Insights from the Endophytic Fungi in Amphisphaeria (Sordariomycetes): A. orixae sp. nov. from Orixa japonica and Its Secondary Metabolites

Endophytic fungi are a remarkably diverse group of microorganisms that have imperceptible associations with their hosts for at least a part of their life cycle. The enormous biological diversity and the capability of producing bioactive secondary metabolites such as alkaloids, terpenoids, and polyketides have attracted the attention of different scientific communities, resulting in numerous investigations on these fungal endophytes. During our surveys of plant-root-based fungi in the mountain areas of Qingzhen, Guizhou Province, several isolates of endophytic fungi were identified. In this study, a novel endophytic fungus was discovered in the roots of a medicinal plant (Orixa japonica) in Southern China and introduced as a new species (Amphisphaeria orixae) based on morphological evidence and molecular phylogenetic analysis (combined ITS and LSU sequence data). To the best of our knowledge, A. orixae is the first reported endophyte as well as the first hyphomycetous asexual morph in Amphisphaeria. A new isocoumarin, (R)-4,6,8-trihydroxy-5-methylisochroman-1-one (1), and 12 known compounds (2–13) were isolated from the rice fermentation products of this fungus. Using 1D- and 2D-NMR, mass spectrometry, and ECD studies, their structures were identified. The antitumor activity of these compounds was tested. Unfortunately, none of the compounds tested showed significant antitumor activity.


Introduction
Endophytic fungi are usually found in the internal tissues or organs of plants, but they do not cause noticeable tissue damage or symptoms. These fungi are often found in a wide array of plant species. At times, endophytic fungi are able to convert to a saprotrophic lifestyle when the host plant undergoes senescence [1]. Even though endophytic fungi can be found in a broad range of hosts, some fungi may be specific to certain hosts and have been extensively researched to gain an understanding of their biological properties. These fungi have also been studied as a source of novel and natural bioactive compounds. Each plant contains one or more endophytic microorganisms that have the potential to produce compounds similar to those of the host plant, and the secondary metabolites they produce are often characterized by novel structures that make them a hot research topic in the field of natural products [2,3]. As new microbial resources, endophytic fungi are an important source of natural bioactive products. The exploitation of endophytic fungi can help alleviate the problems of plant resource shortages and ecological imbalances.

Plant Material
The fresh and healthy whole plant of Orixa japonica was collected from Jiulong Mountain, Qingzhen City, Guizhou Province, China (106 • 30 7 E 26 • 40 36 N). Jiulong Mountain is located in the middle of the Yunnan-Guizhou Plateau, with a hilly landscape and a subtropical monsoonal humid climate, with an average annual temperature of 20 • C. For ease of transport to the laboratory and labeling with relevant metadata (including the date, habitat, location, and host), the materials were enclosed in sealed bags. Fungal isolation was carried out within 24 h of collection.

Isolation of Fungal Endophytes
The fresh and healthy materials were washed with running tap water for at least 10 min. The materials were surface-sterilized to eliminate epiphytic microorganisms in a benchtop by immersing them in 75% (v/v) ethanol for 3 min, then rinsed with sterilized distilled water for 2 min, then soaked with 10% (v/v) NaClO for 2 min, and finally rinsed with sterile distilled water three times continuously. The materials were dried on sterilized filter papers and then cut into small cubes (ca. 3 mm long segments) and placed on fresh potato dextrose agar (PDA) containing an antibiotic (50 µg/mL penicillin). Samples were incubated in a constant-temperature incubator (28 • C). The plates were observed daily, and mycelia from the edges of fungal colonies were transferred to fresh PDA plates to obtain pure cultures. To induce spore production, polypropylene (PP) was mixed with PDA medium and inoculated with the fungal mycelium. The mycelium was gently swept with a sterilized brush, and variable-temperature incubation was applied alternatively between 24 h at 28 • C and 24 h at 4 • C [14]. Specimens of the dried cultures were deposited at the Herbarium of Guizhou Academy of Agricultural Sciences (Herb. GZAAS). Living cultures were deposited at the Guizhou Culture Collection (GZCC).

Morphological Study
Micro-morphological characters were photographed using an ECLIPSE Ni-U compound microscope (Nikon, Tokyo, Japan) fitted with an EOS 90D digital camera (Canon, Tokyo, Japan). Tarosoft (R) Image Frame Work was used to measure different morphological features (including conidiogenous cells, mycelia, conidia, and conidiophores), while Adobe Illustrator CS6 (Adobe Systems, San Jose, CA, USA) was used for the processing of figures and pictures.

DNA Extraction, PCR Amplification, and Sequencing
Using sterile scalpels, fresh mycelia of fungi were scraped. Genomic DNA was extracted from the scraped mycelium by using the Biospin Fungus Genomic DNA Extraction Kit (BioFlux, Shanghai, China) following the manufacturer's instructions. Two gene regions were amplified with universal primers, viz., the internal transcribed spacer region of ribosomal DNA (ITS: ITS5/ITS4) [15] and the partial large subunit nuclear ribosomal DNA (LSU: LR0R/LR5) [16]. The method used for PCR amplification of ITS and LSU by polymerase chain reaction is described by Lu et al. [17]. The quality of PCR products was observed by using ethidium bromide staining in 1% agarose gel electrophoresis. Successfully amplified PCR products were sent to Sangon Biotech (Shanghai, China) for purification and sequencing (with the same primers).

Sequence Alignment and Phylogenetic Analyses
The raw forward and reverse reads were edited for ambiguous bases at both ends and assembled using DNASTAR Lasergene SeqMan Pro v.7.1.0 (44.1). In order to compare species and build databases, newly acquired sequences were utilized as queries in BLAST searches of the NCBI GenBank nucleotide database. Each dataset was aligned using MAFFT v.7 [18]. Trimal was used to trim each alignment [19]. Alignment was checked manually using BioEdit [20].
For phylogenetic inferences, two genetic markers, ITS and LSU, were applied ( Table 1). The phylogenetic tree was extrapolated using 58 taxa according to recent publications [21][22][23][24]. Maximum likelihood trees (ML) were constructed using IQ-Tree v.2 [25]. The combined datasets were analyzed and divided into groups based on genetic markers. One thousand ultrafast bootstrap replicates were used to estimate branch support. MP analysis was performed with the CIPRES Science Gateway platform using the PAUP on XSEDE (4.a168) tool. ModelTest, implemented in MrMTgui [26], was used to identify the most suitable evolutionary model for Bayesian inference analysis using the Akaike Information Criterion (AIC). One thousand rapid bootstrap replicates were used to estimate bootstrap support. Posterior probabilities (PPs) were evaluated in MrBayes v.3.1.2 [27] with Markov chain Monte Carlo sampling (MCMC). The number of generations for each dataset was determined independently and is explicitly stated in the legend of each tree. The top 25% of the trees represent the aging stage of the analysis and were therefore discarded, while the rest of the trees were used to compute PPs for the majority-rule consensus tree. Convergence was declared for all Bayesian inference trees when the average standard deviation was 0.01. The FigTree v1.4.0 program was used to draw the figures of the trees [28]. The new family's placement was evaluated using the approximately unbiased (AU) test implemented in CONSEL [29]. Topologies with p values less than 0.05 in the AU test were regarded as being rejected. Note: Newly generated sequences are highlighted in bold, "T" indicates a type strain, and "-" indicates that were data not available in GenBank.

Fermentation, Extraction, and Separation
The strain was incubated on PDA medium at a constant temperature of 28 • C for 7 days, then cut into small pieces (about 3 × 3 mm) with a scalpel and transferred to a 250 mL conical flask containing 100 mL of liquid medium (20 g of maltose, 10 g of sodium glutamate, 0.5 g of KH 2 PO 4 , 20 g of mannitol, 0.3 g of MgSO 4 -7H 2 O, 3 g of yeast paste, 10 g of glucose, and 1 L of tap water), and incubated on a 28 • C shaker (150 rpm) for about 10 days. Then, 5 mL of the seed liquid that had been made with 50 g of rice and 55 mL of distilled water was transferred to a sterilized plastic container with a capacity of 200 mL. The sterilized bags were then cultured for 65 days at a constant 28 • C temperature. A total of 1000 bags were cultured.
The crude extract was obtained by extracting the fermented product in a 10:1 ratio of ethyl acetate to methanol and then removing the solvent under reduced pressure. The crude extract was first dissolved in a methanol solution, and then an equal amount of a petroleum ether solution was added, and the petroleum ether extract (212 g) and methanol extract (380 g) were obtained after partitioning. In order to enrich the alkaloids, the methanol layer of the extract was dissolved with an appropriate amount of 2% HCl solution, then the acidic water was extracted three times with an equal volume of ethyl acetate, the ethyl acetate layer was discarded, and the pH was adjusted to about 10 with 25% aqueous ammonia. The mixture was extracted three times with an equal volume of chloroform, and the chloroform layer was combined and concentrated to obtain 2 g of extract (Fr.1). The remaining solution was adjusted to neutral pH, concentrated under reduced pressure, and then separated by silica gel column chromatography (Qingdao Marine Chemistry Co. Ltd., Qingdao, China) with dichloromethane/methanol (100:0→0:100, v/v) to obtain six fractions (Fr.2-Fr.7). Fr.1 (2 g) was further separated using Sephadex LH-20 (Amersham Pharmacia Biotech AB) (MeOH) into three fractions (Fr.1.1-Fr.1.3). Fr.1.1 (500 mg) was further separated by semipreparative HPLC (SHIMADZU Essentia LC-16P, GH0525010C18 column 10 × 250 mm, 5 µm) using CH 3 OH/H 2 O (55:45) to obtain compound 5 (4.5 mg, 3 mL/min, t R = 18.1 min) and compound 13 (6 mg, 3 mL/min, t R = 30.1 min), Fr.1.2 (320 mg) was further separated by semi-preparative HPLC using CH 3 OH/H 2 O (40:60) to obtain compound 10 (4.4 mg, 3 mL/min, t R = 6.2 min) and compound 11 (3.2 mg, 3 mL/min, t R = 7.19 min), and Fr.1.3 (70 mg) was further separated by semi-preparative HPLC using CH 3 CN/H 2 O (40:60) to obtain compound 12 (9.5 mg, 3 mL/min, t R = 8.5 min). Fr.2 (20 g) was separated by silica gel column chromatography (200-300 mesh) to obtain three fractions (Fr.2.1-Fr.2.3). Fr.2.2 (4 g) was further separated by Sephadex LH-20 and semi-preparative HPLC using

Antitumor Bioassay
The following established in vitro human cancer cell lines were used: human breast cancer cells (MCF-7), human hepatocellular carcinoma cells (HepG2), and pancreatic cancer cells (PANC-1). Adriamycin was used as a positive control, and antitumor activity was measured for compounds 1, 3, 4, 7, 9, 12, and 13. Cell counting kit-8 was obtained from GLPBIO (USA). MCF-7 and PANC-1 cells in the logarithmic growth phase were digested and added to 96-well plates at a density of 0.6 × 10 4 cells per well and incubated at 37 • C in a 5% CO 2 incubator for 12 h. After the cells were plastered, culture solutions containing 0, 50, 100, and 200 µM of the drug at different concentrations were prepared by performing DMSO gradient dilution in a volume of 100 µL. Each group of five replicate wells was incubated in the cell culture incubator for 24 h; the plate was washed twice with 100 µL of PBS, and then 100 µL of culture solution containing 10% CCK-8 was added. The OD value was measured at a 450 nm wavelength. It was calculated by the following equation.

Phylogenetic Analysis of ITS and LSU Sequences
The aligned sequence matrix comprised ITS (617 bp) and LSU (850 bp) sequence data, including 56 ingroup taxa and two outgroup taxa. The dataset has 1467 characters, of which 884 characters are constant, 160 variable characters are quasi-informative, and 423 characters are quasi-informative. ML, MP, and Bayesian analyses of the combined dataset resulted in the reconstruction of plant genetics with essentially similar topologies, and the ML tree is shown in Figure 1. The tree is rooted with Achaetominum macrosporum (CBS 532.94) and Chaetomium elatum (CBS 374.66). The genus Amphisphaeria contained 25 taxa retrieved from GenBank and two new strains generated in this study. The two new isolates are recognized as one new species, viz., Amphisphaeria orixae, and it formed a sister clade with A. camelliae (HKAS 107021 and MFLUCC 20-0122) and A. uniseptata (CBS 114967).  [22,23]. Most of the species of Amphisphaeria are found on grasses, wood    [22,23]. Most of the species of Amphisphaeria are found on grasses, woody branches, and some monocotyledons as saprobes in terrestrial habitats [30]. The unicellular ascospores of Amphisphaeria members typically have J+ or J− apical rings, solitary or aggregated ventral membranes under undeveloped perithecia or absent perithecia; and a coelomycetous asexual morphology with a light-to dark-brown color, oval to fusiform shape, and 1-3-noded ventral conidia [21][22][23][24]. The species of Amphisphaeria that have been reported are sexual morphs or coelomycetous asexual morphs, and no studies have reported hyphomycetous asexual morphs in this genus so far. In addition, Amphisphaeriaceous taxa have not been recorded as phytopathogens [30]. Based on phylogenetic analysis and morphological evidence, we introduce the first endophytic fungus of Amphisphaeria in this study. It is distinguished as a hyphomycetous asexual morph.
lar ascospores of Amphisphaeria members typically have J+ or J− apical rings, solitary or aggregated ventral membranes under undeveloped perithecia or absent perithecia; and a coelomycetous asexual morphology with a light-to dark-brown color, oval to fusiform shape, and 1-3-noded ventral conidia [21][22][23][24]. The species of Amphisphaeria that have been reported are sexual morphs or coelomycetous asexual morphs, and no studies have reported hyphomycetous asexual morphs in this genus so far. In addition, Amphisphaeriaceous taxa have not been recorded as phytopathogens [30]. Based on phylogenetic analysis and morphological evidence, we introduce the first endophytic fungus of Amphisphaeria in this study. It is distinguished as a hyphomycetous asexual morph. Amphisphaeria orixae X. J. Wang, Y. Z. Lu & Z. Zhang, sp. nov., Figure 2. Index Fungorum number: IF 900170; Facesoffungi number: FoF 13911. Etymology-Name referring to the host "Orixa japonica", using the genitive case meaning "of Orixa".

Antitumor Bioassay
The in vitro cytotoxic activity of the compounds against MCF-7, HepG2, and PANC-1 was analyzed using the CCK-8 assay with Adriamycin as a positive control ( Figure 5). The results demonstrated that none of the tested compounds exhibited significant antitumor activity compared to Adriamycin (Table 3). Although compound 3 demonstrated the best antitumor activity against MCF-7, with an IC 50 value of 51.9 µM, it displayed weak activity against the other two tumor cells. The compounds tested showed considerable differences in their IC 50 values compared to the positive control, indicating that the compounds do not have significant antitumor activity. The IC 50 values for Adriamycin were found to be 2.03 µM, 3.85 µM, and 3.44 µM when tested on MCF-7, HepG2, and PANC-1, respectively.
Additionally, the inhibitory effect of Adriamycin on tumor cells was observed to be dosedependent, as the cell viability decreased with the increase in the drug concentration. For the assay, the concentrations of Adriamycin were set at 0.2, 0.5, 1, 2, 5, 10, 20, 50, and 100 µM, whereas for the tested compounds, they were set at 5, 20, 50, 100, and 200 µM for MCF-7 tumor cells and 50, 100, and 200 µM for HepG2 and PANC-1 tumor cells.

Discussion
Endophytic fungi are an important source for discovering naturally active substances [45]. In this study, a new endophytic fungus, viz., Amphisphaeria orixae, was isolated from the medicinal plant O. japonica. The fungi in Amphisphaeria are usually saprobes on woody branches and some monocotyledons, including grasses in terrestrial, mangrove, and freshwater habitats [21][22][23][24]30]. It is noteworthy that A. orixae is the first endophyte and first hyphomycetous asexual morph in Amphisphaeria, which broadens the morphological characteristics and lifestyles of this genus. The asexual sporulation of the new species Amphisphaeria orixae is relatively complex and includes phialidic and thallic conidiogenesis. This discovery is important for future taxonomic studies on this genus, as the generic boundaries of the family Amphisphaeriaceae have traditionally been based on sexual characteristics, and the delimitation of genera is still ambiguous and challenging. Furthermore, thirteen secondary metabolites, including a new isocoumarin, were isolated from the rice fermenta-tion product of this fungus. All 13 compounds obtained in this study were isolated from the genus Amphisphaeria for the first time. Among them, compounds 1-4 are coumarins, and compounds 5-13 are quinoline alkaloids. The experimental strain was isolated from the roots of O. japonica. Interestingly, compounds 5, 7-9, and 12-13 were also obtained from the roots of the O. japonica, as reported by Liu et al. and Huang et al. [10,46], which provided evidence that the endophytic fungus is able to produce the same or similar compounds as the host. Moreover, these compounds are known to have significant antifungal, insecticidal, and food-repelling activities from the reported literature, with kokusaginine (5) having LC 50 values of 16.66µg/mL and 5.32µg/mL against Bursaphelenchus xylophilus and Meloidogynein congnita, respectively, and lunidonine (7) not only killed Meloidogynein congnita and Anopheles sinensis but also showed a strong food-repelling effect on Ostrinia furnacalis [10,46]. These findings indicate that Amphisphaeria orixae may be a promising source of bioactive compounds with potential applications in agriculture and medicine. In summary, the discovery of this new endophytic fungus and its secondary metabolites further highlights the importance of exploring the microbial diversity of medicinal plants for the discovery of novel biologically active compounds.
Author Contributions: Conceptualization, Z.Z. and Y.L.; methodology, X.W., P.Z., L.Z., J.M. and J.Z.; software, X.W., P.Z. and L.Z.; investigation, X.W.; writing-original draft preparation, X.W.; writingreview and editing, Z.Z., Y.L. and D.N.W.; project administration, Z.Z. and Y.L.; funding acquisition, Z.Z. and Y.L. All authors have read and agreed to the published version of the manuscript. Data Availability Statement: All data analyzed in this study are available within the manuscript and are available from the corresponding authors upon request.

Conflicts of Interest:
The authors declare no conflict of interest.