Isolation, identification, and antibacterial evaluation of endophytic fungi from Gannan navel orange

Gannan navel orange is a famous brand in China but the isolation of its endophytic fungi was rarely reported. In this study, a total of 54 strains of endophytic fungi were successfully isolated from the pulp, peel, twig, and leaf of Gannan navel orange; they were successfully identified to belong to 17 species of 12 genera. All these strains were fermented using potato-dextrose agar (PDA) medium, and their secondary metabolites were then extracted with ethyl acetate (EtOAc). The antibacterial assays of Escherichia coli (E. coli), methicillin-resistant Staphylococcus aureus (MRSA), and Xanthomonas citri subsp. citri (Xcc) were also performed for the EtOAc extracts of these strains. As a result, the extracts of both Geotrichum sp. (gc-1-127-30) and Diaporthe biconispora (gc-1-128-79) demonstrated significant antibacterial activities against Xcc, and the MIC value for the extract of Colletotrichum gloeosporioides against MRSA was low to 62.5 μg/mL. Moreover, the chemical components of the extracts of Colletotrichum sp., Diaporthe biconispora, and Annulohypoxylon atroroseum were primarily investigated, and they successfully led to the isolation of 24 compounds involving a new botryane sesquiterpene. Among the isolated products, compound 2 showed significant inhibitory activities toward SA, MRSA, E. coli, and Xcc with MIC values of 12.5, 3.1, 125, and 12.5 μg/mL, respectively. This study revealed that the endophytic fungi of Gannan navel orange showed high potency to produce secondary metabolites with significant antibacterial effects.


. Introduction
China is one of the most important countries for oranges production with an amount of 7.6 million tons in (USDA, 2022. Gannan, located in the south of Jiangxi province, is a critical cultivation area for orange in China. The Newhall navel orange (Citrus sinensis Osbeck cv. Newhall) produced in Gannan, namely Gannan navel orange, is honored as a national geographic indication product in China (Chen et al., 2009;Zhang et al., 2022b).
Plant endophytes have been disclosed to be an extremely important resource for the discovery of new microorganisms (Cabral et al., 1999). In recent years, numerous studies had disclosed that a huge number of endophytic fungi were present in plants, constructing a mutually beneficial symbiotic relationship with their hosts (Collinge et al., 2022). Moreover, most of them showed the capability to help the host to tolerate the external environments (Manzur et al., 2022), promote the growth of plants (Abdalla and Matasyoh, 2014), and biosynthesize some bioactive ingredients to protect the host plants (Mattoo and Nonzom, 2021). Although a large number of studies have been conducted to illustrate the interaction between endophytes and host plants, systematic investigations on the Gannan navel orange and its endophytes are still rare.
Bacterial infection is becoming to be one of the most intractable infectious diseases because of its emerging resistance to the existing antibiotics. Nowadays, the pathogenic bacterium resistant to widely used antibiotics is considered as one of the most serious threats to global public health, which is responsible for serious infections due to high rates of morbidity and mortality in clinical treatment. Among the frequently encountered bacteria, pathogenic Escherichia coli usually causes severe diarrhea worldwide. There are nearly 1.7 billion cases of diarrheal disease reported every year (Yang S. C. et al., 2017), and 9.4% of them are caused by diarrheagenic E. coli (Indhuprakash et al., 2021), which has resulted in about 760,000 death of children every year from 2011 to (Chowdhury et al., 2015. Methicillin-resistant Staphylococcus aureus (MRSA) is well known as a predominant nosocomial pathogenic bacterium, which represents a crucial crisis in public health systems and poses a serious threat to human life, especially in developed countries (Rebecca, 2008). Therefore, the discovery of novel therapeutic agents that are effective against pathogenic bacteria is of great interest to both medicinal scientists and the pharmaceutical industry to maintain public health in future.
The endophytic fungus is now serving as a promisingly strategic natural bioresource for the discovery of novel natural products with interesting chemical structures and versatile biological activities (Fan et al., 2022). In particular, it was well-respected as a treasure house for developing novel antibacterial agents (Cruz et al., 2020). However, the endophytic fungus of Gannan navel oranges has been rarely investigated. In one of our previous studies, we had isolated and identified an endophytic fungus Aspergillus aculeatus GC-09 from Gannan navel orange, the antibacterial screening disclosed that its crude EtOAc extract showed significant antifungal activity against Penicillium italicum with a MIC of 31.3 µg/mL, which was comparable to the positive control prochloraz (Zhang et al., 2022a). Except that, there were no reports on the endophytic fungi of Gannan navel oranges.
In order to recognize the abundance and distribution of endophytic fungus species of Gannan navel oranges and illustrate their antibacterial potential as a particular interest, a comprehensive investigation on endophytic fungi from Gannan navel orange was performed in the present study. As a result, 54 strains of endophytic fungi were successfully isolated from the healthy pulp, peel, twig, and leaf of Newhall navel orange for the first time. The molecular identification through the ITS rDNA sequences of the isolated endophytic fungi successfully distinguished them to be 17 species of 12 genera. Moreover, the antimicrobial screening against MRSA, E. coli, and X. citri subsp. citri (Xcc) was carried out, revealing that a few fermentation extracts of the isolated endophytic fungi showed significant antibacterial activity. To the best of our knowledge, this study is the first systematic report on the biodiversity, phylogeny, and antibacterial activity of endophytic fungi associated with Gannan navel orange.

. . Isolation and purification of fungal endophytes
The isolation of endophytic fungi from collected plant parts was according to a standard procedure established previously (Li et al., 2020). The pulp, peel, twig, and leaf of Gannan navel orange were used to isolate the corresponding endophytic fungi. The samples were washed with sterile water and dried with sterile filter paper. Then, the cleared tissues were cut into small pieces using a sterile blade and surface-sterilized by sequential immersion in 75% ethanol for 1 min, 1.3 mol/L sodium hypochlorite (3-5% available chlorine) for 30 s, and they were rinsed three times with sterile water between each step simultaneously. These tissue pieces were placed on potato dextrose agar (PDA) plates with 0.4 µg/mL ampicillin and kanamycin to isolate the fungal endophytes. The tissue pieces were observed for mycelia growth. Morphologically distinct isolates were similarly sub-cultured several times to purify and obtain pure isolates. As soon as the fungal mycelium grows out of the tissue slice, the mycelium is transferred to fresh PDA plates for purification and culture. The pure isolates were numbered and kept in a storage tube at −20 • C for further study.

. . ITS sequence processing and molecular identification
The identification of endophytic fungi was based on molecular and morphological analysis. The molecular identification was carried out using the internal transcript spacer regions (ITS1 and ITS4) and the intervening 5.8S rRNA region sequencing. Polymerase chain reaction (PCR) was performed to amplify the ITS region of the fungal isolates using the universal ITS primers, ITS1 (5 ′ -TCCGTAGGTGAACCTGCGG-3 ′ ) and ITS4 (5 ′ - 10 µM of each primer, 0.5 µL 2 × Taq PCR Master Mix, and 8 µL ddH 2 O. 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 as well as a final extension at 72 • C for 10 mins. 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. The ITS sequences were aligned to determine the identity of the corresponding fungal ITS sequences by using BLAST to search from the GenBank database. The endophytic fungus was identified by the percentage identity, and the species level of the targeted endophytic fungus was successfully distinguished with a sequencing identity of more than 99%, whereas the genus level was assigned if sequence identity was between 95 and 99%. Notably, when the sequence identity was determined ≤95%, the related endophytic fungus could be only identified at the family or ordinal level by referring to the suggestion of Landeweert et al. (2003).
The sequencing results were analyzed in the SeqMan module of DNAStar 5.0 software for peak plot analysis (Burland, 1999), the sequence fragments were proofread and saved in fasta format and compared with known sequences in Gene bank using BioEdit v7.1 (Hall, 1999(Hall, , 2011. Then, the sequences were cut and edited, and a matrix was constructed based on the aforementioned results. The sequence output was saved in fas format for phylogenetic tree construction. Bayesian inference was performed by MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003) with the Markov Chain Monte Carlo algorithm and treated the random initial trees as BI settings. Four chains were run, generating 1,000,000 generations per chain. Sampling was conducted every 1,000 generations, and the first 2,500 trees were discarded as burn-in. The remaining trees were used to construct the majorityrule consensus trees. ClustalW Multiple was initially used to perform alignments by BioEdit. Bootstrap (Felsenstein, 1985) values of internal nodes were obtained with 1,000 replicates. Bayesian inference (BI) was operated with the best-fit model (K80+ G) which was selected based on the AIC criterion using MrModeltest 2.0. Calophoma aquilegiicola, Didymella glomerata, Leptosphaeria microscopica, and Paraboeremia litseae were selected as outgroup species.

. . Preparation of EtOAc extracts of di erent endophytic fungi
Submerged cultivation of the endophytic fungi was carried out to produce secondary metabolites. The strain of the agar with the fungal colony was added to a conical flask (500 mL) with 250 mL potato dextrose broth (PDB) at 28 • C for 7 days under constant shaking (150 rpm). After cultivation, the secondary metabolites of each endophyte were extracted by ultrasonic with EtOAc.

. . Isolation of secondary metabolites
The endophytic fungi Colletotrichum sp., Diaporthe biconispora, and Annulohypoxylon atroroseum were cultured on an autoclaved rice solid medium (5 × 3 L Erlenmeyer flasks, each containing 300 g of grains and 360 mL of distilled water) for 30 days at 28 • C. After cultivation, the mycelia and rice solid medium were extracted three times with EtOAc crude extract were obtained. We obtained the main compounds of these two strains that using silica gel chromatography, reversed-phase C 18 column chromatography, and Sephadex LH-20 column chromatography were isolated.

. . Antibacterial activity
The EtOAc extracts of each endophytic fungi were evaluated for their antibacterial activity. MIC values were determined by the methodology of micro broth dilution in Mueller-Hinton broth (MHB) medium according to CLSI guidelines, the positive control was kanamycin, vancomycin, and CuSO 4 . Briefly, 180 µL bacteriological solution (1.25 × 10 6 CFU/mL) with resazurin as an indicator was added into each well of the first row in the 96well plate, whereas 100 µL bacteriological solution (1.25 × 10 6 CFU/mL) with resazurin as indicator was added to the other wells in the 96-well plate. A total of 20 µL tested extracts in DMSO solution with a concentration of 10 mg/mL were added to 180 µL bacterial liquid. Then, the method of double dilution was adopted in the 96-well plates. The change of the indicator in the 96-well plates was observed, and the lowest concentration of the drug preventing visible growth of the pathogen was taken as the MIC value (Wang et al., 2022). Similarly, the monomer compounds were also tested.

. . Diversity of cultural endophytic fungi from Gannan navel orange
In this study, 54 strains of endophytic fungi were successfully isolated from different tissues of Gannan navel orange, which included the pulp, peel, twig, and leaf. Among these endophytic fungi strains, 2 strains (3.70%) were isolated from pulp, 44 strains (81.48%) were purified from the peel, 5 strains (9.26%) originated from twig, and 3 strains (9.26%) were isolated from leaf (Supplementary Table S1). The results indicated that there were abundant endophytic fungi presenting in the Gannan navel orange. Moreover, this study also suggested that the peel of Gannan navel orange might be the most suitable host place for the endophytic fungi.
. . ITS identification of the endophytic fungi of Gannan navel orange PDA culture medium for the 203 purified strains of endophytic fungi, 54 strains showing different morphological features were selected for the molecular biology identification via the sequence analysis of the rDNA ITS (internal transcribed spacer) region.
From the results, all of the endophytic fungi strains isolated from Gannan navel orange could be assigned to 17 representative morphotypes, as shown in Figure 1. Subsequently, ITS rDNA sequences of each morphotype were generated and submitted to the NCBI database to obtain the corresponding sequence numbers (Table 1). Moreover, a phylogenetic tree of the endophytic fungi of Gannan navel orange was successfully constructed as depicted in Figure 2. Moreover, the GenBank accession numbers, homolog sequences, sequence identities, and closest accession numbers of the identified endophytic fungal isolates were systematically cataloged and shown in Table 1. Therefore, according to the results of morphological and molecular identification, 17 representative morphotypes were successfully identified belonging to 12 genera. Moreover, it is worth noting that 14 endophytic fungi from the 17 ones were identified as species ( Figure 1). Furthermore, all the isolated endophytic fungi were identified to be 2 phyla (Ascomycota and Basidiomycota), 5 classes (Sordariomycetes, Saccharomycetes, Agaricomycetes, Eurotiomycetes, and Dothideomycetes), 9 orders (Hypocreales, Saccharomycetales, Polyporales, Diaporthales, Glomerellales, Eurotiales, Glomerellales, Xylariales, and Botryosphaeriales), 10 families (Nectriaceae, Dipodascaceae, Polyporaceae, Diaporthaceae, Glomerellaceae, Aspergillaceae, Xylariaceae, Apiosporaceae, Hypoxylaceae, and Botryosphaeriaceae), 12 genera, and 17 species in total.

. . Antibacterial screening of the EtOAc extracts from endophytic fungi cultures
With the fermentation products of the endophytic fungi from Gannan navel orange in hand, the antimicrobial screening for the discovery of potential strain-producing biologically interesting secondary metabolites was then conducted. The antimicrobial results of the tested extracts are depicted in Table 2. The results revealed that the EtOAc extracts of 8 endophytic fungi exhibited obvious inhibitory activities against Gram-negative bacterium E. coil, whereas the other 9 EtOAc extracts showed no noticeable antimicrobial activities. Notably, the aforementioned 8 antibacterial strains belonged to 7 genera of Fusarium, Geotrichum, Fusarium, Diaporthe, Colletotrichum, Penicillium, Annulohypoxylon, and Neofusicoccum.
Moreover, the antibacterial activities against the Grampositive bacterium MRSA and plant pathogenic bacterial Xcc were also conducted for the 17 EtOAc extracts of the isolated endophytic fungi. As a result, 6 EtOAc extracts of endophytic fungi exhibited significant inhibitory activities against MRSA, involving Fusarium solani, Fusarium proliferatum, Diaporthe biconispora, Colletotrichum gloeosporioides, Colletotrichum sp., and Fusarium graminearum. Notably, among them, Colletotrichum gloeosporioides (gc-1-129-133) demonstrated the highest antibacterial activity with a MIC value of 62.5 µg/mL, which was closely comparable to that of the clinical drug vancomycin (positive control, MIC = 1.3 µg/mL), showing significant potential for the discovery of novel antibacterial drugs. Similarly, some EtOAc extracts of the endophytic fungi exhibited considerable inhibitory activities against the plant pathogenic fungus Xcc, which were Fusarium solani, Geotrichum sp., Polyporus arcularius, Diaporthe biconispora, Colletotrichum gloeosporioides, Penicillium oxalicum, Colletotrichum sp., and Penicillium paneum. Interestingly, both the EtOAc extracts of Geotrichum sp. (gc-1-127-30) and Diaporthe biconispora (gc-1-128-79) demonstrated very potent antibacterial activity against Xcc with MIC values as low as 62.5 and 31.3 µg/mL, respectively, strongly suggesting that these two endophytic fungi might be severed as promising natural bioresources for the discovery of novel antibacterial agents.
Taken together, the endophytic fungus Fusarium solani showed the best antibacterial activities against all of the tested bacteria involving E. coli (MIC = 500 µg/mL), MRSA (MIC = 500 µg/mL), and Xcc (MIC = 250 µg/mL), which were much more potent than other sixteen endophytic fungi from Gannan navel orange (Table 2). Notably, the attractable results also came from the endophytic fungus Fusarium solani, which showed comprehensive antibacterial activities against all of the Gram-negative bacterium (E. coli), Gram-positive bacterium (MRSA), and pathogenic fungus (Xcc), suggesting that the EtOAc extract of the endophytic fungus Fusarium solani might be developed as promising biocontrol agent with broad-spectrum antibacterial effect.

. . Isolation of the EtOAc extracts
Based on the antibacterial effects of all EtOAc extracts tested, three endophytic fungi Colletotrichum sp., D. biconispora, and A. atroroseum were selected for the discovery of antibacterial novel secondary metabolites by column chromatography method.
Colletene A (1) was obtained as a colorless oil with the chemical molecular formula of C 17 H 28 O 4 , which was successfully established on the basis of (+)-HRESIMS m/z 319.1887 [M + Na] + (calcd for C 17 H 28 NaO 4 , 319.1880), implying four degrees of hydrogen deficiency. The 1 H NMR data (Table 3)     oxygen carbon (δ C 35.1, 57.0, and 71.7), and five non-protonated carbons (two quaternary carbons at δ C 38.7 and 46.6, two olefinic carbons at δ C 135.7 and 144.8, a carbonyl carbon at δ C 170.7). These results strongly suggested that compound 1 might be a sesquiterpene derivative. The established functional groups chemo-logically accounted for two degrees of hydrogen deficiency. Therefore, the remaining two degrees of hydrogen deficiency necessitated that compound 1 should possess a bicyclic ring system. In order to construct the bicyclic skeleton of compound 1, the 2D NMR spectra involving both the HMBC and 1 H-1 H COSY (Figure 4) spectra were performed and elucidated. First, the 1 H-1 H COSY correlations of H 3 -11/H-2/H 2 -3/H-4/H-5 informatively suggested the presence of one independent spin system. The key HMBC correlations from H 2 -10 to C-2 and C-9, H 3 -14 to C-7, C-9, and C-15, H-5 to C-1, C-12, and C-13, H 2 -7 to C-5, C-9, C-12, C-13, C-14, and C-15 could readily conclude the existence of rings A-B, which formatted as a 6/5 fused bicyclic ring system. The aforementioned result also coincides with its hydrogen deficiency. Moreover, the obvious HMBC correlation from H-4 to C-16 further confirmed that the carbonyl group should be attached at the C-4 position. Therefore, the planar structure of 1 was completely established, which suggested that compound 1 should be a new botryane sesquiterpene.
. /fmicb. .  The relative configuration of 1 was determined by the careful analysis of its NOESY spectrum (Figure 4). The key NOESY signals between H-4 to H-2 and H 3 -13 as well as H 3 -13 to H 3 -14 informatively suggested that these protons were on the same side and assigned as α-orientation. Then, the NOESY correlations of H-5 to H 2 -15 demonstrated that these protons were β-orientated. Notably, the absolute configuration of 1 could not be determined because of its weak Cotton effects. Therefore, the chemical structure of 1 was finally established as depicted in Figure 4, and named as colletene A.
All the isolated compounds were evaluated for antibacterial activities against SA, MRSA, E. coli, and Xcc. Notably, the phenolic compound 2 obtained from Colletotrichum sp. showed significant inhibitory effects toward all the tested bacterial strains of SA, MRSA, E. coli, and Xcc with MIC values of 12.5, 3.1, 125, and 12.5 µg/mL, respectively (Table 4). Satisfactorily, these results are consistent with our previous screening experiment for the EtOAc crude extract of Colletotrichum sp. Moreover, compounds 19 and 22 isolated from A. atroroseum also showed weak antibacterial activities against SA, MRSA, and Xcc.
The significant health benefits and potent biological activities of Gannan navel orange suggested that its endophytic fungi might also be interesting. In the present study, a total of 54 endophytic fungi were isolated from Gannan navel orange fruits. Meanwhile, we also tested the antibacterial activity of the EtOAc extracts of most of the isolated endophytic fungi against Gram-negative bacterium E. coli, MRSA, and Xcc as three representative pathogenic bacteria for the first time. Therefore, this study will provide some valuable information for the wide utility of endophytic fungi of Gannan navel oranges and facilitate the development of the Gannan navel oranges industry as well.
In this study, we have successfully isolated and identified 17 strains of endophytic fungi from the Gannan navel orange (C. sinensis Osbeck cv. Newhall). To the best of our knowledge, this is the first comprehensive investigation on the isolation and identification of endophytic fungi from healthy Gannan navel orange tissues. As shown in Table 1, most of the endophytic fungi such as Fusarium solani (Kurt et al., 2020), Geotrichum sp. (Zhao et al., 2020), Fusarium proliferatum (Hyun et al., 2000), Diaporthe biconispora , Colletotrichum .
Notably, the Polyporus arcularius, Penicillium paneum, Nigrospora chinensis, Nigrospora sphaerica, Annulohypoxylon atroroseum, and Fusarium graminearum were discovered from the family of Citrus for the first time, and they might be served as characteristic fungi of Gannan navel orange, although more evidences should be provided to support this statement in future study. Moreover, this study has not only first investigated the endophytic fungi of Gannan navel orange, but also provided some valuable information for biologists to explore the potent relationship between the endophytic fungi and their host plants. The investigation of its rarely studied endophytic fungi might broaden the microcosmic knowledge and illustrate their pharmaceutical potency. Therefore, this study greatly enriched the species diversity of endophytic fungi of Frontiers in Microbiology frontiersin.org . /fmicb. .

FIGURE
The structures of compounds -.
Gannan navel orange, and it laid a good foundation for the subsequent research on the endophytic resource of Gannan navel orange. Endophytic fungi have been extensively investigated due to their remarkable ability for the production of a variety of secondary metabolites with pronounced pharmaceutical activity and interesting chemical structures (Newman and Cragg, 2020). In addition, endophytic fungi usually play important roles in the protection of host plants and produce different bioactive natural products under different certain conditions (Wang et al., 2023), which makes them much more interesting for natural product discovery and plant-microbial interaction (Schulz et al., 2002). In this study, we tested the inhibitory effect of secondary metabolites of endophytic fungi of navel orange against three different pathogens, and many of them showed considerable antimicrobial effects. The results strongly suggested that the endophytic fungal types of Gannan navel orange are rich and diverse. Moreover, this study also provides scientific data for the exploitation of endophytic fungal resources of Gannan navel orange. Metabolites from endophytic fungi have great potential for the discovery of pharmaceutical agents, and the present study provided important information for the discovery of antibacterial natural compounds from the endophytic fungi of Gannan navel orange.
In particular, many countries have encountered huge economic losses because of the severity of CBC caused by Xcc. The traditional treatment of the disease with copper-based chemical sprays and streptomycin has led to the emergence of resistant Xanthomonas strains (Martins et al., 2020). The development of alternative biocontrol agents has now become a major challenge for scientists. In the antibacterial activity screening, the crude extracts of Colletotrichum sp., D. biconispora have better inhibitory effects against Xcc. Therefore, we have performed the chemical composition investigation and an anti-Xcc assay of individual compounds isolated from Colletotrichum sp. and D. biconispora. The active compound 2 might be responsible for the antibacterial effects of the EtOAc extracts. The current results suggested that it might be a prospective and promising way to discover anti-Xcc pesticides from Citrus plants itself based on the protective effect of endophytic fungi on the host plant.
Furthermore, we also selected A. atroroseum for the chemical study, which successfully resulted in the isolation of nine compounds involving isocoumarin, azaphilones, tetralones, and steroids, but none of these compounds showed significant antibacterial activity against SA, MRSA, and E. coli. More bioassay-guided isolation strategies should be performed in our future study to identify highly potent antibacterial compounds from the isolated endophytes. Moreover, the biological evaluations of Gannan navel orange endophytic fungi in this study were limited by the experimental platforms, other biological activities such as the antiviral, antiinflammatory, antifungal, and various enzyme inhibitory effects should be carried out to fully illustrate the pharmaceutical

. Summary
In conclusion, the identification of endophytic fungi from Gannan navel orange, the antibacterial screening of their crude fermentation products, and the isolation of bioactive compounds from the EtOAc extracts of two bioactive endophytic fungi were carried out in the present study. A total of 54 strains belonging to 17 species of 12 genera were successfully isolated from Gannan navel orange for the first time. The EtOAc extract of Fusarium proliferatum (gc-1-128-74-1) exhibited excellent antibacterial activity against E. coli, and the EtOAc extracts of both Geotrichum sp. (gc-1-127-30) and Diaporthe biconispora (gc-1-128-79) demonstrated significant antibacterial activity against Xcc. In addition, The MIC value of the EtOAc extract of Colletotrichum gloeosporioides against MRSA was comparable to the positive control vancomycin. Furthermore, 24 compounds involving a new compound were isolated from the EtOAc extracts of Colletotrichum sp., D. biconispora, and A. atroroseum. In addition, compound 2 showed significant inhibitory activities toward SA, MRSA, E. coli, and Xcc with MIC values of 12.5, 3.1, 125, and 12.5 µg/mL. The present results indicated that the endophytic fungi of Gannan navel orange were abundant and had great potential to produce antibacterial compounds against E. coli, SA, MRSA, and Xcc deserving more attention in future studies.

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

Author contributions
HW conducted the experiment and collected the experimental data. ZL guided the molecular experimental and phylogenetic analysis. YC and KQ evaluated the activities of the extracts. FD and QX revised the manuscript. HL performed the experiments of compounds isolation. JZ and HT conceived and designed the experiments. All authors contributed to the article and approved the submitted version.

Funding
Financial support for this research was provided by the National Natural Science Foundation of China (82173711)