Endophytic Fungi Associated with Coffee Leaves in China Exhibited In Vitro Antagonism against Fungal and Bacterial Pathogens

Coffee endophytes have been studied for almost 74 years, and several studies have demonstrated coffee-endophytic fungi with antibacterial and antifungal potential for human and plant pathogens. In this study, we isolated and identified a total of 235 strains of endophytic fungi from coffee leaf tissues collected in four coffee plantations in Pu’er city, Yunnan province, China. Molecular identification was carried out using maximum likelihood phylogenetic analysis of nuclear ribosomal internal transcribed spacer (ITS1-5.8S rDNA-ITS2) sequences, while the colonization rate and the isolation frequency were also calculated. Two pathogenic fungi (Alternaria alternata and Penicillium digitatum) and two pathogenic bacteria (Pseudomonas syringae and Salmonella enterica subsp. enterica) were used for screening the antagonistic activities of 61 strains of coffee-endophytic fungi by a dual-culture test assay while maximum likelihood phylogenetic analysis confirmed their natural classification. This is the first study of coffee-leaf-endophytic fungal diversity in China, and the results revealed that coffee-endophytic fungi from this study belong to the Ascomycota, distributed among two classes, 10 orders, and 17 families. Concurrently, endophytic fungi isolates distributed in Arthrinium, Biscogniauxia, Daldinia, Diaporthe, and Nigrospora showed strong antagonistic activities against the pathogens. For the pathogens Alternaria alternata and Pseudomonas syringae, Nigrospora XCE-7 showed the best inhibitory effects with inhibition rates of 71.76% and 61.11%, respectively. For the pathogen Penicillium digitatum, Daldinia ME-9 showed the best inhibitory effect with a 74.67% inhibition rate, while Biscogniauxia PTE-7 and Daldinia T5E-1-3 showed the best inhibitory effect with a rate of 60.42% against the pathogen Salmonella enterica subsp. enterica. Overall, our study shows the diversity of coffee endophytes in four coffee-growing areas in Pu’er city, Yunnan province, China, and their potential use as biological control agents against two fungal and two bacterial pathogens.


Introduction
Coffee is one of the most important crops around the world that grow in tropical and subtropical areas, and it is the second-largest export commodity after crude oil [1,2]. Coffee is susceptible to diseases and pests, but most coffee diseases are caused by pathogenic root growth in crops such as barley and tobacco [47,48]. The endophytic fungus Acremonium alternatum controls the damage caused by the moth Plutella xylostella in beans and induces resistance against Leveillula taurica (the powdery mildew pathogen) in tomatoes [49]. Ek-Ramos et al. [49] showed that the cotton fungal endophyte Phomopsis sp. reduces caterpillar herbivory activity in cotton plants. The endophytic fungi Aspergillus terreus and Penicillium citrinum reduce the pathogenic Sclerotium rolfsii and induce increases in the biomass yield of sunflower plants [50]. Many studies have revealed that endophytic fungi, such as Ampelomyces quisqualis, Aureobasidium pullulans, Beauveria bassiana, Botryosphaeria sp., Candida oleophila, Coniothyrium minitans, Gliocladium catenulatum, Metarhizium anisopliae, Paecilomyces lilacinus, Pestalotiopsis microspora, Phlebiopsis gigantea, Purpureocillium lilacinum, Trichoderma asperellum, T. atroviride, T. gamsii, T. polysporum, and Verticillium lecanii, can be used as biocontrol agents in commercial products [51][52][53][54][55][56]. Therefore, endophytic fungi represent an effective agent in managing diseases and promoting plant growth; they also provide great opportunities for researching new products.
Studies of endophytic fungi in coffee were pioneered by Rayner in 1948 [57]. Since 1948, many studies have been published on coffee endophytes but no research has been carried out on the antagonistic effects of coffee endophytes as evaluated by a dual-culture assay. Beauveria bassiana can be established in coffee seedlings through different inoculation methods and the plants can resist coffee berry borers [58]. Fernandes et al. [59] showed that the fungal endophytes isolated from the healthy leaves of Coffea arabica could produce bacteriostatic, antifungal, and antitumor bioactive substances. Trichoderma sp. isolated from healthy coffee roots by Mulaw et al. [60] was demonstrated to have an antagonistic effect on the fungus Fusarium sp., which causes vascular wilt diseases. Monteiro et al. [61] isolated endophytes from Coffea arabica, and the results showed that Acremonium, Muscodor, and Simplicillium have antibacterial activities against Aspergillus ochraceus, while six different endophytic fungal strains can inhibit the growth of Fusarium verticillioides. The endophyte Simplicillium coffeanum isolated from coffee branches by Gomes et al. [62] has antimicrobial activities against Aspergillus niger, A. ochraceus, A. sydowii, and A. tubingensis. Trichoderma harzianum (CRF1) and T. viride (CRF-2), isolated from coffee rhizosphere soil by Ranjini et al. [63], can control Myrothecium roridum in vivo, a fungus that causes stem necrosis and leaf spot of coffee seedlings.
Both bacteria and fungi cause diseases in coffee [7]. Alternaria alternata was identified in Brazil in 1999 as the causative agent of necrotic spots on coffee leaves [64]. This pathogen is more serious when present in other fruits and vegetables, and the current control methods include fungicides, synthetic chemical fungicides, and biological control [65]. Penicillium digitatum is a destructive postharvest pathogen causing green mold in fruit, especially citrus fruits [66]. Fungicides are still the main control agent of postharvest green mold, and it is necessary to develop novel and safer strategies for effectively controlling plant diseases [67]. Bacterial halo blight disease caused by Pseudomonas syringae has affected coffee plantations in Brazil and Ethiopia. Due to only a few efficient commercial products being available on the market to control this disease and the low efficiency of the chemical control on field conditions, biological control and disease-resistant variety development should be the most promising management methods [68][69][70]. Salmonella enterica subsp. enterica is a common pathogen of animals, humans, or plants [71]. Salmonella can adhere to plant surfaces before actively infecting the interior of different plants, leading to the colonization of plant organs [72,73] and suppression of the plant immune system [74]. In addition, Salmonella originating from plants reduces its virulence toward animals [75]. Therefore, plants act as an alternative host for Salmonella pathogens and have a role in transmitting back to animals. Moreover, Salmonella can adapt to survive in different environments outside the host organism, such as low pH or high temperatures [76].
The microfungal diversity of the Greater Mekong Subregion (GMS) has been relatively well-studied, but their secondary metabolites have not been well-studied [77]. Since endophytic fungi have become the source of new antibacterial and antifungal agents, with some examples producing valuable secondary metabolites, this study was planned to identify and examine the antagonistic abilities against two fungal and two bacterial pathogens of endophytic fungi, which were isolated from healthy coffee leaves from four coffee-growing areas in Pu'er city, Yunnan province, China.

Sampling Location and Leaf Collection
Pu'er city has the largest coffee planting area, with the highest yield, and the bestquality coffee production in Yunnan province, China [78]. This study was conducted on mature and healthy leaf samples collected from four coffee-planting areas in Pu'er city, Yunnan province, China ( Figure 1, Table 1). All sites were >30 km and <210 km apart. More than 50 healthy leaves were collected from each coffee plant host, placed in clean plastic bags, kept in iceboxes, brought to the laboratory, and refrigerated at 4 • C until further processing. role in transmitting back to animals. Moreover, Salmonella can adapt to survive in different environments outside the host organism, such as low pH or high temperatures [76]. The microfungal diversity of the Greater Mekong Subregion (GMS) has been relatively well-studied, but their secondary metabolites have not been well-studied [77]. Since endophytic fungi have become the source of new antibacterial and antifungal agents, with some examples producing valuable secondary metabolites, this study was planned to identify and examine the antagonistic abilities against two fungal and two bacterial pathogens of endophytic fungi, which were isolated from healthy coffee leaves from four coffee-growing areas in Pu'er city, Yunnan province, China.

Sampling Location and Leaf Collection
Pu'er city has the largest coffee planting area, with the highest yield, and the bestquality coffee production in Yunnan province, China [78]. This study was conducted on mature and healthy leaf samples collected from four coffee-planting areas in Pu'er city, Yunnan province, China ( Figure 1, Table 1). All sites were >30 km and <210 km apart. More than 50 healthy leaves were collected from each coffee plant host, placed in clean plastic bags, kept in iceboxes, brought to the laboratory, and refrigerated at 4 °C until further processing.

Coffee Leaves Processing and Fungal Endophytes Isolation
The isolation process of endophytic fungi refers to the slightly modified method of Tibpromma et al. [79] and Du et al. [80]. Sterile water, 70% alcohol, 1% sodium hypochlorite, a sterile scalpel, sterile tissue paper, sterile fine-tip forceps, and potato dextrose agar (PDA) plates (supplemented with amoxicillin) were prepared. Ten healthy leaves were randomly selected for each host and each leaf was washed under running tap water and then air-dried. Working under an aseptic laminar hood, a hole puncher was used to randomly punch the leaves to obtain 5 mm size pieces (10 pieces/leaf) and these were washed in sterile water for one minute, dried on sterile tissue paper, soaked in 70% alcohol for 30 s, transferred to 1% sodium hypochlorite and soaked for one minute, then soaked in 70% alcohol for 30 s, and finally transferred to sterile water for one minute to remove chemicals. Afterwards, they were dried on sterile tissue paper; then, five pieces were placed on 90 mm-diameter PDA plates. A total of 30 leaf pieces were placed on six PDA plates for each host and each location. All PDA plates were incubated at 28 • C and observation of the culture plates was performed every day to check emerging hypha from the edge of the leaves. After colonies had grown, a small piece of mycelium with agar was cut and transferred to new PDA plates to obtain a pure culture [26,81,82]. There was a total of 270 coffee leaf pieces, including Arabica coffee (150 pieces × 5 plants × 1 place), Catimor (90 pieces × 1 plant × 3 places), and Yellow Bourbon (30 pieces × 1 plant × 1 places). A total of 235 pure cultures were isolated in this study ( Table 2).

DNA Extraction, PCR Amplification, and ITS Gene Sequencing
The Biospin Fungus Genomic DNA Extraction Kit-BSC14S1 (BioFlux, Beijing, China) was used for DNA extraction from pure cultures grown on PDA for 7-10 days. The extraction process followed the protocol of the manufacturer. Internal transcribed spacer (ITS) regions 1 and 2 of the nuclear ribosomal DNA operon, including the 5.8S region, were amplified by the primers ITS5 and ITS4 [83]. The methods of Lu et al. [84] were followed for the polymerase chain reaction (PCR). Amplification reactions were performed in a 25 µL reaction volume which contained 2 µL DNA, 1 µL of each reverse and forward primers and 8.5 µL ddH2O, 12.5 µL 2 × FastTaq Premix (mixture of FastTaq TM DNA Polymerase, buffer, dNTP Mixture, and stabilizer) (Beijing Qingke Biological Technology Co., Ltd., Beijing, China). The amplified PCR products were sent to Bioer Technology Co., Ltd., Hangzhou, and Beijing Kinco Biotechnology Co., Ltd. Kunming Branch, China for sequencing.

Molecular Identification and Phylogenetic Analyses
A total of 235 sequences were obtained, and the newly generated sequences were merged forward and reversed by Geneious 9.1.2 (https://www.geneious.com/ (Auckland, New Zealand), accessed on 30 June 2022). The BLAST search of the sequences was carried out and the top five high-similarity results were recorded. Among our isolates, resistant genera based on published papers and less frequently isolated genera were selected (61 isolates) for the dual-culture assay. The closest match of sequences for the 61 isolates that were used for dual-culture assay were retrieved from GenBank. Multiple alignments were prepared using the online tool MAFFT version 7 (https://mafft.cbrc.jp/alignment/server/, accessed on 1 June 2022) [85], manually edited in BioEdit v. 7.0.5.2 [86], and then their format converted from FASTA to PHYLIP by ALTER online tool (https://www.sing-group.org/ALTER/, accessed on 1 April 2022) [87]. Phylogenetic analyses of the aligned sequences were conducted using the maximum likelihood method [88]. Maximum likelihood trees were generated via RAxML-HPC version 8 on XSEDE (8.2.12) [89,90] in the CIPRES Science Gateway platform [91] using the GTR+I+G model of evolution. The resulting trees were visualized with FigTree version 1.4.0 and annotated in Microsoft 365 PowerPoint. Sixty-one generated sequences were deposited in GenBank and accession numbers were obtained.

Diversity Analysis
The doughnut chart in Excel 365 was used to show the family, order, and class distribution of endophytic fungi. The colonization rate (CR) and the isolation frequency (IF) of the calculated endophytic fungi: CR = NS/NL × 100%, IF = NS/NT × 100%, NS = number of strains isolated from the location, NL = number of leaf pieces prepared from the location, NT = all number of isolates from all location. Then, the taxonomic diversity based on the species/genera (S/G) ratio was calculated to evaluate the endophyte diversity of each host. In addition, the relative abundances of the species were calculated and the similarity between the fungal communities was estimated using Sorensen's index [92]. The species diversity of different hosts in each sampling site was calculated by the Shannon diver-

Tests for Antagonism: Endophytic Fungi vs. Pathogenic Fungi/Bacteria
Endophytes were selected for dual-culture testing in vitro based on the information reported in the literature [95,96] and the less frequent strains from our results. The living cultures of the 61 endophytic fungi were deposited in the Kunming Institute of Botany Culture Collection (KUMCC). The 61 endophytic fungi were tested for dual-culture assays in 9 cm Petri dishes with two fungal and bacterial pathogens (i.e., Alternaria alternata, Penicillium digitatum, Pseudomonas syringae, and Salmonella enterica subsp. enterica) ( Table 3) from China General Microbiological Culture Collection Center (CGMCC). These pathogens were selected for our experiments because they are common and cause different diseases on numerous hosts (Table 3) and have been used in biological control tests [97][98][99][100][101][102][103]. The PDA was supplemented with amoxicillin antibiotic and nutrient agar (NA) was prepared without amoxicillin antibiotic. The steps used are given in Figure 2. The PDA was supplemented with amoxicillin antibiotic and nutrient agar (NA) was pared without amoxicillin antibiotic. The steps used are given in Figure 2.

Isolation and Identification of Endophytic Fungi
A total of 235 fungal endophytes were isolated from 270 coffee leaf pieces obta from four coffee plantations in Yunnan province, China. All endophytes were used fo molecular identification, based on the blast result of ITS sequences, and the closest sp information was obtained. Overall, 235 endophytes belong to Ascomycota (100%) and distributed among two classes, 10 orders, and 17 families (Figure 3). At the class l 91% are members of the Sordariomycetes, while 9% are members of the Dothideomyc

Isolation and Identification of Endophytic Fungi
A total of 235 fungal endophytes were isolated from 270 coffee leaf pieces obtained from four coffee plantations in Yunnan province, China. All endophytes were used for the molecular identification, based on the blast result of ITS sequences, and the closest species information was obtained. Overall, 235 endophytes belong to Ascomycota (100%) and are distributed among two classes, 10 orders, and 17 families (Figure 3). At the class level, 91% are members of the Sordariomycetes, while 9% are members of the Dothideomycetes. At the order level, the Glomerellales (42.6%) is the largest group, while the Amphisphaeriales (0.4%) and the Hypocreales (0.4%) are reported as the least important. At the family level, the Glomerellaceae (42.6%) is the most common family, while the Cucurbitariaceae (0.4%), Naviculisporaceae (0.4%), Nectriaceae (0.4%), Podosporaceae (0.4%), Pestalotiopsidaceae (0.4%), Sordariaceae (0.4%), and Shiraiaceae (0.4%) are the less important families.
Amphisphaeriales (0.4%) and the Hypocreales (0.4%) are reported as the least important. At the family level, the Glomerellaceae (42.6%) is the most common family, while the Cucurbitariaceae (0.4%), Naviculisporaceae (0.4%), Nectriaceae (0.4%), Podosporaceae (0.4%), Pestalotiopsidaceae (0.4%), Sordariaceae (0.4%), and Shiraiaceae (0.4%) are the less important families.  Table 2 shows that 235 species are distributed in 21 genera. Colletotrichum is the most common genus, composed of 100 isolates, and found in each location and on each host. This is followed by Xylaria (composed of 29 isolates) and Daldinia (composed of 25 isolates). Seven genera (Fusarium, Kretzschmaria, Naviculispora, Neurospora, Pestalotiopsis, Pyrenochaetopsis, and Shiraia) have a low frequency of occurrence; all of these are represented by one isolate and are distributed in two coffee plantations. In addition, 21 genus names could not be determined from only the blast results of ITS, which indicated these as unidentified fungal endophytes, unidentified fungal species, or an undetermined species in the Sordariomycetes.

Phylogenetic Analyses
Phylogenetic analyses were carried out on the 61 endophytes that were used for the dual-culture testing. Our ITS sequences were combined with the sequences of close relatives in GenBank to construct an ITS phylogenetic tree that was composed of 167 sequences. The ML analysis of the combined dataset yielded a best scoring tree with a final ML optimization likelihood value of − 13283.937651. The alignment has 684 distinct alignment patterns, with 32.41% completely undetermined characters and gaps. Parameters for the GTR+I+G model: estimated base frequencies A = 0.246497, C = 0.253353, G = 0.238501,  Table 2 shows that 235 species are distributed in 21 genera. Colletotrichum is the most common genus, composed of 100 isolates, and found in each location and on each host. This is followed by Xylaria (composed of 29 isolates) and Daldinia (composed of 25 isolates). Seven genera (Fusarium, Kretzschmaria, Naviculispora, Neurospora, Pestalotiopsis, Pyrenochaetopsis, and Shiraia) have a low frequency of occurrence; all of these are represented by one isolate and are distributed in two coffee plantations. In addition, 21 genus names could not be determined from only the blast results of ITS, which indicated these as unidentified fungal endophytes, unidentified fungal species, or an undetermined species in the Sordariomycetes.

Phylogenetic Analyses
Phylogenetic analyses were carried out on the 61 endophytes that were used for the dual-culture testing. Our ITS sequences were combined with the sequences of close relatives in GenBank to construct an ITS phylogenetic tree that was composed of 167 sequences.   The phylogenetic tree includes two classes, seven orders, and nine families. Five orders (Amphisphaeriales, Diaporthales, Glomerellales, Sordariales and Xylariales) and seven families (Apiosporaceae, Diaporthaceae, Glomerellaceae, Graphostromataceae, Hypoxylaceae, Lasiosphaeriaceae and Xylariaceae) belong to the class Sordariomycetes. Another two orders (Botryosphaeriales and Capnodiales) and two families (Cladosporiaceae and Phyllostictaceae) belong to the class Dothideomycetes (Figure 4).
Since the blast result of XCE-26 has a high similarity with both Podospora intestinacea (MN341351, 99.40%) and Cercophora thailandica (NR_164483, 99.12%), we were not able to determine the genus name and it was treated as Lasiosphaeriaceae sp.

Diversity of Coffee Fungal Endophytes
In Table 4

Dual-Culture Assay
To evaluate the antagonistic abilities of coffee endophytes, 61 coffee endophytes along with four pathogens were selected for a dual-culture assay in our study. The results are shown in Table 5. We had some dual-culture plates grown for 10 days with an inhibition rate greater or equal to 60%. For bacterial pathogens, only two endophytes exhibited significant values of growth inhibition against Pseudomonas syringae (1.3333) and Salmonella enterica subsp. enterica (1.10603) ( Figure 5). In contrast, the effect of fungal pathogens ( Figure 6) was appreciably greater than that of bacteria. For example, 13 endophytes exhibited significant values of growth inhibition against Alternaria alternata (3.15535) and 10 endophytes exhibited significant values of growth inhibition against Penicillium digitatum (3.15410). Table 5. Results of the dual-culture assay of 61 selected endophytes against the four pathogens. The type of fungal interaction for each isolate against each pathogen and the percentage of growth inhibition (I%) ± standard deviation (SD) is indicated.  Nigrospora sp. XCE-7 (Figure 5b) was able to inhibit the growth of Pse. syringae with an inhibition rate of 61.11%, and Daldinia sp. ME-9 (Figure 5c) showed an inhibition rate of 60.00%. The Pse. syringae control plate is shown in Figure 5a Nigrospora sp. XCE-7 (Figure 5b) was able to inhibit the growth of Pse. syringae with an inhibition rate of 61.11%, and Daldinia sp. ME-9 (Figure 5c) showed an inhibition rate of 60.00%. The Pse. syringae control plate is shown in Figure 5a Biscogniauxia sp. PTE-7 ( Figure 5e) and Daldinia sp. T5E-1-3 (Figure 5f) showed the same inhibition rate of 60.42% against the growth of S. enterica subsp. enterica. PTE-7 produced a yellow mycelium after it was in contact with bacterial pathogens. The S. enterica subsp. enterica control plate is shown in Figure 5d.  Daldinia sp. ME-9 (Figure 6q) inhibited the growth of Pen. digitatum with an inhibition rate of 74.67%, and this was followed by Nigrospora sp. T5E-7 (Figure 6t) with an inhibition   Illustration of an antagonist test using the dual-culture technique with two fungal pathogens and an inhibition rate greater than or equal to 60%. The two pathogens were cocultivated with the coffee fungal endophytes on PDA plates and incubated for ten days at 28 °C. (a) Alternaria alternata control plate; (b-k) Coffee endophytes inhibit A. alternata; (l) Penicillium digitatum control plate; (m-y) Coffee endophytes inhibit Pen. digitatum. Figure 6. Illustration of an antagonist test using the dual-culture technique with two fungal pathogens and an inhibition rate greater than or equal to 60%. The two pathogens were cocultivated with the coffee fungal endophytes on PDA plates and incubated for ten days at 28 • C. (a) Alternaria alternata control plate; (b-k) Coffee endophytes inhibit A. alternata; (l) Penicillium digitatum control plate; (m-y) Coffee endophytes inhibit Pen. digitatum.

Discussion
The results of our study agree with the results of previous studies [59,81,93,104], which showed that Colletotrichum and Xylaria are the dominant endophytes in coffee leaves. However, the species richness and diversity of endophytes as determined in the present study are different and higher than those reported by Santamarıá and Baymanet [81] in Puerto Rico, Saucedo-García et al. [104] in Mexico, and Oliveira et al. [93] in Brazil for coffee leaf endophytes. Species of Colletotrichum are common saprobes, pathogens, and endophytes on a range of economically important plant hosts in tropical regions [105,106], and species of Xylaria are recognized as saprotrophic fungi and as endophytic fungi of many plants [107]. Other unique and less frequently encountered species were reported, most likely due to different climates, altitude, humidity, and sampling period. Herein, nine genera are reported for the first time to be isolated as coffee endophytes. These are Annulohypoxylon, Biscogniauxia, Kretzschmaria, Naviculispora, Neurospora, and Pyrenochaetopsis, and they were isolated from Coffea arabica in the Xiang Yuan Shu He coffee plantation. Fusarium sp. and Shiraia sp. were isolated from Catimor in the Qi Xiang coffee plantation, and Nemania sp. was isolated from Yellow Bourbon in the Xiao Ao Zi coffee plantation. In this study, only the PDA medium was used, and it is possible that some species were not capable of growing on PDA.
Arthrinium is known for its antifungal capacity [110]. The species A. aureum and A. phaeospermum have the potential to inhibit the growth of Fusarium oxysporum and F. niveum and thus can be applied in biological controls [111]. Our isolate Arthrinium sp. (XCE-10) showed an inhibition rate of 64.89% against the growth of Penicillium digitatum.
Biscogniauxia sp. (O-811) isolated from the fresh fruiting bodies of wild mushrooms acts against the rice blast disease Magnaporthe oryzae by producing an inhibitory compound [112], while our endophytic strain Biscogniauxia sp. (PTE-7) can inhibit the growth of the fungal pathogens Alternaria alternata and Penicillium digitatum while also inhibiting the bacterial pathogen Salmonella enterica subsp. enterica with inhibition rates of 61.11%, 67.56%, and 60.42%, respectively.
Studies have shown that some endophytic species belonging to the genus Diaporthe can produce metabolites that protect the host from infection or act as a biological control for disease. For example, the endophytic fungus Diaporthe phaseolorum isolated from tomatoes can produce 16 different compounds, and it has an inhibitory effect on the growth of Xanthomonas vesicatoria, which causes bacterial spot disease in tomatoes [115]. Dhakshinamoorthy et al. [116] showed that the endophytic fungus Diaporthe caatingaensis from Buchanania axillaris leaves can produce the bioactive metabolite Camptothecin (CPT). While our endophyte Diaporthe sp. ME-7 has inhibitory effects on the growth of the fungal pathogens Alternaria alternata (63.43%) and Penicillium digitatum (64.89%), it did not show any apparent inhibitory effects on bacterial pathogens.
Nigrospora oryzae was isolated as an endophytic fungus from the leaves of Coccinia grandis and its secondary metabolites can be used as drugs to control diabetes. They also exhibit strong antifungal activities against the plant pathogen Cladosporium cladosporioides [117]. Our four Nigrospora isolates T5E-13, T5E-7, XCE-7, and XCE-25 showed antagonistic abilities for all three pathogens. The best isolate among all of these was XCE-7 which showed the highest inhibition rate of 71.76% against Alternaria alternata.
Our study is the first report on coffee-leaf-endophytic fungal diversity in China. We also tested the antagonistic abilities of endophytic fungi present in coffee leaves against major bacterial and fungal pathogens. Two isolates (Nigrospora sp. XCE-7 and Daldinia sp. ME-9) showed antagonism against the growth of Alternaria alternata, with an inhibition rate of over 70%. Three isolates (Daldinia sp. ME-9, Nigrospora sp. T5E-7, and Daldinia sp. T5E1-3) displayed antagonism against the growth of Penicillium digitatum, with an inhibition rate of over 70%. It is necessary to carry out further research on secondary metabolites and identification of the four coffee endophytes ME-9, T5E-1-3, T5E-7, and XCE-7 to the species level.

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