, and diversity of culturable endophytic fungi. In this study, 984 fungal colonies (isolation rate% [IR] = 78.03 %) were isolated from 1261 tissue segments of N. fordii plants. The 984 isolates were assigned to 124 strains in accordance with their culture characteristics on potato dextrose agar (PDA) (Fig. 1 d) and ITS rDNA sequence analyses. They included 51 (41.13%), 48 (38.04%), and 76 (61.29%) strains from rhizome, corm, and leaf tissue segments, respectively (Table 1). Except for 12 unidenti ed fungi without high similarity in the GenBank database, all 112 isolates were categorized at the species, genus, or family level ( Table 1).
At least 39 fungal genera were identi ed in accordance with the diversity and sequence data of the 112 isolates recovered from N. fordii plants. Among the 39 genera, 36 were a liated with phylum Ascomycota and included 715 isolates (72.66%). Three (19.00%) strains were classi ed as Basidiomycota, comprising Epulorhiza, which is a liated with Tulasnellaceae; and Phanerochaete cf., which is a liated with Phanerochaetaceae.
Further analyses revealed that most isolates belonged to four classes, including Eurotiomycetes, Dothideomycetes, Sordariomycetes, and Leotiomycetes.
The exclusion of 12 unidenti ed fungi (2.85%) failed to show that the endophytes were widely distributed. The Shannon-Weiner diversity index (H' ) was estimated on the basis of taxonomic units or morphological characters. ITS sequences showed that the corm segment presented the highest fungal species diversity (2.686), followed by the rhizome (1.923) and leaf (1.976) segments. The corme segment showed a higher Pielou Evenness index (J ) (0.835) than the rhizome (0.565) and leaf (0.600) segments (Table 1).
In the present data, endophytic fungi were highly abundant and diverse in the rhizomes of N. fordii and that the most ubiquitous phylum of fungi was Ascomycota, which is reportedly among the most prevalent group of eukaryotes globally 25,26,27 . Eurotiomycetes was the most prevalent class of endophytic fungi, followed by Dothideomycetes. Moreover, 47.87% of endophytic fungi were present in the corm and rhizome of N. fordii, and 52.13% of the fungal isolates were found in the leaves of N. fordii. The genera of Epulorhiza, Alternaria, Phoma, Sclerostagonospora, Torula, Ilyonectria, Purpureocillium, Amesia, Corallomycetella, and Daldinia colonized rhizomes, whereas Periconia, Dictyosporium, Volutella, and Lecanicillium were exclusively detected in corms. In addition, Arthrinium (26.83%) was the most common fungal genus in N. fordii that was abundant in the leaves but was less abundant in corms and rhizomes. The dominant genera of N. fordii also included Colletotrichum (10.26%, mainly colonizing the leaves) and Tulasnellaceae (16.97%, mainly colonizing the rhizomes).
Antimicrobial activity of culturable endophytic fungi from N. fordii. A total of 20 fungal isolates that effectively inhibited pathogen growth were screened out by using the agar diffusion method. The antimicrobial fungi belonged to the genera Penicillium, Aspergillus, Epicoccum, Alternaria, Fusarium, Bipolaris, Cylindrocarpon, Phoma, Lecanicillium, Amesia, Sclerostagonospora, and Arthrinium and included ascomycete and fungal endophyte species (Table 2). Among these fungi, 13 strains (9.33%) showed antibacterial activity against Escherichia coli, 14 strains (12.30%) demonstrated activity against Staphylococcus aureus, and 2 strains (2.33%) presented activity against Candida tropicalis. Notably, Penicillium sp. (1151, Fig. 2 a-b) showed the highest activity against all pathogens. The diameter of the inhibition zone (ZI) against E. coli was 34.698 mm (Fig. 2 c), which was 1.42 times that against streptomycin and 1.31 times that against tetracycline. The diameter of the ZI against S.aureus was 28.478 mm (Fig. 2 d) and was 1.22 and 1.15 times that against the positive control, respectively. However, no activity against C. tropicalis was observed. In addition, Epicoccum sp. (1243) and Phoma sp. (1244, Fig. 1  Comparison with the positive control group revealed that the antibacterial activities of the strains against the two pathogens exceeded 70%.
Interestingly, the fungi have different antimicrobial activity which isolated from different parts of N. fordii. And those that were mainly distributed in the rhizome. However, further studies are required to characterize the dynamic changes in endophytic communities 28 and uncultured fungi 29 and to con rm fungal tissue speci city 30 in N. fordii. The results suggested that endophytic fungi from N. fordii. are potential sources of natural antimicrobial products.
Seed germination trials. This study aimed to obtain isolates with high seed germination activities from N. fordii plants. The seeds of the orchid plant Bletilla striata were selected as the experimental subject for seasonal reasons.
The results showed that Arthrinium sp. (1130 and 1232) and Tulasnellaceae sp. (1217, Fig. 1 d) had higher seed germination activity than the CK group. After 28 days, tthe germination rate of the seeds treated with the two endophytic Arthrinium species increased by 0.37% compared with that of the CK group. After 3 weeks treatment with the two Arthrinium isolates increased the germination of B. striata seeds up to the emergence of rst leaf by 27.63% and 2.72%, respectively. Arthrinium sp. (1130) promoted seed germination up to the elongation of the secand leaf stage (0.63%) after 4 weeks, and the dominant seed germination stage was the emergence of the second leaf stage (33.54%). The germination rate during this stage increased by 33.14% compared with that under treatment with the CK. After 4 weeks, Arthrinium sp. (1232) promoted seed germination up to the emergence of the second leaf stage (0.39%) and stage 4 was the dominant stage (33.54%). The germination rate in the treatment group increased by 18.90% compared with that in the CK group. After 7 weeks, the two strains of Arthrinium promoted seed germination up to the appearance of root stage by 73.17% and 21.05%, respectivelybut. Without symbiotic fungi, seed development of B.striata was arrested at stage 7 ( Fig. 4. a). The above data indicated that the two Arthrinium isolates had a certain capability to promote seed germination.
Tulasnellaceae sp. (1217) had the best germination-promoting activity (Fig. 3 a-f). After 3 weeks, the rate of seed germination under treatment with Tulasnellaceae sp. was 97.89%, which was signi cantly higher than that under treatment with CK (85.21%). After 7 weeks of sowing, roots appeared in 52.41% of B. striata seeds, and the uninoculated control showed a germination rate of zero (Fig. 4 b). B. striata seedlings in the experimental group had more roots and larger leaves than the seedlings in other groups. The fresh weight and plant height of the B. striata seedlings in the experimental group were 1.22 and 3.34 times higher than those of the seedlings in the control group. Most of the seedlings demonstrated root germination. The total germinated root number and root length of the treated seedlings were 13.55 and 6.07 times those of the control, respectively.

Discussion
Endophytes affect the the quality and quantity of the crude drugs through speci c fungus-host interactions 31,32 . They are widely considered as valuable natural resources with diverse applications in a variety of areas, such as agriculture and biotechnology 33 . Most endophytic fungi from wild orchids have been studied. However, studies on endophytic fungi from Nervilia plants, particularly studies fungi related to biological activity, have rarely been conducted.
In this study, 112 strains of endophytic fungi belonging to tow phyla, ve classes, 16 orders, 28 family and 39 genera were obtained from N. fordii. Consistent with the ndings of Song et al. 34 , Arthrinium, Colletotrichum, and Tulasnellaceae were found to be the dominant endophytic fungi of N. fordii and accounted for more than 10% of the total endophytes. The quantities of endophytic fungi varied across different plant tissues from N. fordii. The corm of N. fordii. harbored the highest number of endophytic fungi. The variation in the quantities of endophytic fungi across different plant tissues might be ascribed to environmental differences between the above-and below-ground parts of plants; for example, the roots are highly susceptible to infestation by soil microorganisms due to their long-term presence and close interaction in the soil 35 . Isolates Ascomycota sp. (1192 and 1299, Fig. 1 d) and Venturiaceae sp. (1300) had darkly pigmented and septate hyphae of thick walls. These strains are often referred to as dark septate fungi (DSE) 36 and are isolated from corms. Mandyam & Jumpponen suggested that DSE-plant symbioses are multifunctional and play unique roles in terrestrial ecosystems 37 . They participate in nutrient acquisition and in the resultant positive host growth responses 38, 39 . The isolation of 20 strains exhibiting strong antimicrobial activity indicated that these plants and their endophytes could be potential sources of novel natural antimicrobials. These strains belonged to 12 genera and one to fungal species, thus illustrating the diversity of the distribution of endophytic fungal genera with antibacterial activity. Phoma, Fusarium and Penicillium, which have been isolated from medicinal orchids, exhibit antibacterial activity. Several endophytic fungi with high antioxidant capacities, such as Alternaria, Fusarium, Xylaria, and Penicillium, have been isolated from Dendrobium 40,41 . Similarly, Lecanicillium, Phoma, and Ilyonectria isolated from Dysosma versipellis possess antibacterial activity 3,42 . Penicillium sp. (1151) with the best antibacterial activity was screened. The secondary metabolites produced by Penicillium sp. have consistently shown good biological activity in antibacterial activities. The well-known antibacterial drugs penicillin and griseofulvin were isolated from Penicillium chrysogenum 43 and Penicillium griseofulvum 44 , which are commonly found in nature. Furthermore, endophytic fungi of Penicillium fungal species that have been isolated from some herbal plants, such as Pogostemon cablin 45 , Withania somnifera 46 , and Panax ginseng Meyer 47 have also been shown to possess a variety of antibacterial activities.
Orchidaceae are typical mycorrhizal plants. Their seeds are tiny, without endosperm and must depend on suitable mycorrhizal fungi to germinate in natural conditions 48 . Symbiotic seed germination has been practically used in orchid recovery projects worldwide and is considered as an effective way for orchid conservation 49,50 . Seed germination trials con rmed the capability of Arthrinium sp. (1130 and 1232) and Tulasnellaceae (1217) to enhance the germination of B. striata, among which 1217 had the best seed germination-promoting activity. Previous studies have demonstrated that the majority of orchid mycorrhizal symbionts fall under the broad category of Rhizoctonia-like fungi, whose members include Tulasnellaceae, and constitute an important class of symbiotic germinating fungi 51,52 . Among the reported orchids, fungi of the Tulasnellaceae can promote seed germination in several species of orchids (approximately 40 species), such as Dendrobium, Epidendrum secundum, Chiloglottis, Dichromanthus and so on 53,54,55 .
Endophytic fungi were highly abundant in N. fordii plants and exhibited a wide range of biological activities. However, their ecological function, relevant metabolic pathways, and secondary metabolites need extensive investigation. These species are potential viable sources for the exploration of novel natural products and the actual propagation of other orchid species.