Endophytic fungus Biscogniauxia petrensis produces antibacterial substances

Widespread drug resistance and limited antibiotics challenge the treatment of pathogenic bacteria, which leads to a focus on searching for new antimicrobial lead compounds. We found the endophytic fungus Biscogniauxia petrensis MFLUCC14-0151 from the medicinal plant Dendrobium harveyanum had antibacterial activity for the first time. This work aimed to reveal the capacity of Biscogniauxia petrensis MFLUCC14-0151 against foodborne pathogenic bacteria and identify its bioactive substances. Bioassay-guided isolation led to the discovery of six infrequent active monomers, including (10R)-Xylariterpenoid B (1), Xylariterpenoid C (2), Tricycloalternarene 1b (3), Tricycloalternarene 3b (4), Funicin (5) and Vinetorin (6) from MFLUCC14-0151 for the first time. The results of antibacterial tests showed that (10R)-Xylariterpenoid B and Xylariterpenoid C exhibited inhibitory activities against Streptococcus agalactiae with MIC values ranging from 99.21 to 100.00 μM, and against Streptococcus aureus with MIC values ranging from 49.60 to 50.00 μM. Tricycloalternarene 1b and Tricycloalternarene 3b showed inhibitory effects on Streptococcus agalactiae with MIC values ranging from 36.13 to 75.76 μM. Unexpectedly, Funicin and Vinetorin exhibited remarkable antagonistic activities against Streptococcus agalactiae with MIC values of 10.35 and 10.21 μM, respectively, and against Streptococcus aureus with MIC values of 5.17 and 20.42 μM, respectively. In conclusion, we suggest that the isolated compounds Funicin and Vinetorin may be promising lead compounds for natural antibacterial agents.


INTRODUCTION
Endophytes are microorganisms living in the internal tissues of the host plants without causing any overt symptoms (Stone, Bacon & White, 2000). The host plants provide essential spaces and nutrients for endophytes, meanwhile the endophytes provide protection and survival value for host via producing various active substances. The medicinal plants were reported to be rich in endophytes with approximately one million species awaiting exploring (Gakuubi et al., 2021). Furthermore, the endophytes in medicinal plants possessed the ability to synthesize same or similar bioactive ingredients as those produced by the host (Prajapati, Goswami & Rawal, 2021). For example, the anti-tumor drugs paclitaxel, camptothecin, and the platelet activator inhibitor ginkgolide B, initially extracted from the medicinal plants have also been produced by their endophytes viz Taxomyces andreanae, Paenibacillus polymyxa and Fusarium oxysporum, respectively (Stierle, Strobel & Stierle, 1993;Pu et al., 2015;Cui et al., 2012). Searching for unique bioactive components from endophytes in unusual niches has important scientific and pharmaceutical values.

Fungal and bacterial strains
Biscogniauxia petrensis MFLUCC14-0151 was isolated and identified by our research group (Ma et al., 2020), and deposited at the China General Microbiological Culture Collection Center (CGMCC 40341;No. 3,Yard 1,Beichen West Road,Chaoyang District,Beijing). The foodborne pathogenic bacteria Streptococcus agalactiae GBS-1, Streptococcus aureus SA-1 and Escherichia coli EC-1 were obtained from the Engineering Research Center of the Utilization for Characteristic Bio-pharmaceutical Resources in Southwestern, Ministry of Education, Guizhou University. The fungus strain was maintained at 4 C on potato dextrose agar (PDA) and incubated at 28 C on Martin modified (MM) medium. The tested pathogenic bacteria were cultivated in nutrient broth (NB) medium and nutrient agar (NA) medium at 37 C.

Strain cultivation and fermentation
The fungus strain was cultured on PDA at 28 C for a week. Three pieces of mycelial agar plugs with a diameter 6 mm were inoculated into the Erlenmeyer flasks (500 mL), each containing 200 mL of Martin modified (MM) medium to obtain the seed culture. Fermentation was carried out in 250 Erlenmeyer flasks (volume 1 L), each containing 200 g of rice and 150 mL of distilled H 2 O, and then autoclaving at 120 C for 30 min. Each flask was inoculated with 10 mL of seed culture and incubated at 28 C under static condition for 2 months.

Extract preparation
The crude extract was obtained using the method with some modifications (Wang et al., 2020). The whole cultures (60 kg) were extracted thrice with 120 L of methanol, and the organic solvent was evaporated to a small volume (5 L) under vacuum. The extract (900 g) was suspended in 10 L of distilled H 2 O and partitioned successively by extracting thrice with 2-fold volume of EtOAc (20 L) and n-BuOH (20 L), respectively. The EtOAc solution was evaporated under reduced pressure to obtain a crude extract (56 g).

Antibacterial activity
The antagonistic activities of the crude extract against indicator bacteria viz Streptococcus agalactiae GBS-1, Streptococcus aureus SA-1 and Escherichia coli EC-1 strains were determined using agar diffusion method with some modifications (Ngamsurach & Praipipat, 2021). The analysis plates used in the assay were prepared via transferring 1 mL of each indicator bacterial suspension (10 8 CFU/mL) into 100 mL NA medium (50-55 C). The indicator bacteria and the medium were mixed homogeneously, and then the mixture was poured into a petri-dish (20 mL). The Oxford cups were added and the plate was left for approximately 2 h to solidify. The Oxford cups were removed, and followed by adding 10 mL of samples into the well and incubation for 24 h at 37 C. The samples were prepared by dissolving the tested components in dimethyl sulfoxide (DMSO) to obtain a solution with a concentration of 2 mg/mL. Ampicillin (AMP) with a concentration of 500 mg/mL was used as the positive control, and DMSO as the negative control. All tests were performed in triplicates. The diameters of the inhibition zones were measured after 48 h incubation.

Structure elucidation of monomers
A total of 5 mg of compounds were dissolved in 600 mL of deuterium reagent in the NMR tubes, and then the 1 H NMR and 13 C NMR spectra were recorded on a Bruker 500 MHz NMR apparatus (Bruker, Bremerhaven, Germany). The spectra data were processed and analyzed with MestReNova-9.0.1 software. The known compounds were identified by comparing its nuclear magnetic resonance spectral data ( 1 H-NMR and 13 C-NMR) with those of compounds reported in NMR Carbon Spectrum Database of Organic Compounds (http://www.nmrdata.com).

Determination of the minimum inhibitory concentration
The minimum inhibitory concentration (MIC) measurement was conducted to evaluate the antibacterial activities of compounds 1-6 against three tested pathogenic bacteria (GBS-1, SA-1 and EC-1 strains) via broth microdilution method with some modifications (Chen et al., 2019). The monomers were dissolved in DMSO (1 mg/mL), and then the solution was diluted at concentrations of (500, 250, 125, 62.5, 31.3, 15.6, 7.81, 3.91, 1.95, 0.98 µg/mL) by twofold dilutions in a 96-well plate. The tested bacterial suspension was adjusted to a density of bacterial cells of 10 6 CFU/mL. To each well, 100 µL of each bacterial suspension was inoculated and incubated at 37 C for 24 h. The MIC values were recorded as the lowest concentrations of the monomers that had no visible bacterial turbidity according to the guidelines (M27-A3) (CLSI 2008) (Iraji et al., 2020). The tests using DMSO as negative control and Ampicillin (AMP) as positive control were carried out in parallel. Each treatment was repeated three times.

Statistical analysis
All experiments were conducted in three times and expressed as mean ± standard deviation. The data was analyzed using a t test and the differences among groups were evaluated by Two-way analyses of variance (ANOVA) using the GraphPad Prism software (version 5; GraphPad, San Diego, CA, USA). P < 0.05 and P < 0.01 were considered significant and highly significant differences, respectively.

Antibacterial activities of crude extract
In order to determine whether the crude extract showed antagonistic activities against foodborne pathogenic bacteria, Streptococcus agalactiae GBS-1, Streptococcus aureus SA-1 and Escherichia coli EC-1 were used as tested strains to detect the inhibitory effects of the crude extract on their growth. We observed that the crude extract had obvious bacteriostatic zones against all tested pathogenic bacteria contrasted with the negative control DMSO (Fig. 1A). The inhibitory activities of the crude extract against three tested bacteria were GBS-1 > EC-1 > SA-1 with the diameter of inhibition zones were 19.67, 17.67 and 16.33 mm, respectively, but none of them were as strong as the positive control AMP (P < 0.05) (Fig. 1B). It indicated that Biscogniauxia petrensis MFLUCC14-0151 produced one or more antibacterial active ingredients.

Three sub-fractions showed antagonistic activities
To determine the distributions of active constituents against tested pathogenic bacteria, the crude extract was divided into different fractions and their bioactivities were tracked. The antibacterial activities of six fractions (A1-A6) obtained from the crude extract were tested. The results suggested that fractions A4 and A5 had antagonistic activities against all tested pathogenic bacteria contrasted with the negative control DMSO ( Fig. 2A), but their inhibitory activities were far less than those of positive control AMP (P < 0.05 and P < 0.01) (Fig. 2B). The fraction A3 performed inhibitory effects on Streptococcus agalactiae GBS-1 and Escherichia EC-1, without Streptococcus aureus SA-1 (P < 0.05 and P < 0.01) (Figs. 2A and 2B). Those results confirmed that diverse active substances produced by Biscogniauxia petrensis MFLUCC14-0151, and provided transparent guidance for the subsequent targeted separation and purification of active monomers.

Monomers showed antibacterial activities
To evaluate the antibacterial potential of monomers against pathogenic bacteria. The minimum inhibitory concentrations (MICs) of compounds 1-6 against Streptococcus agalactiae GBS-1, Streptococcus aureus SA-1 and Escherichia coli EC-1 were determined. The results showed that all monomers exhibited different antagonistic activities (Table 1)

DISCUSSION
Although the fungi in Biscogniauxia genus are generally regarded as plant pathogenic fungi, they might be endophytic fungi before transforming into pathogen microorganisms in the host, and possess the potential to synthesize and metabolize active constituents which are similar with those produced by the host plant in activity or structure. The metabolites derived from Biscogniauxia fungi had multiple bioactivities, such as antifungal, anti-inflammatory, anti-cancer and anti-Alzheimer's disease (Jantaharn et al., 2021;Zhao et al., 2021b). The antibacterial activity of Biscogniauxia was rarely reported. In our study, Biscogniauxia petrensis MFLUCC14-0151 from the medicinal plant Dendrobium harveyanum was found to repress foodborne pathogenic bacteria Streptococcus agalactiae GBS-1, Streptococcus aureus SA-1, and Escherichia coli EC-1 for the first time. Furthermore, the active substances were isolated, purified and identified as (10R)-Xylariterpenoid B, Xylariterpenoid C, Tricycloalternarene 1b, Tricycloalternarene 3b, Funicin and Vinetorin.
As far as we know, the six scarce monomers were isolated from Biscogniauxia genus for the first time and their biological activities were originally reported. The (10R)-Xylariterpenoid B, a sesquiterpene, was only discovered in the marine fungus Graphostroma sp. MCCC 3A00421, which had weak anti-inflammatory effect (Niu et al., 2017). Our results showed that (10R)-Xylariterpenoid B had weak antagonistic activity against Streptococcus agalactiae GBS-1 and moderate inhibitory effect on Streptococcus aureus SA-1 and Escherichia coli EC-1. Xylariterpenoid C, a sesquiterpene, which was only isolated from Xylariaceae fungus, exhibited no significant cytotoxicity (Wu et al., 2014). However, we found that Xylariterpenoid C had weak and moderate antagonistic activities against Streptococcus agalactiae GBS-1 and Streptococcus aureus SA-1, respectively. Tricycloalternarene 1b and Tricycloalternarene 3b are mixed terpenoids, which were initially discovered in Alternaria alternata (Liebermann et al., 1997). Tricycloalternarene 1b and Tricycloalternarene 3b showed anti-tumor effects on mouse Lewis lung carcinoma cells 3LL and human neuroblastoma SH-SY5Y. (Yuan, Huang & Zhao, 2013). In addition, the compounds of Tricycloalternarene type have also been discovered in fungi Septoria sp., Ulocladium sp., Aspergillus sp., Colletotrichum capsici and Didymella sp. (Sugawara et al., 1998;Wang et al., 2013Wang et al., , 2016Zhang et al., 2018;Li et al., 2018). The antibacterial tests indicated that Tricycloalternarene 1b and Tricycloalternarene 3b had antagonistic activities against Streptococcus agalactiae GBS-1. Funicin, a diphenyl ether substance, was previously discovered in Aspergillus sp. with remarkable antimicrobial activities against Trichophyton asteroides, Trichophyton rubrum, and Trichophyton interdigitale (Hamasaki et al., 1980). In the study, Funicin displayed stronger inhibitory activities against Streptococcus agalactiae GBS-1 and Streptococcus aureus SA-1 with MIC values ranging from 5.17 to 10.35 mM. Vinetorin, a xanthone containing chlorine atoms, was discovered in lichen and higher plant Hypericum ascyron, but its biological activity has not been reported (Feige & Lumbsch, 1993;Hu, Yip & Sim, 1999). Our data revealed that Vinetorin presented significant inhibition effects on Streptococcus agalactiae GBS-1 and Streptococcus aureus SA-1 with MIC values ranging from 10.21 to 20.42 mM. In general, Funicin and Vinetorin were main bioactive substances and can be used as lead molecules of antimicrobial agents for foodborne pathogens. Using Funicin and Vinetorin as prodrugs, their bioactivities and yields are improved, and their toxicities are weakened via chemical modification. Meanwhile, it is awaiting revealing the antibacterial mechanism of these active compounds.

CONCLUSIONS
In summary, we found for the first time that the endophytic fungus Biscogniauxia petrensis exhibited antibacterial activity. Six infrequent monomers were isolated from the Biscogniauxia petrensis and their chemical structures were determined by spectroscopic analyses. Funicin and Vinetorin were the main bioactive substances and showed inhibitory effects on Streptococcus agalactiae and Streptococcus aureus with MIC values ranging from 5.17 to 20.42 mM. These data confirmed that the endophytic fungus B. petrensis is a new source of antibacterial substances, and Funicin and Vinetorin can be considered as lead compounds of antibacterial inhibitors.

ADDITIONAL INFORMATION AND DECLARATIONS Funding
This work was supported by the National Natural Science Foundation of China (No. 31670027, 32170019, 32160667 and 31901947). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.