Front. Plant Sci. Frontiers in Plant Science Front. Plant Sci. 1664-462X Frontiers Media S.A. 10.3389/fpls.2024.1494855 Plant Science Original Research Antagonistic effects of endophytic fungi from Camellia reticulata pedicels on yeasts: implications for antimicrobial mechanism of nectar Huang Rong 1 † Meng Qingxin 1 † Xun Lijie 1 2 Wu Xiaoman 1 Yue Dan 1 Zhao Wenzheng 1 Dong Xia 3 Gong Xueyang 1 * Dong Kun 1 * 1 Yunnan Provincial Engineering and Research Center for Sustainable Utilization of Honey Bee Resources, Eastern Bee Research Institute, College of Animal Science and Technology, Yunnan Agricultural University, Kunming, China 2 Institute of Sericulture and Apiculture, Yunnan Academy of Agricultural Sciences, Mengzi, China 3 College of Food Science and Technology, Yunnan Agricultural University, Kunming, China

Edited by: Marie-Joelle Virolle, Centre National de la Recherche Scientifique (CNRS), France

Reviewed by: Ann C. Lawrie, RMIT University, Australia

Hany H. A. El-Sharkawy, Agricultural Research Center, Egypt

*Correspondence: Xueyang Gong, xueyangg11@126.com; Kun Dong, dongkun19722004@aliyun.com

†These authors have contributed equally to this work and share first authorship

 15 11 2024 2024 15 1494855 11 09 2024 30 10 2024 Copyright © 2024 Huang, Meng, Xun, Wu, Yue, Zhao, Dong, Gong and Dong 2024 Huang, Meng, Xun, Wu, Yue, Zhao, Dong, Gong and Dong

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Endophytic fungi are extensive in plant tissues and involved in the defense against stress from harmful microbes. The interaction between pedicel endophytic fungi and nectar yeasts is critical for maintaining nectar homeostasis. This study used Camellia reticulata as the research subject. High-throughput sequencing revealed that the community composition of endophytic fungi in the pedicel is dominated by Ascomycota and Basidiomycota. Their abundance varies at different taxonomic levels, showing sample variability. In total, 27 endophytic fungal isolates were isolated and screened from the pedicel under laboratory conditions. They exhibited antagonistic effects against three nectar yeasts (Metschnikowia reukaufii, Cryptococcus laurentii, and Rhodotorula glutinis) and displayed morphological and physiological diversity. The isolates were classified into the phylum Ascomycota and further categorized into the genera Alternaria, Trichoderma, Fusarium, and Dactylaria. The endophytic fungus D23, which effectively antagonizes nectar yeasts, was identified as Alternaria alternata. This fungus produces various secondary metabolites, including antibiotics such as penicillin G, grandiomycin, and cephalosporin C. The metabolic pathways involved include the biosynthesis of plant secondary metabolites, phenylpropanoids, amino acids, nucleotides, and antibiotics. The endophytic fungal community in C. reticulata pedicel is rich and diverse, making it a valuable material for screening antagonistic strains. This study provides a theoretical basis for the antagonistic effects of endophytic fungal metabolites from the pedicel of C. reticulata against nectar yeasts, highlighting their significance in maintaining nectar stability and reproductive fitness in cross-pollinated plants.

 Camellia reticulata endophytic fungi nectar yeasts antagonistic activity antimicrobial mechanism Alternaria alternata 32060241 National Natural Science Foundation of China10.13039/501100001809 section-in-acceptance Plant Symbiotic Interactions Introduction

Nectar is an energy reward from plants for pollinators. Yeast commonly inhabits the nectar of most plants. Increased pollinator visits enhance yeast density in the nectar. This increase in density and subsequent metabolic activity alters the chemical composition of the nectar, particularly affecting sugar and amino acid levels (Canto and Herrera, 2012; Peay et al., 2012). However, such changes may weaken the nectar’s appeal to specific pollinators and reduce visitation rates (Canto and Herrera, 2012; Schaeffer et al., 2017). Yeasts in plant nectar, primarily classified under Ascomycota and Basidiomycota, include genera such as Metschnikowia, Cryptococcus, Rhodotorula, Saccharomyces, and Sporobolomyces (Herrera et al., 2009; Pozo et al., 2012; Jacquemyn et al., 2013). Without effective antimicrobial mechanisms, nutrient-rich nectar likely supports high microorganism proliferation, diminishing its value for plant reproduction. Researchers have investigated the chemical components and defensive functions of nectar worldwide. They found that factors like pH, high osmotic pressure, and various secondary metabolites or proteins contribute to the antimicrobial properties of the nectar (Pusey, 1999; Brysch-Herzberg, 2004; Herrera et al., 2011; Álvarez-Pérez et al., 2013). However, research into the antimicrobial substances in nectar and their functions remains exploratory, and the role of endophytes is also under examination.

Endophytic fungi are crucial for the plant microenvironment, establishing a dynamic balance through colonization and operational mechanisms. They form communities with significant ecological functions (Philippot et al., 2013). These fungi enhance plant stress resistance, promote growth (Compant et al., 2005; Zhang et al., 2006), and suppress pathogen populations by spatial localization. They produce various antimicrobial metabolites that help plants resist microbial stress (Zhang et al., 2006; Pieterse et al., 2014). For instance, Stinson et al. (2003) isolated Gliocladium sp. from Eucryphia cordifolia. This fungus produces volatile organic compounds, such as cyclooctatetraene and 3-methyl-1-butanol, with strong antimicrobial effects against plant pathogens like Pythium ultimum and Verticillium dahliae. Richardson et al. (2015) isolated two Massarinaceae species from Pinus strobus, producing phenolic compounds that inhibit Bacillus subtilis and Microbotryum violaceum. Hiremani et al. (2020) isolated two species of Nigrospora sphaerica from Gossypium arboreum, producing volatile organic compounds effective against Corynespora cassiicola. Recent research has found that endophyte colonization can alter the composition of volatile metabolites in flowers, attract more pollinators, and enhance the reproductive adaptability of plants, providing new insights into the resistance mechanism of endophyte-assisted nectar against yeast (González-Mas et al., 2023). However, studies on the antagonistic effects of endophytic fungi against nectar yeasts are rare.

Camellia reticulata has been cultivated in Yunnan for a long time and is a rare plant protected in China (Feng, 1980). Its seed-extracted oil is rich in unsaturated fatty acids and antioxidants and has nutritional value comparable to olive oil, which makes it a geographical indication product in China (Qin et al., 2023). Due to self-incompatibility, C. reticulata heavily depends on pollinators for fruit set and seed production. The plant has a long flowering period, produces abundant nectar, and faces significant microbial contamination during pollination. Preliminary studies in our laboratory found that nectar from flowers blooming for many days maintains low microbial concentration. However, yeast abundance significantly increases during ex vivo fermentation, deteriorating nectar’s physicochemical properties (unpublished data). Importantly, the antimicrobial mechanism within C. reticulata nectar remains unclear. The bacteriostatic effects of endophytic fungi colonizing plant tissues may provide new explanations for their antimicrobial mechanisms. Pedicels, which are plant tissues connecting to flowers, play a crucial role in spatial distribution and serve as important conduits for nutrient supply (Zhou, 2015). The endophytic fungi colonize the pedicel near the flowers for extended periods, making them valuable microbial resources that can antagonize nectar yeasts in an auxiliary role. However, there is limited understanding of the community composition and functions of endophytic fungi colonizing C. reticulata pedicels. Accordingly, this study focuses on C. reticulata pedicels to analyze its endophytic fungal communities, screen isolates inhibiting nectar yeast, and analyze their metabolic products. Specific research objectives include (1) high-throughput sequencing to analyze endophytic fungi diversity in C. reticulata pedicels; (2) isolation of endophytic fungi from pedicels; (3) screening of endophytic fungi with antagonistic activity against nectar yeasts and evaluating these isolates’ inhibitory effects; (4) morphological and molecular biological identification of these isolates; (5) evaluation of minimum inhibitory concentration (MIC) and antibacterial potential of ethyl acetate extract from efficient isolate D23; and (6) non-targeted metabolomics analysis of metabolic products and antimicrobial substances from efficient isolate D23. This study analyzed endophytic fungi diversity in C. reticulata pedicels and screened for fungi antagonizing nectar yeast. Our results provide insights into mechanisms by which cross-pollinated plants resist yeast stress, regulate nectar stability, and maintain reproductive fitness.

 Materials and methods Sampling information and plant sample collection

In January and February 2024, C. reticulata plant samples were collected from Shaba Forestry Farm (98°5’86’’ E, 24°9’57’’ N) in Tengchong, Yunnan Province. This site has a tropical monsoon climate, with an average annual temperature of 14.6°C, approximately 2,167 h of sunshine per year, an average annual precipitation of 1,531 mm, and an average altitude of 2,149 m. During sampling, healthy C. reticulata plants were selected. Flower buds and the lower 6 cm of stems were cut using sterilized scissors (sterilized with 75% alcohol) as plant samples. These samples were placed in sterile bags and transported to the laboratory at 4°C for further processing.

 Sample surface sterilization

Plant samples were washed in flowing tap water. Under sterile conditions, flower buds and stems were trimmed to leave only pedicels. Following the method of Cardoso et al. (2020), pedicels were subjected to stringent surface sterilization. They were first rinsed with sterile distilled water and then immersed in 75% ethanol for 1 min. Pedicels were washed four times with sterile distilled water and dried with sterile filter paper. Next, they were soaked in 5% sodium hypochlorite solution for 2 min, rinsed four times with sterile distilled water, and dried again with sterile filter paper. To verify sterilization effectiveness, 100 µL of the sterile water from the final wash was plated on Luria broth (LB) and tryptic soy agar (TSA) media and incubated at 30°C for 72 h. The absence of microorganism growth confirmed that sterilization was successful and that pedicels were suitable for subsequent endophytic fungal screening and identification.

 Diversity analysis of endophytic fungi

Surface-sterilized pedicels were ground in liquid nitrogen, and total DNA from the pedicel microbiota was extracted using the E.Z.N.A.® Soil DNA Kit (Omega Bio-tek, Norcross, GA, U.S.). DNA concentration and purity were measured with a NanoDrop 2000 machine, and DNA quality was assessed using 1% agarose gel electrophoresis. PCR amplification of the ITS1-ITS2 region of genomic DNA was performed using endophytic fungal primers ITS1F (5’-CTTGGTCATTTAGAGGAAGTAA-3’) and ITS2R (5’-GCTGCGTTCTTCATCGATGC-3’) (Gardes and Bruns, 1993). PCR products were recovered using the AxyPrep DNA Gel Recovery Kit, and the target fragment size was confirmed with 2% agarose gel electrophoresis. Quantification was conducted using the QuantiFluor™-ST blue fluorescence quantification system. Purified PCR products were sent to Shanghai Majorbio Biomedical Technology Co., Ltd. for Illumina high-throughput sequencing. Sequences from raw data were optimized, and OTU clustering analysis of non-redundant sequences was performed using the Usearch software platform (similarity: 97%). Diversity analysis was conducted using the I-Sanger bioinformatics cloud platform.

 Isolation, purification, and preservation of endophytic fungi

Sterilized pedicels were cut into small pieces using a sterile scalpel and transferred to the potato dextrose agar (PDA) medium. They were incubated at 28°C in an incubator for 3-7 days. Once fungal mycelium grew at the edges of the plant tissue, hyphae were isolated from the medium based on colony characteristics using a scalpel and transferred to a fresh PDA medium for purification. After several rounds of purification to achieve uniform colony morphology, colonies were transferred to a PDA slant and stored at 4°C for preservation, with subculturing every 30 days to prevent isolate degradation. Additionally, surface mycelium was scraped using an inoculation spatula, placed in a 50% glycerol cryopreservation tube, mixed thoroughly, and frozen at -80°C for long-term storage.

 Screening of isolates antagonizing nectar yeasts

Three types of nectar yeasts provided by the China General Microbiological Culture Collection Center were used for testing: Metschnikowia reukaufii, Cryptococcus laurentii, and Rhodotorula glutinis. The yeast strains were first revived using the malt extract broth (MEB) medium. After 24 h, 1 mL of each strain suspension was taken and diluted with sterile phosphate buffered saline (PBS) to a 0.5 McFarland standard (approximately 108 CFU/mL). The prepared yeast suspension was evenly spread onto a PDA medium using a sterile cotton swab for testing. Additionally, 5 mm diameter agar discs medium covered with fungal mycelium were punched from the PDA medium. After overnight incubation at 28°C, isolates exhibiting inhibition zones were initially selected. The endophytic fungi initially screened with antagonistic activity against nectar yeast were then inoculated into potato dextrose broth (PDB) medium and cultured at 135 rpm and 28°C for 5 days in a shaking incubator. Afterward, each endophytic fungal culture (10 mL) was centrifuged at 12,000 rpm for 15 min to obtain the supernatant, which was then filtered through a 0.22 μm membrane to produce a sterile fermentation extract for each isolate. Oxford cups with a diameter of 5 mm were placed on the medium surface, and 100 μL of the fermentation extract was added to each cup. PDB medium served as the blank control (CK), and Amphotericin B was used as the positive control (+). All media were wrapped with sealing film and incubated at 28°C for 24 h. Finally, the inhibition zone diameters for each isolate were measured.

 Identification of endophytic fungi

Firstly, morphological characteristics of endophytic fungi exhibiting antagonistic activity against nectar yeast were recorded, including colony color, size, surface features, and pigment production. DNA was extracted from selected endophytic fungi using the E.Z.N.A.® Soil DNA Kit, following the manufacturer’s instructions. PCR amplification was performed with fungal primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGAATGC-3’) (White et al., 1990). Amplification results were analyzed by 1% agarose gel electrophoresis (150V/100mA for 20 min). Target DNA fragments were recovered using a QIAquick agarose gel extraction kit. The purified PCR products were sequenced using the ABI3730XL system. Sequencing data were compared to the NCBI database using BLAST. The accession number and percent identity for the strain that showed the highest alignment score with the isolate were obtained.

 Analysis of isolate metabolites showing effective antagonism against nectar yeasts Extraction of secondary metabolites of isolate

First, the endophytic fungus D23 was activated and rejuvenated. The fungus was transferred to PDA medium and incubated at 28°C for 4 days. After spores and mycelium matured, 5 mm mycelial discs were prepared and inoculated into a PDB medium. The culture was incubated at 28°C and 135 rpm for 3 days to obtain a fungal liquid culture. Next, 200 g of rice and 250 mL of MEB medium were added to each 2L wide-mouthed bottle. After autoclaving and cooling, 100 mL of fungal liquid culture was inoculated, mixed well, and incubated at 28°C for 30 days. Once the rice was fully colonized by mycelium, it was transferred to a sterilized tray, dried completely at 45°C, and then crushed. To extract the compounds, 100 g of the powdered rice was mixed with 500 mL of ethyl acetate. The mixture was extracted in a 45°C water bath for 3 h, then filtered, and the organic phase was collected. The extraction was repeated with ethyl acetate for each batch of powder a total of three times. The combined organic phases were transferred to a rotary evaporator to evaporate the solvent at 45°C. After evaporation, the organic solvent was recovered, and the crude extract was dissolved in methanol, dried in a ventilated cabinet, and stored at 4°C for future use.

 MIC estimation of isolate metabolites against nectar yeasts

MIC values were determined using the broth dilution method following Clinical and Laboratory Standards Institute (CLSI) guidelines, with Amphotericin B as the positive control (Wang et al., 2023). In the first row of a 96-well plate, 180 µL of yeast suspension (1.25 × 106 CFU/mL) was added, while 100 µL was added to the remaining wells. A crude extract solution of D23 in DMSO (10 mg/mL) was prepared, and 20 µL was added to the first row of the wells. A two-fold serial dilution was then performed across the plate, and turbidity in each well was observed to determine the lowest D23 concentration required to inhibit yeast growth.

 LC-MS/MS analysis of metabolites composition in the isolate

Non-targeted metabolomics LC-MS/MS was conducted to analyze active components in the ethyl acetate extract of D23. The MSConvert tool in the Proteowizard software package (v3.0.8789) was used to convert the original mass spectrometry downloaded file into mzXML format. Peak detection, filtering, and alignment were performed using the R XCMS package (v3.12.0), resulting in a quantitative metabolite list. Systematic errors were corrected using support vector regression based on QC samples, and substances with relative standard deviation (RSD) > 30% in QC samples were filtered out. Substance identification was conducted using public databases such as HMDB, MassBank, LipidMaps, mzCloud, and KEGG, with parameters set to < 30 ppm. Metabolite identification was conducted at Shanghai Majorbio Biomedical Technology Co., Ltd.

 Results Community structure of endophytic fungi in the pedicel of <italic>C. reticulata</italic> OTU analysis of endophytic fungi in pedicel of <italic>C. reticulata</italic>

After the final rinse of the pedicel, plating on LB and TSA media followed by 72 h of incubation revealed no colony growth, indicating successful tissue sterilization. Consequently, the isolated fungi were derived from the endophytic colonization of the pedicel and not from the environment.

OTUs of endophytic fungi from the pedicel were classified based on 97% similarity, resulting in an average of 72, 061 effective sequences with a mean length of 238 bp. Sequence lengths primarily ranged between 201 and 280 bp, aligning with expected amplification lengths and demonstrating good concentration ( Figures 1A, B ). A total of 1,034 OTUs was obtained, distributed across 4 phyla, 24 classes, 77 orders, 161 families, and 267 genera. This indicates that the endophytic fungi in the pedicel of C. reticulata exhibit rich community diversity at various taxonomic levels.

Rarefaction curves for (A) Sobs and (B) Shannon Indices, and relative abundance of endophytic fungi in C. reticulata pedicels at the (C) phylum, (D) order, and (E) family levels.

 Abundance analysis of endophytic fungi in <italic>C. reticulata</italic> pedicel

The endophytic fungal communities detected in C. reticulata pedicels included Ascomycota, Basidiomycota, and unclassified_k_Fungi ( Figure 1C ). Ascomycota and Basidiomycota together accounted for 90% of the abundance. The proportion of Ascomycetes in each group reached 60%, indicating it is a dominant phylum. These endophytic fungi were distributed across 15 orders, with varying proportions and strain uniformity in each sample. Pleosporales, Capnodiales, unclassified_p_Ascomycota, Tremellales, unclassified_c_Dothideomycetes, Xylariales, and Chaetothyriales were the dominant orders, representing over 80% of each sample ( Figure 1D ). These fungi were also distributed across 30 families, but dominant families differed among samples ( Figure 1E ). In some samples, dominant families accounted for over 20%, including Cladosporiaceae, unclassified_p_Ascomycota, Phaeosphaeriaceae, Didymosphaeriaceae, and Lophiotremataceae. However, only some endophytic fungi were classified to the family level, with others classified only to the order, class, phylum, or kingdom level. This suggests that C. reticulata pedicels harbor a diverse range of fungal species, warranting further exploration.

 Screening of endophytic fungi antagonizing nectar yeasts in <italic>C. reticulata</italic> pedicels

After rigorous surface sterilization, 118 endophytic fungal isolates were isolated from C. reticulata pedicels based on different colony characteristics. Of these, 27 isolates were preliminarily screened for antagonistic activity against the three nectar yeasts, indicated by inhibition zones. Among these, 20 isolates were inhibitory to M. reukaufii, 25 to C. laurentii, and 22 to R. glutinis. Isolates B1, B2, B10, B18, B27, D14, D23, E23, E52, E53, E57, E78, E79, and E80 exhibited antagonistic activity against all three nectar yeasts ( Table 1 ).

Preliminary screening results of endophytic fungi with antagonistic activity (indicated by inhibition zone) against nectar yeasts in C. reticulata pedicels.

Isolates Nectar yeasts
Metschnikowia reukaufii Cryptococcus laurentii Rhodotorula glutinis
B1 + + +
B2 + + +
B6 – + +
B10 + + +
B18 + + +
B24 + + –
B26 + + –
B27 + + +
D9 – + +
D14 + + +
D15 – + +
D23 + + +
E17 + + –
E23 + + +
E31 + + –
E35 + + –
E37 – + +
E40 – + +
E46 – + +
E52 + + +
E53 + + +
E57 + + +
E61 – – +
E69 + – +
E78 + + +
E79 + + +
E80 + + +

In addition, endophytic fungi with antagonistic activity against nectar yeast were again screened using fermentation substances. Our results showed that fermentation substances from isolates with preliminary antagonistic activity also exhibited antagonistic activity against yeasts ( Table 2 ). Inhibition zone measurements revealed that endophytic fungus D23 had the strongest inhibitory effect on all three yeasts, with inhibition zone diameters of 16.37 ± 0.09 mm for M. reukaufii, 15.03 ± 0.65 mm for C. laurentii, and 13.97 ± 0.46 mm for R. glutinis.

Inhibition zone diameters of endophytic fungi with antagonistic activity against nectar yeasts (mean ± S.E., unit: mm).

Isolates Inhibition zone diameter (mm)
Metschnikowia reukaufii Cryptococcus laurentii Rhodotorula glutinis
B1 7.27 ± 0.41 6.90 ± 0.29 9.20 ± 0.28
B2 10.83 ± 0.24 10.43 ± 0.63 9.37 ± 0.33
B6 – 5.47 ± 0.05 6.50 ± 0.08
B10 7.97 ± 0.17 8.80 ± 0.42 8.37 ± 0.12
B18 – – 6.47 ± 0.37
B24 8.00 ± 0.14 7.73 ± 0.09 –
B26 7.73 ± 0.39 7.87 ± 0.74 –
B27 8.13 ± 0.17 8.07 ± 0.48 8.13 ± 0.29
D9 – 8.33 ± 0.12 8.63 ± 0.12
D14 8.30 ± 0.37 7.93 ± 0.25 8.90 ± 0.29
D15 – 9.30 ± 0.14 8.27 ± 0.25
D23 16.37 ± 0.09 15.03 ± 0.65 13.97 ± 0.46
E17 9.30 ± 0.29 9.27 ± 0.17 –
E23 – 8.90 ± 0.37 8.17 ± 0.09
E31 6.83 ± 0.29 10.87 ± 0.29 –
E35 9.00 ± 0.16 8.20 ± 0.29 –
E37 – 12.10 ± 0.37 8.40 ± 0.16
E40 – 12.37 ± 0.45 8.17 ± 0.25
E46 – 11.47 ± 0.47 11.33 ± 0.54
E52 10.83 ± 0.25 10.33 ± 0.41 11.17 ± 0.54
E53 8.87 ± 0.25 7.97 ± 0.34 8.23 ± 0.31
E57 6.77 ± 0.21 8.13 ± 0.21 9.77 ± 0.52
E61 – – 8.40 ± 0.08
E69 7.83 ± 0.25 – 7.93 ± 0.12
E78 11.13 ± 0.25 9.23 ± 0.42 11.10 ± 0.50
E79 9.17 ± 0.21 7.80 ± 0.16 9.27 ± 0.26
E80 9.77 ± 0.52 9.00 ± 0.16 11.37 ± 0.54
Amphotericin B 24.30 ± 3.48 20.27 ± 0.88 21.73 ± 0.34
Identification of endophytic fungi antagonizing nectar yeasts Morphological characterization of the isolates

Isolates isolated from the C. reticulata pedicel showed diverse colony features, including color and hyphal characteristics. In early growth stages, colonies were mainly white or gray, with some light yellow or light pink. As colonies grew, colors gradually changed to gray-green, gray-brown, black, or orange. Some produced pigments altering the medium’s color to gray-brown, gray-yellow, light yellow, brown, reddish-brown, brown-yellow, and black ( Figure 2 ).

Colony morphology of endophytic fungi within the C. reticulata pedicel under cultivation conditions of 28°C on PDA medium.

 Molecular identification of isolates

Endophytic fungi isolated from the C. reticulata pedicel were all Ascomycota, classified into the Eurotiomycetes, Sordariomycetes, and Dothideomycetes classes. They were further categorized into genera such as Trichoderma, Fusarium, Daldinia, Nodulisporium, Hypoxylon, Colletotrichum, Coniochaeta, Epicoccum, Alternaria, Endomelanconiopsis, and Botryosphaeria ( Table 3 ). Among them, fungus D23, with efficient antagonism against nectar yeast, was identified as Alternaria alternata.

Molecular identification of endophytic fungi in C. reticulata pedicel.

Isolates Species Length (bp) Closest number ldentity (%)
B1 Aspergillus niger 575 PQ041979.1 99.83
B2 Penicillium citrinum 533 OK103925.1 100
B6 Trichoderma atroviride 602 LC785690.1 100
B10 Coniochaeta sp. 535 JX838853.1 99
B18 Hypoxylon griseobrunneum 524 OW985412.1 99.61
B24 Phaeoacremonium scolyti 581 KC166687.1 99.65
B26 Phaeoacremonium scolyti 581 KC166687.1 99.65
B27 Penicillium implicatum 571 MF687276.1 100
D9 Daldinia bambusicola 544 KU940155.1 99.81
D14 Alternaria tenuissima 549 MZ047512.1 100
D15 Trichoderma harzianum 598 MF109019.1 99.83
D23 Alternaria alternata 550 OQ727506.1 100
E17 Daldinia eschscholtzii 553 MZ724905.1 99.82
E23 Colletotrichum citri 577 MZ724781.1 99.82
E31 Trichoderma gamsii 592 PP469586.1 99.66
E35 Colletotrichum fructicola 558 OQ511318.1 99.82
E37 Botryosphaeria dothidea 994 LC317472.1 99.35
E40 Daldinia eschscholtzii 554 KJ466979.1 99.82
E46 Colletotrichum siamense 557 MZ066745. 99.82
E52 Fusarium acuminatum 542 MK583544.1 99.45
E53 Alternaria solani 548 KX452728.1 99.82
E57 Endomelanconiopsis microspora 566 MH862651.1 99.46
E61 Nodulisporium sp. 685 EF694672.1 98.93
E69 Colletotrichum camelliae 556 ON025203.1 99.28
E78 Penicillium citrinum 539 KY921947.1 99.81
E79 Epicoccum nigrum 527 OP696974.1 99.81
E80 Fusarium acuminatum 557 MZ724839.1 99.27
Antagonistic activity of <italic>A. alternata</italic> against nectar yeasts Determination of <italic>A. alternata</italic> MIC against nectar yeasts

The MIC of A. alternata was below 500 µg/mL for all three tested nectar yeasts, showing significant potential to inhibit them, similar to Amphotericin B. Specifically, A. alternata exhibited the highest antimicrobial activity against M. reukaufii, with a MIC of 125 µg/mL, followed by a MIC of 250 µg/mL against C. laurentii and 500 µg/mL against R. glutinis. For Amphotericin B, MIC values for the three nectar yeasts were 1.95, 3.91, and 3.91 µg/mL, respectively.

 Compositional analysis of the <italic>A. alternata</italic> metabolites

The ethyl acetate extract of A. alternata was analyzed using LC-MS/MS, identifying 4036 metabolites categorized into nine classes: organic acids, lipids, carbohydrates, nucleic acids, peptides, vitamins and cofactors, steroids, hormones and transmitters, and antibiotics. Amino acids had the highest proportion, followed by carboxylic acids, monosaccharides, steroid hormones, nucleotides, fatty acids, vitamins, cofactors, neurotransmitters, nucleosides, and eicosanoids. In addition, aminoglycosides, beta-lactams, quinolones, and other antibiotics were also present ( Figure 3 ).

Compositional analysis of metabolites of A. alternata using KEGG database annotation.

Six antibiotics were detected in positive ion mode: cephalosporin C, cyclohexanimide, griseofulvin, mitomycin, nalidixic acid, and streptomycin. Additionally, seven antibiotics related to antimicrobial activity were detected in negative ion mode: ampicillin, chloramphenicol, fusiform acid, geldermycin, neomycin, penicillin G, and grandiomycin. These antibiotics mainly belong to beta-lactams, polyketides and nonribosomal peptides, quinolones, aminoglycosides, and other protein families ( Table 4 ).

Major antibiotics present in the metabolites of A. alternate.

ID Metabolite Category Formula
pos_704 cephalosporin C beta-lactams C16H21N3O8S
pos_776 cyclohexanimide others C15H23NO4
pos_1056 griseofulvin polyketides and nonribosomal peptides C17H17ClO6
pos_1316 mitomycin others C15H18N4O5
pos_1412 nalidixic acid quinolones C12H12N2O3
pos_1855 streptomycin aminoglycosides C21H39N7O12
neg_391 ampicillin beta-lactams C16H19N3O4S
neg_495 chloramphenicol others C11H12Cl2N2O5
neg_710 fusiform acid others C31H48O6
neg_728 geldermycin polyketides and nonribosomal peptides C29H40N2O9
neg_1135 neomycin aminoglycosides C23H46N6O13
neg_1293 penicillin G beta-lactams C16H18N2O4S
neg_1419 grandiomycin aminoglycosides C14H24N2O7

Metabolic pathways involving A. alternata metabolites were mainly related to ABC transporters, biosynthesis of phenylpropanoids, alkaloids derived from the shikimate pathway, nucleotide metabolism, carbon metabolism, tyrosine metabolism, pyrimidine metabolism, and degradation of aromatic compounds ( Figure 4 ).

Major pathways involved in the metabolism of A. alternata, revealed by KEGG database annotations.

 Discussion

Ascomycota and Basidiomycota were the dominant phyla of endophytic fungi found in the pedicel of C. reticulata. Previous studies have shown that in C. oleifera, Ascomycota is the most abundant phylum across all tissues, followed by Basidiomycota (Ercolani, 1991). Similarly, most plants also have Ascomycota as the dominant endophytic phylum, with Basidiomycota as the second most common, such as in Aechmea, Fagopyrum tataricum, and Lunasia amara (Rajamanikyam et al., 2017; Istikorini and Hartoyo, 2019; Leroy et al., 2019; Chen et al., 2024). However, most fungal species in C. reticulata pedicel were only classified to the order level, indicating the potential for discovering new species. Additionally, significant differences in the abundance of endophytic fungi at the family level indicate a diverse distribution of species in C. reticulata. No clear patterns were found in the dominant fungal families in C. reticulata pedicel compared to other Camellia species, possibly due to differences in the plant’s growth environment, which significantly influences endophytic fungal diversity.

Endophytic fungi are widely existed in plant tissues and an increasing number of these fungi have been isolated and identified (Hardoim et al., 2015). Their metabolites can effectively control diseases in agricultural and forestry crop (Abbasi et al., 2002). For instance, Penicillium vinaceum, isolated from Crocus sativus, secretes quinazoline alkaloids and exhibits significant antimicrobial activity against various pathogens (Zheng et al., 2012). Similarly, Phomopsis cassiae, isolated from Cassia spectabilis, produces five different sesquiterpenes and demonstrates strong antimicrobial activity against two plant-pathogenic bacteria (Silva et al., 2006). Nectar is rich in various nutrients, and yeast is commonly found within it. Yeast can significantly alter the composition and concentration of sugars and amino acids in nectar, accelerate flower aging, reduce attractiveness to pollinators, and impact the reproductive fitness of cross-pollinated plants (Herrera et al., 2008; Canto and Herrera, 2012). Given the antibacterial properties of endophytic fungi, they may assist nectar in antagonizing yeast proliferation. In this study, numerous endophytic fungi with inhibitory effects on yeasts have been screened from the C. reticulata pedicel, opening avenues for further research into the role of these endophytes in maintaining nectar stability. However, although research has shown that endophytes can change their colonization sites through rhizosphere competence and motility (Hardoim et al., 2015), the hypothesis that endophytic fungi within the pedicels could migrate to the nectar and influence its composition remains unconfirmed.

Endophytic fungi with antagonistic activity against nectar yeasts, isolated from the pedicel of C. reticulata under laboratory conditions, belong to Ascomycota phylum. Numerous species from the genera Trichoderma, Penicillium, and Fusarium are recognized as effective biocontrol agents. Fungi with antagonistic properties identified in previous studies predominantly belong to the Ascomycota, aligning with the findings of this study (Arnold and Lutzoni, 2007). The genus Trichoderma is a highly competitive biocontrol agent, with extensive research and applications documented (Peng et al., 2021). Trichoderma sp. protects several crops from diseases (Youssef et al., 2016; Swain et al., 2018). Penicillium, another endophytic genus, produces various antimicrobial compounds that have been used in crop disease and nematode control (Chen et al., 2022). Su et al. (2010) reported antibacterial activity in the culture filtrate of Aspergillus endophytes from tea trees, effective against Camellia brown spot disease. Furthermore, this study identified two antagonistic endophytic fungi only at the genus level, highlighting the potential for new species discovery in C. reticulata.

The identified A. alternata exhibited superior inhibition of nectar yeasts, affecting M. reukaufii, C. laurentii, and R. glutinis. Secondary metabolites from the genus Alternaria include pyrones, quinones, alkaloids, steroids, and terpenes, all of which demonstrate significant antimicrobial activity (Tantry et al., 2018; Lee et al., 2019; Chen et al., 2020). Specifically, capsaicin from A. alternata has been shown to exert antimicrobial effects against Staphylococcus aureus, Escherichia coli, and Aspergillus niger (Devari et al., 2014). Additionally, naphthalene and pyrone compounds from marine-derived A. alternata inhibit Candida albicans and Fusarium oxysporum (Shaaban et al., 2012). Alternaramide, a cyclic peptide from marine-derived A. alternata, is effective against Bacillus subtilis and Staphylococcus aureus (Kim et al., 2009). The identified metabolites in this study include antibiotics with antimicrobial properties, such as chloramphenicol, penicillin G, grandiomycin, and cephalosporin C, thus expanding the known data on A. alternata metabolites. Moreover, key metabolic pathways in A. alternata encompass the biosynthesis of plant secondary metabolites, phenylpropanoids, amino acids, antibiotics, and alkaloids. This analysis provides a basis for further research into its applications and mechanisms.

Importantly, nectar generally contains antimicrobial substances that are transported through the phloem and xylem during the nectar secretion process, enhancing pollination services and safeguarding rewards for flowers and/or pollinators (Adler, 2000; Stevenson et al., 2017). The nectar of C. reticulata is abundant and it depends on pollinators for fertilization. Endophytic fungi colonize the pedicels and produce a wealth of secondary metabolites, including various antibiotics. Under laboratory conditions, some of these fungi have demonstrated promising inhibitory effects against nectar yeasts. Our research findings provide insights into potential strategies that cross-pollinated plants may employ to cope with yeast stress, regulate nectar stability, and maintain reproductive fitness. However, further research is needed to determine whether metabolites secreted by endophytic fungi are transported from the phloem sieve tubes to the nectar to exert their effects, along with the underlying mechanisms and potential applications in antagonistic nectar yeasts.

 Conclusion

Endophytic fungal diversity in pedicels of C. reticulata was high, primarily comprising Ascomycota and Basidiomycota. Twenty-seven isolates of endophytic fungi that exhibited antagonistic activity against three types of nectar yeasts were isolated and cultured. These isolates exhibited diverse morphologies and were identified into genera such as Trichoderma, Fusarium, and Alternaria. Among them, isolate D23, identified as A. alternata, demonstrated the highest antimicrobial potential. Its metabolites included antibiotics like penicillin G, grandiomycin, and cephalosporin C, and it participated in metabolic pathways related to phenylpropanoid and alkaloid biosynthesis. This research provided insights into the antagonistic effects of endophytic fungal metabolites against nectar yeasts in C. reticulata pedicels, emphasizing the importance of endophytes in assisting nectar maintain stability and support reproductive fitness in cross-pollinating plants.

 Data availability statement

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

 Author contributions

RH: Formal analysis, Software, Visualization, Writing – original draft, Writing – review & editing. QM: Software, Visualization, Writing – original draft, Writing – review & editing. LX: Formal analysis, Writing – original draft. XW: Methodology, Writing – original draft. DY: Formal analysis, Writing – original draft. WZ: Methodology, Writing – original draft. XD: Conceptualization, Writing – original draft. XG: Writing – review & editing. KD: Data curation, Funding acquisition, Project administration, Supervision, Writing – review & editing.

 Funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by National Natural Science Foundation of China (32060241 and 31572339), the China Agriculture Research System of the Ministry of Finance and the Ministry of Agriculture and Rural Affairs (CARS-44-KXJ13), and the Reserve Talents Training Program for Young and Middle-Aged Academic and Technical Leaders in Yunnan (2018HB041).

 Acknowledgments

We gratefully acknowledge the support of the Forestry Bureau of Tengchong, Yunnan Province, and the Tengchong Camellia Resource Bank at Shaba Forestry Farm for their contributions to this project, as well as MJEditor (www.mjeditor.com) for their English editing services during the preparation of this manuscript.

 Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

 Publisher’s note

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