Research Article
Print
Research Article
Discovering fungal communities in roots of Zoysia japonica and characterising novel species and their antifungal activities
expand article infoHaifeng Liu‡, Hyeongju Choi‡, Narayan Chandra Paul‡, Hiran A. Ariyawansa§, Hyunkyu Sang‡
‡ Chonnam National University, Gwangju, Republic of Korea
§ National Taiwan University, Taipei, Taiwan

Corresponding author: Hyunkyu Sang ( hksang@jnu.ac.kr )

Academic editor: Yasmina Marin-Felix
© 2025 Haifeng Liu, Hyeongju Choi, Narayan Chandra Paul, Hiran A. Ariyawansa, Hyunkyu Sang.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Liu H, Choi H, Paul NC, Ariyawansa HA, Sang H (2025) Discovering fungal communities in roots of Zoysia japonica and characterising novel species and their antifungal activities. IMA Fungus 16: e138479. https://doi.org/10.3897/imafungus.16.138479
Open Access

Abstract

Turf-grasses are economically important horticultural crops, which have been utilised by humans to improve the environment for more than a thousand years. Turf-grasses are widely distributed in landscapes, slopes and sport fields, such as golf courses. Endophytic fungi are a resource of unexplored fungal diversity with potential bioactive compounds. In this study, culture-independent ITS amplicon sequencing and culture-dependent isolation methods were used to reveal fungal community in roots of the turf-grass Zoysia japonica. A total of 317 OTUs were identified from root samples of Z. japonica by analysis of ITS amplicon reads. Fungal community was dominated by Sordariales (32.45%), followed by Chaetothyriales (18.16%), unknown taxa in Sordariomycetes (14.63%) and Pleosporales (12.48%). During isolation, 151 endophytic fungal strains were obtained from roots of Z. japonica and a variety of taxa were found by ITS amplification and sequencing. Moreover, 11 endophytic fungal species were further characterised in this study, based on morphological characterisation and multi-loci phylogenetic analysis, including Niesslia dimorphospora, a newly-recorded species in Korea and 10 novel species (Dactylaria hwasunensis sp. nov., Lophiostoma jeollanense sp. nov., Magnaporthiopsis zoysiae sp. nov., Poaceascoma endophyticum sp. nov., P. koreanum sp. nov., P. magnum sp. nov., P. zoysiiradicicola sp. nov., Stagonospora endophytica sp. nov., Setophoma zoysiae sp. nov. and Pseudorhypophila poae sp. nov.). Antifungal activities of these species were tested against the turf-grass brown patch pathogen Rhizoctonia solani AG2-2(IIIB), with S. zoysiae being the best antagonist. In addition, butanol extract from mycelia of S. zoysiae strongly inhibited R. solani AG2-2(IIIB) in vitro and in planta. The results of this study expand the biodiversity of endophytic fungi and revealed potential biological resources for future turf-grass management and bioactive compound exploitation.

Key words:

Bioactivity, endophytic fungi, ITS amplicons, phylogeny, turf-grass

Introduction

Zoysiagrasses (Zoysia spp. Willd.) are mat-forming perennial warm-season grasses belonging to the family Poaceae, native to the western Pacific Rim and the Indian Ocean (Tothill and Hacker 1983; Soreng et al. 2015). Zoysiagrasses gained popularity amongst growers for their low maintenance inputs and tolerance to multiple biotic and abiotic stresses. A total of 11 species are reported in the genus Zoysia, but only Z. japonica, Z. matrella and Z. pacifica and their interspecific hybrids have been widely used as turf-grass including on golf courses, athletic fields, home lawns and other recreational sites (Chandra et al. 2017). Zoysia japonica is one of the most important turf-grass species distributed mainly in Asia, North and South America and Australia (Loch et al. 2017). Due to its popularity, some biotechnologies, such as conventional breeding and molecular transformation have been used to improve Z. japonica (Ge et al. 2006; Yang et al. 2023).

Endophytes colonising intra- or intercellular plant tissues play multifaceted roles in plant-microbe interactions, ranging from promotion of plant growth to biocontrol of pathogens and enhancement of biotic/abiotic tolerance (Sridhar 2019; Bharadwaj et al. 2020). Application of endophytes is increasingly envisioned to reduce the use of agrochemicals and lower production costs (Tiwari et al. 2023). In recent years, endophytic fungi have attracted great attention from researchers and are increasingly being investigated for their ability to secrete various bioactive compounds (secondary metabolites), simulating plant response to environmental stresses, facilitating nutrient uptake and acting as biocontrol agents (Poveda et al. 2020, 2021; Jha et al. 2023). For example, endophytic Trichoderma spp. are well known to enhance hosts’ tolerance to multiple stresses and antagonise the growth of a wide range of plant pathogens (Ripa et al. 2019; Rajani et al. 2021). Xia et al. (2019) reported that endophytic fungal species Aspergillus nidulans, Coniothyrium aleuritis, Fusarium oxysporum, Fusarium proliferatum, Pichia guilliermondii and Trichoderma spirale significantly increased tomato fruit yield. According to Fávaro et al. (2012), the endophyte Epicoccum nigrum isolated from sugarcane (Saccharum officinarum) was able to increase root biomass and inhibit several pathogens in vitro. In turf-grass, the most frequently studied endophytes belong to the genus Epichloë (anamorphs in Neotyphodium) (Meyer et al. 2015). Epichloë sp. was reported to increase the root growth, metabolic activity and nutrient uptake of ryegrass (Lolium perenne), thereby improving its survival in less fertile soil (Chen et al. 2020). It was also found that ryegrass with the presence of the endophyte Epichloë showed increased resistance to pathogens Drechslera siccans and Fusarium spp. (Wiewióra et al. 2015). Currently, more than 80 commercial turf-grass cultivars contain endophytes and new grass-endophyte combinations are being developed to reduce pesticide use and lower turf maintenance input (Clay 1990; Wiewióra et al. 2015). However, endophytes have not been found in warm-season grasses (Held and Potter 2012).

Although many studies on endophytes have been carried out to capitalise on their potential applications in enhancing agricultural productivity, only a small fraction of endophytes have been isolated and studied and most endophytes remain largely unexplored. Thus, new approaches such as metagenomic and amplicon sequencing have provided more insights into the diversity of endophytes and simplified the analysis of endophytic microbial communities. For fungi, the internal transcribed spacer (ITS) region has been recognised as the standard barcode and either the ITS1 or the ITS2 region is used for high-throughput sequencing to analyse environmental fungal diversity (Monard et al. 2013). Using this technique, Porras-Alfaro et al. (2008) revealed root associated fungi (RAF) in a grass Bouteloua gracilis, which was dominated by dark septate fungi (DSF) in Pleosporales. Khidir et al. (2010) found the presence of RAF in several grass species and a similar cohort of fungal dominants was shared by grasses in semi-arid landscapes. In addition to using a high-throughput sequencing technique alone, an increasing number of studies are incorporating culture-dependent isolation for improved results. For example, Abeywickrama et al. (2023) elucidated fungal community composition associated with Astrugalus sinicus and Vicia villosa by culture-dependent methods and culture-independent amplicon analyses, which is meaningful for green manure practices. Manzotti et al. (2020) analysed the fungal root endophytes community structure of tomato with similar integrative methods and revealed pathogenicity of endophytic fungi by isolation and re-inoculation, suggesting that equilibrium and evenness within the microbiome community are essential for plant health.

Due to the significance of root-inhabiting endophytic fungi to turf-grasses and because little is known about the endophytes associated with the warm-season turf-grass Z. japonica, this study was designed to explore the structure of fungal community in roots of Z. japonica through culture-independent and culture-dependent approaches, describe unexplored species and investigate their potential antifungal activities (Fig. 1).

Figure 1. 

Sampling region of Zoysia japonica from a golf course (left) and schematic diagram of this study (right).

Materials and methods

Sampling and ITS amplicon sequencing

Healthy plants of Z. japonica were sampled from three different sites on a golf course located in Hwasun, South Jeolla Province, Korea. The three samples were washed of soil and the roots of each sample were cut with sterilised scissors. Then the roots were surface disinfected with 2% sodium hypochlorite for 2 min and rinsed three times in sterile distilled water. Genomic DNA from 100 mg of each root sample was extracted using a FavorPrep Plant Genomic DNA Extraction Mini Kit (Favorgen Biotech Corp, Ping-Tung, Taiwan) according to the manufacturer’s instructions. Primer set ITS3 (5’-GCATCGATGAAGAACGCAGC-3’)/ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) was used to amplify the ITS2 region of the internal transcribed spacer. Amplicon sequencing was performed using an Illumina MiSeq platform by the Life is Art of Science (LAS) Laboratory (Gimpo, Korea).

Analysis of ITS amplicon sequencing

Quality control of the raw reads from ITS amplicon sequencing of the three Z. japonica root samples was performed with FastQC v.0.12.1 (Andrews 2018). Primer sequences in the reads were removed by Cutadapt v.4.0 (Martin 2011). Resulting reads were further processed using a Vsearch v.2.22.1 pipeline (Rognes et al. 2016). First, paired-end reads were merged, truncated to an equal length (300 bp) and quality filtered. Chimeras were removed after de novo detection, followed by removal of singletons. Reads were subsequently clustered into operational taxonomic units (OTUs) with a threshold of 0.97 after full-length dereplication. Taxonomic annotation of the OTUs was conducted using a SINTAX algorithm (Edgar 2016) against the ITS reference database UNITE (Nilsson et al. 2019). OTUs annotated to Zoysia sp. were removed and only fungal OTUs were retained for data analysis.

The abundance distribution of fungal OTUs in the three root samples was summarised by alpha diversity (within-sample diversity) metrics including Shannon and Simpson indices. Composition of the fungal community in the samples was plotted, based on the relative abundance of OTUs at different taxonomic levels. Dominant fungal OTUs in each sample were identified by analysing the OTUs with relative abundance in the top 20 at the order and genus levels. Sequences of these OTUs were extracted and phylogenetic trees were constructed using the Maximum Likelihood method. The above data analysis and visualisation were performed using the packages vegan v.2.6-4 (Oksanen et al. 2022), phyloseq v.1.42.0 (McMurdie and Holmes 2013) and ggplot2 (Wickham 2011) in R v.4.2.2 software (R Core Team 2019).

Fungal isolation

The remaining root samples of Z. japonica were used for fungal isolation. Briefly, surface-disinfected roots were cut into small pieces (5 mm long) and placed on potato dextrose agar (PDA) amended with 50 μg ml-1 of ampicillin and kanamycin. Plates were incubated in darkness at 25 °C for 3–7 days. Fungal colonies developed from plant tissues were sub-cultured on fresh PDA to obtain pure cultures. All the fungal strains were maintained in the Molecular Microbiology Lab., Department of Integrative Food, Bioscience and Biotechnology, Chonnam National University, Gwangju, Republic of Korea.

Fungal DNA extraction and polymerase chain reaction

Fungal genomic DNA was extracted by a modified CTAB method (Cubero et al. 1999) using fresh mycelia grown on PDA. Amplification of gene fragments of ITS, SSU, LSU, TEF1, RPB1, RPB2, TUB2 and MCM7 was performed using primer pairs of ITS4/ITS5 (White et al. 1990), NS1/NS4 (White et al. 1990), LR0R/LR5 (Vilgalys and Hester 1990), EF1-983F/EF1-2218R (Rehner and Buckley 2005), RPB1-Ac/RPB1-Cr (Stiller and Hall 1997; Matheny et al. 2002), RPB2-5f/RPB2-7cR (Liu et al. 1999), T1/Bt2b (Glass and Donaldson 1995; O’Donnell and Cigelnik 1997) and MCM7-709/MCM7-1348 (Schmitt et al. 2009), respectively. The PCR products were purified with ExoSap-IT reagent (GE Healthcare, USA) before paired-end Sanger sequencing by Macrogen Inc. (Seoul, Korea).

Phylogenetic analysis

The resulting sequences of fungal strains in this study were subjected to BLASTn searches in the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) with an additional option (sequences from type materials). Related reference sequences of different genera were accessed, based on recent relative publications (Cai et al. 2006; Zhang et al. 2011, 2023; Quaedvlieg et al. 2013; Luo et al. 2016; Thambugala 2017; Liu et al. 2019; Marin-Felix et al. 2020; Andreasen et al. 2021; Crous et al. 2022a; Wong et al. 2022; Tanney et al. 2023). Accession numbers of the reference strains were shown in Suppl. material 1. A newly-developed tool OFPT (One-click Fungal Phylogenetic Tool) by Zeng et al. (2023) was used for phylogenetic analysis in this study. Complete procedures including sequence retrieving, multi-sequence alignment, sequence trimming, sequence concatenating, model testing and construction of Maximum Likelihood (ML) and Bayesian trees were accomplished by this tool. Briefly, nucleotide substitution models of the datasets were tested by ModelFinder (Kalyaanamoorthy et al. 2017). For the ML analysis, 1,000 bootstrap replicates were performed by IQ-TREE (Nguyen et al. 2015) with the best-fit substitution model. For the Baysian method, Markov Chain Monte Carlo (MCMC) analysis was performed by MrBayes 3.2.7 (Ronquist et al. 2012) with parameters of 50,000,000 generations and a sampling frequency of every 100th generation. The first 25% of the samples were discarded during the calculation of convergence diagnostics. Consensus BI trees with posterior probabilities (PP) were generated after the standard deviation of the runs fell below 0.01. Sequence alignments of phylogenetic analyses were shown in Suppl. material 2.

Morphological characterisation

Colonial characteristics of the fungal strains were recorded after incubation on PDA at 25 °C in darkness for 7 days. To stimulate sporulation, fungal strains were inoculated on malt extract agar (MEA) and oatmeal agar (OA) and incubated at 25 °C. Colonies of strains that had not sporulated after 7 days were scratched and exposed to UV light for 2 h and again incubated at 25 °C for further observation. Conidial morphology was observed under an optical microscope (Olympus, Tokyo, Japan) equipped with a differential interference contrast (DIC) module.

In vitro dual culture assay

Antifungal activity of fungal strains against turf-grass brown patch pathogen Rhizoctonia solani AG2-2(IIIB) KACC 40151 was tested by in vitro dual culture assay. Mycelia plugs (5 mm diameter) of endophytic fungi and pathogen R. solani AG2-2(IIIB) were placed on PDA (3 cm apart). PDA plates inoculated with only pathogen were used as control. The experiment was repeated twice. Mycelial growth of colonies of pathogens with (a) and without (b) endophytic fungi was measured after incubation at 25 °C for 2 d. Inhibition rate of mycelia growth was calculated as follows: Inhibition rate (%) = 100 − (a/b × 100). Endophytic fungus with the highest inhibition rate was chosen for further investigation of antifungal activity against pathogens R. cerealis KACC 40154, R. solani AG2-2(IV) KACC 40132, Clarireedia jacksonii CMML 20-31, Pythium ultimum KACC 40705, Sclerotinia sclerotiorum KACC 40457, Botrytis cinerea CMML 20-BC04, Fusarium oxysporum CMML 21-1 and Colletotrichum gloeosporiodes KACC 40003.

Crude extraction of fungal metabolites

Mycelia of the selected endophytic fungus grown in potato dextrose broth (PDB) for 2 weeks were collected and used as raw material for metabolite extraction. First, a mycelial sample (2 g) was mixed with 8 ml of different solvents (methanol, ethyl acetate, hexane, acetone and butanol) and incubated in a shaker at room temperature for 24 h. Crude extracts were then obtained after centrifugation. Subsequently, antifungal activity against R. solani AG2-2(IIIB) was tested on each crude extract using the paper disc method. Briefly, agar plugs (5 mm) of R. solani AG2-2(IIIB) were inoculated on the centre of PDA plates, sterile paper discs were then loaded with 20 μl of the crude extracts, air-dried thoroughly and placed on the surface of pathogen-inoculated PDA plates. Mycelial growth inhibition was observed after incubation at 25 °C for 2 d. The experiment was performed three times.

Mycelial viability

To test the viability of pathogen R. solani AG2-2(IIIB) with or without crude extracts of the selected endophytic fungus, mycelia were picked, placed on glass slides and stained with neutral red (0.1 μg ml-1, DaeJung, Siheung, Korea) or Evans blue (0.5 μg ml-1, Alfa Aesar, Haverhill, USA). After incubation for 5 min at room temperature, the mycelia were washed three times with sterile distilled water and examined under the microscope.

Pot assay

In planta antifungal activity against R. solani AG2-2(IIIB) was tested using butanol extract of the selected endophytic strain on creeping bentgrass. Mycelia of R. solani AG2-2(IIIB) grown in PDB for 3 d were collected and pulverised for inoculation. Approximately 2-week-old creeping bentgrass grown in pots was used for pathogen treatments. Butanol extract (5 ml) of the fungal strain was evaporated and re-dissolved in the same volume of distilled water, then sprayed on the pathogen treated pots. Distilled water was treated as a negative control and the same volume of the fungicide azoxystrobin (20 μg ml-1) was treated as a positive control. Three pots were used for each treatment. All the pots were placed in a greenhouse at 25 °C with a light period of 16 h per day until disease was noted.

Statistical analysis

Mycelial growth of pathogens in dual culture assays was measured to calculate mycelial growth inhibition rates. Data were used for multiple comparison by the least significant difference (LSD) test (P ≤ 0. 05) in R software (R Core Team 2019).

Results

ITS amplicon analysis

A total of 317 fungal OTUs were obtained from Z. japonica root samples, based on amplicon sequencing of the ITS2 region. Sample size-based rarefaction curves show a saturated trend (Fig. 2a), indicating that the community diversity was adequately captured in the three samples. Alpha diversity measurements were similar amongst the samples, ranging from 2.16–2.61 and 0.76–0.88 for Shannon and Simpson indices, respectively (Fig. 2b, c).

Figure 2. 

Alpha diversity of fungal communities in Zoysia japonica root samples a rarefaction curves for OTUs at different sequence reads b measurements of Shannon indices c measurements of Simpson indices.

Fungal OTUs from root samples of Z. japonica were classified into seven phyla, including Ascomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota, Glomeromycota, Monoblepharomycota and Rozellomycota, with Ascomycota being the most abundant (94.15% on average) phylum (Fig. 3a). A total of 40 orders were found with different relative abundance, including Agaricales, Annulatascales, Archaeosporales, Atractiellales, Atractosporales, Auriculariales, Blastocladiales, Branch06 (unclassified order in Sordariomycetes), Cantharellales, Capnodiales, Chaetothyriales, Chytridiales, Cladochytriales, Cystobasidiales, Entrophosporales, Eurotiales, Exobasidiales, Glomerales, Glomerallales, Gyalectales, Helotiales, Hypocreales, Lecanorales, Magnaporthales, Malasseziales, Marthamycetales, Microascales, Monoblepharidales, Orbiliales, Paraglomerales, Pleosporales, Saccharomycetales, Savoryellales, Sebacinales, Sordariales, Spizellomycetales, Tubeufiales, Xylariales and two unknown orders (Fig. 3b).

Figure 3. 

Fungal communities in Zoysia japonica root samples a relative abundance at phylum level b relative abundance at order level.

The composition of OTUs were similar in the three samples at the family and genus levels. A total of 49 families and 64 genera were found in the OTUs and unknown taxa in Sordariales dominated the fungal community at both the family and genus levels (Fig. 4). Other classified genera of the OTUs were Ambispora, Atractiella, Biatora, Budhanggurabania, Cladophialophora, Colletotrichum, Cortinarius, Cucurbitinus, Curvularia, Dictyosporella, Dominikia, Entrophospora, Exophiala, Funneliformis, Fusarium, Fusidium, Glomus, Helicoma, Kamienskia, Lasiosphaeria, Lophiostoma, Macgarvieomyces, Magnaporthe, Magnaporthiopsis, Marthamyces, Melomastia, Microdominikia, Myrothecium, Naemacyclus, Paraglomus, Oliveonia, Phlyctis, Poaceascoma, Podospora, Pseudophialophora, Rhizophagus, Septoglomus, Serendipita, Slopeiomyces, Stagonospora, Tetraploa, Tirispora and Wettsteinina.

Figure 4. 

Fungal communities in Zoysia japonica root samples a relative abundance at family level b relative abundance at genus level.

In the fungal OTUs, the top 20 relative abundance orders were Sordariales (32.45%), Chaetothyriales (18.16%), unknown order in Sordariomycetes (14.63%), Pleosporales (12.48%), Magnaporthales (9.34%), Capnodiales (4.14%), Glomerales (3.87%), Hypocreales (1.17%), unknown order in Glomeromycota (1.05%), Annulatascales (0.39%), Tubeufiales (0.32%), Lecanorales (0.26%), Microascales (0.25%), Paraglomerales (0.24%), Xylariales (0.20%), unknown order in Sordariomycetes (0.16%), Auriculariales (0.14%), Atractiellales (0.14%), unknown order in Sebacinales (0.13%) and unknown order in Rozellomycota (0.13%, Fig. 5).

Figure 5. 

Top 20 abundance orders of fungal communities in Zoysia japonica root samples with their phylogenetic relationships.

Highest relative abundance was found in unknown genera in Sordariales (30.84%), followed by unknown genera in Herpotrichiellaceae (18.08%) and Sordariomycetes (14.63%). The remaining genera in the top 20 in relative abundance were Wettsteinina (5.39%), Magnaporthe (4.50%), Melomastia (4.14%), Poaceascoma (4.06%), Budhanggurabania (3.76%), unknown genus in Glome­raceae (1.94%), Podospora (1.59%), Rhizophagus (1.42%), Lophiostoma (1.40%), unknown genus in Pleosporales (1.37%), unknown genus in Hypocreales (1.09%), unknown genus in Glomeromycota (1.05%), Pseudophialophora (0.98%), Glomus (0.4%), Dictyosporella (0.39%), Helicoma (0.32) and Biatora (0.26%, Fig. 6).

Figure 6. 

Top 20 abundance genera of fungal communities in Zoysia japonica root samples with their phylogenetic relationships.

Raw reads of ITS amplicon sequencing data in this study were deposited into the sequence read archive (SRA) in NCBI with BioProject number PRJNA1165193.

Diversity of culturable endophytic fungi

A total of 151 fungal strains were isolated from roots of Z. japonica. Based on colony morphology on PDA, 54 strains with different colony morphology (shape, colour, texture etc.) were preliminarily selected for ITS amplification and sequencing. Resulting sequences of these strains were then used for BLASTn search against NCBI database to obtain reference sequences with high similarities. Phylogenetic analysis revealed that these strains belonged to genera Curvularia, Setophoma, Poaceascoma, Preussia, Lophiostoma, Stagonospora, Niesslia, Purpureocillium, Fusarium, Collectotrichum, Pseudorhypophila, Magnaporthiopsis, Nemania, Xylaria, Cladosporium, Cutaneotrichosporon, Irpex, genera in Tubeufiaceae, Magnaporthales and two unknown taxa. (Fig. 7). Amongst these strains, 17 strains (31.48%) were detected as Curvularia spp. and 10 strains (18.51%) were identified as Poaceasoma spp. Strains with relative low similarities against GenBank were used for separate multi-loci phylogenetic analyses.

Figure 7. 

Phylogenetic analysis of fungal strains isolated from Zoysia japonica root samples, based on ITS sequences using Maximum Likelihood method. Bootstrap values (BS) are given at the nodes. Saccharomyces cerevisiae (CBS 1171) was used as the outgroup taxon.

Taxonomy

Niesslia dimorphospora (W. Gams) W. Gams & Stielow (2019).

Fig. 8

Culture characteristics.

Colony reaching 53.82 mm diam. after 7 days in darkness at 25 °C on PDA, surface initially floccose, later slimy, white on front and reverse sides (Fig. 8a).

Figure 8. 

Niesslia dimorphospora (CMML 20-40) a front and reverse sides of colony on PDA b–d globose conidia on a conidiophore e–i ellipsoidal conidia on a conidiophore j globose conidia k ellipsoidal conidia. Scale bars: 10 μm.

Description from living culture CMML 20-40.

Sexual morph: undetermined. Asexual morph: Sporulation abundant on MEA. Phialides 40–75 μm long, 1.5–2.3 μm wide, thick-walled. Conidia smooth-walled, globose, 4.5–6.5 μm diam., or ellipsoidal, slightly curved, 6.5–10.5 × 2.2–3.8 μm (Fig. 8b–k).

Type.

Korea • South Jeolla Province, Hwasun, isolated from roots of Zoysia japonica, October 2020, H. Liu and H. Sang, living cultures CMML 20-40, CMML 20-41 and CMML 20-42.

Notes.

Niesslia dimorphospora typically produce dimorphic conidia (globose and ellipsoidal). In multi-loci phylogenetic analysis using gene sequences of ITS, TEF1, TUB2 and RPB2, three strains (CMML 20-40, CMML 20-41 and CMML 20-42) were clustered into a clade containing ex-type strain of N. dimorphospora (CBS 785.69) and representative strain CBS 361.76 with high statistical support (100%/1.00) (Fig. 9). This is the first record of N. dimorphospora associated with Z. japonica in Korea.

Figure 9. 

Maximum Likelihood phylogenetic tree, based on combined sequences of ITS, LSU, RPB2 and TUB2 from Niesslia species. Bootstrap values (BS) and Bayesian posterior probabilities (PP) are given at the nodes (BS/PP). Strains obtained from this study are in bold blue. Ex-type isolates are marked with T. Simplicillium lanosoniveum (CBS 322.72) was used as the outgroup taxon.

Dactylaria hwasunensis H. Liu & H. Sang, sp. nov.

MycoBank No: 857258
Fig. 10

Etymology.

Name refers to Hwasun County in Korea, where it was isolated.

Description from living culture CMML 20-35.

Sexual morph: undetermined. Asexual morph: Sporulation abundant on MEA. Conidiophore erect, mironematous to macronematous, aseptate or septate, hyaline, 6–35 μm in length, 2.2–2.8 μm in width. Conidiogenous cells terminal, integrated, hyaline 2–2.8 μm wide. Conidia clavate, hyaline, blunt end, 1–5 septate, 10–60 × 2.2–2.8 μm (Fig. 10b–h).

Figure 10. 

Dactylaria hwasunensis sp. nov. (CMML 20-35) a front and reverse sides of colony on PDA b–g conidiophores and conidia h conidia. Scale bars: 10 μm.

Type.

Korea • South Jeolla Province, Hwasun, isolated from roots of Zoysia japonica, October 2020, H. Liu and H. Sang, holotype CMML 20-35H (permanently preserved in a metabolically inactive state), ex-holotype CMML 20-35, ex-isotype CMML 20-88.

Culture characteristics.

Colony reaching 31.81 mm diam. after 7 days in darkness at 25 °C on PDA, white to yellowish, surface smooth, cracked (Fig. 10a).

Notes.

In phylogenetic analysis of genus Dactylaria using sequence data of LSU, the strains used in the present study CMML 20-35 and CMML 20-88 formed a distinct clade sister to clade containing representative strain of D. fragilis (P057) and ex-type strain of D. acaciae (CPC 29771) with a high statistical support (84%/0.95) (Fig. 11). Based on nucleotide sequences, ex-holotype strain of D. hwasunensis (CMML 20-35) differed from ex-type strain of D. acaciae (CPC 29771): LSU sequence identities = 806/818 (98.53%). D. hwasunensis (CMML 20-35) also differed from representative strain of D. fragilis (P057): LSU sequence identities = 844/857 (98.48%). Morphologically, conidial dimensions of D. hwasunensis are larger than D. acaciae (16–37 × 2–2.5 μm; Crous et al. (2016)) and D. fragilis (18–26 × 1.5 µm; De Hoog (1985)). Therefore, Dactylaria hwasunensis sp. nov. was introduced in this study to accommodate CMML 20-35 and CMML 20-88 in the genus Dactylaria.

Figure 11. 

Maximum Likelihood phylogenetic tree, based on LSU sequences from Dactylaria species. Bootstrap values (BS) and Bayesian posterior probabilities (PP) are given at the nodes (BS/PP). Strains obtained from this study are in bold blue. Ex-type isolates are marked with T. Ramularia endophylla (CBS 113265) was used as the outgroup taxon.

Magnaporthiopsis zoysiae H. Liu & H. Sang, sp. nov.

MycoBank No: 857259
Fig. 12

Etymology.

Name refers to its host Zoysia japonica.

Description from living culture CMML 20-39.

Sexual morph: undetermined. Asexual morph: Sporulation observed on OA media. Conidiophores hyaline, single or sometimes branched, septate. Conidiogenous cells erect or curved, hyaline, 2.5–4 μm in width. Conidia ovoid or cylindrical, hyaline, slightly curved, 5.5–14.5 × 3.0–5.2 μm (Fig. 12b–h).

Figure 12. 

Magnaporthiopsis zoysiae sp. nov. (CMML 20-39) a front and reverse sides of colony on PDA b–e sporulation pattern on OA media f conidia. Scale bars: 3 μm.

Type.

Korea • South Jeolla Province, Hwasun, isolated from roots of Zoysia japonica, October 2020, H. Liu and H. Sang, holotype CMML 20-39H (permanently preserved in a metabolically inactive state), ex-holotype CMML 20-39, ex-isotype CMML 20-92.

Culture characteristics.

Colony reaching 31.81 mm diam. after 7 days in darkness at 25 °C on PDA, centre dark, margin white, mycelia frizzy (Fig. 12a).

Notes.

In phylogenetic analysis of Magnaporthiopsis, based on sequences of six genes (ITS, SSU, LSU, RPB1, TEF1 and MCM7), the strains used in the present study CMML 20-39 and CMML 20-92 fell into a distinct clade with a high statistical support (100%/1.00) (Fig. 13), sister to clades of species M. cynodontis, M. agrostidis and M. meyeri-festucae, which are all turf-grass-associated species. Morphologically, the conidial size of these two strains is larger than those of M. agrostidis (4–6 × 1 µm; Crous et al. (2015)) and M. meyeri-festucae (3–5 × 1–2.5 µm; Luo et al. (2017)). Conidia of these two strains is slightly longer than M. cynodontis (7–13 × 2–6.5 µm; Vines et al. (2020)). Therefore, based on phylogenetic analysis and morphological characteristics, Magnaporthiopsis zoysiae sp. nov. was introduced in this study.

Figure 13. 

Maximum Likelihood phylogenetic tree, based on combined sequences of ITS, SSU, LSU, RPB1, TEF1 and MCM7 from Magnaporthiopsis species. Bootstrap values (BS) and Bayesian posterior probabilities (PP) are given at the nodes (BS/PP). Strains obtained from this study are in bold blue. Ex-type isolates are marked with T. Pyricularia grisea (M82) was used as the outgroup taxon.

Setophoma zoysiae H. Liu & H. Sang, sp. nov.

MycoBank No: 857260
Fig. 14

Etymology.

Name refers to its host genus Zoysia.

Description from living culture CMML 20-14.

Sexual morph: undetermined. Asexual morph: Sporulation observed on OA media (Fig. 14b–f). Conidiomata produced on surface of colonies (Fig. 14b, c). Conidia ellipsoidal to subcylindrical, aseptate, 3.0–4.6 × 1.8–2.5 μm (Fig. 14d–f).

Figure 14. 

Setophoma zoysiae sp. nov. (CMML 20-14) a front and reverse sides of colony on PDA b, c conidiomata on OA media d–f conidia. Scale bars: 30 μm (d, e); 4.5 μm (f).

Type.

Korea • South Jeolla Province, Hwasun, isolated from roots of Zoysia japonica, October 2020, H. Liu and H. Sang, holotype CMML 20-14H (permanently preserved in a metabolically inactive state), ex-holotype CMML 20-14, ex-isotype CMML 20-15.

Culture characteristics.

Colony reaching 28.12 mm diam. in darkness after 7 days at 25 °C on PDA, front side white to light pink, reverse side yellow to sandy brown, mycelia dense (Fig. 14a).

Notes.

Phylogenetic analysis was conducted using dataset from combined sequences of ITS, LSU, TEF1, RPB2 and TUB2. The strains CMML 20-14 and CMML 20-15 formed a distinct single branch in the genus Setophoma, supported with a high statistical support (100%/1.00) (Fig. 15), sister to clade containing ex-type strain (CBS 335.29) and representative strains (CBS 335.87, CBS 377.52 and CPC 18417) of S. terrestris. However, conidia of these two strains are smaller than those of S. terrestris (previously Phoma terrestris, 4.5–5.5 × 1.8–2.3 μm; Hassen (1929)). Hence, Setophoma zoysiae sp. nov. was introduced in this study to accommodate CMML 20-14 and CMML 20-15 in the genus Setophoma.

Figure 15. 

Maximum Likelihood phylogenetic tree based on combined sequences of ITS, LSU, TEF1, RPB2 and TUB2 from Setophoma species. Bootstrap values (BS) and Bayesian posterior probabilities (PP) are given at the nodes (BS/PP). Strains obtained from this study are in bold blue. Ex-type isolates are marked with T. Setoseptoria phragmitis (CBS 114802 and CBS 114966) was used as the outgroup taxon.

Stagonospora endophytica H. Liu & H. Sang, sp. nov.

MycoBank No: 857261
Fig. 16

Etymology.

Name refers to endophyte.

Description from living culture CMML 20-37.

Sexual morph: undetermined. Asexual morph: Sporulation observed on MEA. Conidiomata globose, dark brown, 73–105 μm diam. (Fig. 16b–f). Conidiophores reduced to conidiogenous cells. Conidiogenous cells 5–8.5 × 4–7.5 μm, hyaline, smooth, ampulliform, produced from the inner wall of conidiomata (Fig. 16g). Conidia smooth, 1–3 septate, globose or ellipsoidal with obtuse ends, constricted at septa, 15–22 × 7–9 μm (Fig. 16h–l).

Figure 16. 

Stagonospora endophytica sp. nov. (CMML 20-37) a front and reverse sides of colony on PDA b, c conidiomata produced on MEA d–f section of conidiomata g conidiogenous cell (indicated by arrows) with developing conidia h–l conidia. Scale bars: 40 μm (b–d); 20 μm (e); 50 μm (f); 12 μm (g); 10 μm (h–l).

Type.

Korea • South Jeolla Province, Hwasun, isolated from roots of Zoysia japonica, October 2020, H. Liu and H. Sang, holotype CMML 20-37H (permanently preserved in a metabolically inactive state), ex-holotype CMML 20-37, ex-isotype CMML 20-93.

Culture characteristics.

Colony reaching the edge of the PDA plates (90 mm) after 7 days in darkness at 25 °C, front side white to yellowish, centre brown, reverse side faint yellow (Fig. 16a).

Notes.

Phylogenetic analysis of Stagonospora was performed using sequences of ITS, SSU, LSU, RPB2 and TUB2. Strains in the present study CMML 20-37 and CMML 20-93 fell into a distinct single clade, supported by a high statistical support (100%/1.00) (Fig. 17), sister to clade comprising ex-type strain of S. tauntonensis (BRIP 70573) and representative strains of S. tauntonensis (BRIP 70684), S. bicolor (ATCC 42652) and S. poaceicola (NCYUCC 19-0350). In morphology, these two strains differ from S. tauntonensis (Crous et al. 2022b) and S. bicolor (previously Leptosphaeria bicolor; Kaiser et al. (1979)) in having globose conidia and visible contraction at septa of conidia. For S. poaceicola, sexual morph was described and asexual morph of this species remains undetermined. Thus, based on phylogenetic analysis and morphological characteristics, Stagonospora endophytica sp. nov. was introduced in this study.

Figure 17. 

Maximum Likelihood phylogenetic tree, based on combined sequences of ITS, SSU, LSU, RPB2 and TUB2 from Stagonospora species. Bootstrap values (BS) and Bayesian posterior probabilities (PP) are given at the nodes (BS/PP). Strains obtained from this study are in bold blue. Ex-type isolates are marked with T. Massarina cisti (CBS 266.62) was used as the outgroup taxon.

Pseudorhypophila poae H. Liu & H. Sang, sp. nov.

MycoBank No: 857267
Fig. 18

Etymology.

Name refers to its host family Poaceae.

Description from living culture CMML 20-36:

Sexual morph: undetermined. Asexual morph: Sporulation abundant on MEA. Conidiophore erect, 1.5–2.5 μm in width, Conidia solitary or in clusters, pyriform, obovoid or triangular, 4.2–5.6 × 2.5–4.5 μm (Fig. 18b–e).

Figure 18. 

Pseudorhypophila poae sp. nov. (CMML 20-36) a front and reverse sides of colony on PDA b–d conidiophores and conidia e conidia. Scale bars: 10 μm (b–d); 8 μm (e).

Type.

Korea • South Jeolla Province, Hwasun, isolated from roots of Zoysia japonica, October 2020, H. Liu and H. Sang, holotype CMML 20-36H (permanently preserved in a metabolically inactive state), ex-holotype CMML 20-36, ex-isotype CMML 20-89.

Culture characteristics.

Colony reaching 82.88 mm diam. on PDA after 7 days in darkness at 25 °C, white to buff in both front and reverse sides (Fig. 18a).

Notes.

The genus Pseudorhypophila was recently introduced by accommodating four species including Triangularia mangenotii, Zopfiella marina, Z. pilifera and Z. submersa (Harms et al. 2021). In phylogenetic analysis using combined sequences of ITS, LSU, RPB2 and TUB2, strains in the present study CMML 20-36 and CMML 20-89 formed a single clade in the genus Pseudorhypophila supported with a high statistical support (100%/1.00) close to clade comprising ex-type strains of P. pilifera (CBS 413.73) and P. mangenotii (CBS 419.67) (Fig. 19). However, both P. pilifera and P. mangenotii produce sexual morph, which was not observed in strains CMML 20-36 and CMML 20-89. In addition, these two strains differ from P. mangenotii in producing conidia singly or in clusters, whereas the latter produces conidia singly (Harms et al. 2021). Therefore, based on phylogenetic analysis and morphological characteristics, Pseudorhypophila poae sp. nov. was introduced in this study.

Figure 19. 

Maximum Likelihood phylogenetic tree based on combined sequences of ITS, LSU, RPB2 and TUB2 from Pseudorhypophila and relevant genera. Bootstrap values (BS) and Bayesian posterior probabilities (PP) are given at the nodes (BS/PP). Strains obtained from this study are in bold blue. Ex-type isolates are marked with T. Lasiosphaeris hirsuta (SMH 1543) was used as the outgroup taxon.

Lophiostoma jeollanense H. Liu & H. Sang, sp. nov.

MycoBank No: 857262
Fig. 20

Etymology.

Name refers to Jeolla Province in Korea, the place it was isolated from.

Description.

Lophiostoma jeollanense differs from its closest phylogenetic neighbour, L. japonicum (KT573) by unique fixed alleles in three loci: ITS positions 25 (A), 26 (G), 31 (indel), 40 (indel), 70 (C), 91 (C), 93 (G), 114 (G), 132 (T), 134 (A), 136 (C), 138 (T), 142 (G), 364 (G), 365 (A), 368 (T), 383 (T), 407 (C); LSU positions 41 (T), 43 (C), 155 (T), 614 (C); TEF1 positions 42 (C), 127 (T), 128 (C), 129 (C), 162 (T), 222 (C), 225 (C), 240 (T), 249 (T), 318 (T), 336 (C), 342 (C), 351 (T), 372 (C), 399 (T), 405 (C), 408 (T), 442 (G), 465 (C), 477 (G), 492 (C), 528 (C), 537 (C), 663 (T), 669 (C), 672 (G), 693 (C), 705 (C), 708 (T), 735 (T), 748 (G), 780 (G), 792 (C).

Type.

Korea • South Jeolla Province, Hwasun, isolated from roots of Zoysia japonica, October 2020, H. Liu and H. Sang, holotype CMML 20-43H (permanently preserved in a metabolically inactive state), ex-holotype CMML 20-43, ex-isotype CMML 20-90.

Culture characteristics.

Colony reaching 22.24 mm diam. on PDA after 7 days in darkness at 25 °C, surface white to light brown, reverse side yellow, mycelia dense (Fig. 20a).

Figure 20. 

Lophiostoma jeollanense sp. nov. (CMML 20-43) a front and reverse sides of colony on PDA b–e chlamydospore-like structures on OA. Scale bars: 10 μm.

Notes.

Lophiostoma jeollanense did not sporulate on synthetic media. Chlamydospore-like structures within mycelia were observed on OA after two weeks (Fig. 20b–e). In phylogenetic analysis of the genus Lophiostoma, based on combined sequences of ITS, LSU, TEF1 and RPB2, strains CMML 20-43 and CMML 20-90 formed a distinct single clade with a high statistical support (96%/1.00) (Fig. 21), sister to clade comprising an ex-type strain (KT573) and representative strains (KT 686-1, UESTCC 23.0040 and MFLUCC 17-2450) of L. japonicum. Based on nucleotide sequences of three loci, ex-holotype strain of L. jeollanense (CMML 20-43) was different from the ex-type strain of L. japonicum (KT573): ITS sequence identities = 494/513 (96.30%), gaps = 2; LSU sequence identities = 851/855 (99.53%); TEF sequence identities = 835/868 (96.20%). The species L. japonicum (previously Biappendiculispora japonica) was found as a saprophyte on dead stems of unknown herbaceous plants with its sexual morph (Thambugala et al. 2015), whereas strains CMML 20-43 and CMML 20-90 were isolated from roots of Z. japonica as a potential endophyte and only chlamydospore-like structures were observed in these strains. Therefore, Lophiostoma jeollanense sp. nov. was introduced in this study to accommodate CMML 20-43 and CMML 20-90 in the genus Lophiostoma.

Figure 21. 

Maximum Likelihood phylogenetic tree based on combined sequences of ITS, LSU, TEF1 and RPB2 from Lophiostoma species. Bootstrap values (BS) and Bayesian posterior probabilities (PP) are given at the nodes (BS/PP). Strains obtained from this study are in bold blue. Ex-type isolates are marked with T. Oleaginea sichuanensis (CGMCC 3.24427) was used as the outgroup taxon.

Poaceascoma magnum H. Liu & H. Sang, sp. nov.

MycoBank No: 857263
Fig. 22

Etymology.

Name refers to the character of large chlamydospores produced by this fungus.

Description.

Chlamydospores 10–85 μm in length and 15–23 μm in width, hyaline to dark, clavate, sometimes dumb-bell-shaped or gourd-shaped, straight or sometimes curved. Poaceascoma magnum differs from its closest phylogenetic neighbour, L. lochii (BRIP 71546) by unique fixed allels in two loci: ITS positions 49 (G), 57 (G), 65 (C), 67 (C), 70 (C), 71 (A), 73 (G), 76 (T), 77 (C), 79 (C), 95 (C), 133 (T), 137 (A), 143 (C), 152 (T), 157 (C), 158 (A), 162 (G), 163 (indels), 169 (A), 184 (C), 190 (T), 192 (G), 194 (A), 376 (indels), 440 (C), 444 (G), 446 (T), 474 (C), 480 (T), 481 (G), 482 (T), 483 (A), 511 (T), 512 (G), 515 (indel), 528 (A), 542 (T), 549 (indel), 560 (T); LSU positions 99 (G), 138 (G), 206 (G), 208 (A), 291 (T), 693 (C), 695 (C), 696 (indel).

Culture characteristics.

Colony reaching 22.02 mm diam. on PDA after 7 days in darkness at 25 °C, white to grey at the edge, centre tawny, reverse side yellow brown (Fig. 22a).

Figure 22. 

Poaceascoma magnum sp. nov. (CMML 20-47) a front and reverse sides of colony on PDA b–k chlamydospores on MEA. Scale bars: 10 μm.

Type.

Korea • South Jeolla Province, Hwasun, isolated from roots of Zoysia japonica, October 2020, H. Liu and H. Sang, holotype CMML 20-47H (permanently preserved in a metabolically inactive state), ex-holotype CMML 20-47, ex-isotype CMML 20-91.

Notes.

Sporulation was not observed during culture on synthetic media. On MEA, strains CMML 20-47 and CMML 20-91 produced large (10–85 × 15–23 μm), clavate, hyaline to dark, intercalary or terminal chlamydospores (Fig. 22b–k). Phylogenetic analysis using multi-loci of ITS, LSU, SSU and TEF1 revealed that strains CMML 20-47 and CMML 20-91 formed a single clade within the genus Poaceascoma with a strong statistical support (100%/1.00) basal to clade containing ex-type strains of P. lochii (BRIP 71546), P. helicoides (MFLUCC 11-0136), P. herbaceum (GZCC 19-0046) and representative strain of P. helicoides (MFLU 11-0172) (Fig. 26). In comparison of nucleotide sequences of ITS and LSU, ex-holotype strain of P. magnum (CMML 20-47) differed from ex-type strain of P. lochii (BRIP 71546): ITS identities = 491/526 (93.35%), 38 gaps; LSU identities = 889/896 (99.22%). In addition, P. magnum (CMML 20-47) differed from ex-type strain of P. helicoides (MFLUCC 11-0136) in four loci: ITS identities = 435/469 (92.75%), 71 gaps; SSU identities = 914/916 (99.78%); LSU identities = 786/794 (98.99%); TEF1 identities = 879/924 (95.13%). P. magnum (CMML 20-47) also differed from ex-type strain of P. herbaceum (GZCC 19-0046) in these loci: ITS identities = 390/420 (92.86%), 69 gaps; SSU identities = 1021/1025 (99.61%); LSU identities = 891/900 (99.00%); TEF1 identities = 883/924 (95.56%). Morphologically, this fungus differs from other Poaceascoma spp. by producing large and sometimes dark chlamydospores. Therefore, Poaceascoma magnum sp. nov. was introduced in this study to accommodate CMML 20-47 and CMML 20-91.

Poaceascoma endophyticum H. Liu & H. Sang, sp. nov.

MycoBank No: 857264
Fig. 23

Etymology.

Name refers to endophyte.

Description.

Poaceascoma endophyticum differs from its closest phylogenetic neighbour, P. halophilum (MFLUCC 15-0949) by unique fixed alleles in two loci: LSU positions 84 (T), 88 (indel), 280 (C), 484 (C), 534 (T), 654 (T), 691 (T), 766 (indel), 800 (indel); SSU position 174 (indel), 972 (indel).

Culture characteristics.

Colony reaching 29.33 mm diam. on PDA after 7 days in darkness at 25 °C, white ring at the edge, centre brownish, reverse side dark brown with a white edge, mycelia dense (Fig. 23a).

Figure 23. 

Poaceascoma endophyticum sp. nov. (CMML 20-48) a front and reverse sides of colony on PDA b–i chlamydospore-like structures on MEA. Scale bars: 10 μm.

Type.

Korea • South Jeolla Province, Hwasun, isolated from roots of Zoysia japonica, October 2020, H. Liu and H. Sang, holotype CMML 20-48H (permanently preserved in a metabolically inactive state), ex-holotype CMML 20-48, ex-isotype CMML 20-49.

Notes.

Strains CMML 20-48 and CMML 20-49 did not sporulate on synthetic media. Only chlamydospore-like structures were observed on MEA after two weeks, mostly elliptic or oval in shape and 5.5–12.5 μm in width (Fig. 23b–i). In phylogeny, based on multi-loci of ITS, LSU, SSU and TEF1, strains CMML 20-48 and CMML 20-49 clustered into a distinct clade, sister to clade comprising ex-type strains of P. halophilum (MFLUCC 15-0949), P. zoysiiradicicola (CMML 20-50) and representative strain CMML 20-51. Based on nucleotide sequence, ex-holotype strain of P. endophyticum (CMML 20-48) differed from ex-type strain of P. halophilum (MFLUCC 15-0949) in LSU sequence (identities = 830/836, 99.28%). P. endophyticum (CMML 20-48) also differed from P. zoysiiradicicola (CMML 20-50 and CMML20-51) in three different loci: ITS identities = 523/558 (93.73%), 40 gaps; LSU identities = 828/838 (98.81%); TEF1 identities = 824/849 (97.06%). According to (Hyde et al. 2017), colonies of P. halophilum on PDA reaches 20–30 mm diameter after 4 weeks, indicating a slower vegetative growth rate than P. endophyticum. In addition, P. endophyticum differs from P. zoysiiradicicola in producing larger chlamydospore-like structures. Thus, Poaceascoma endophyticum sp. nov. was introduced in this study to accommodate CMML 20-50 and CMML 20-51 in the genus Poaceascoma.

Poaceascoma koreanum H. Liu & H. Sang, sp. nov.

MycoBank No: 857265
Fig. 24

Etymology.

Name refers to Korea, the country from where it was isolated.

Description.

Poaceascoma koreanum differs from its closest phylogenetic neighbour P. lochii (BRIP 71546) by unique fixed alleles in two loci: ITS positions 13 (A), 16 (C), 19 (G), 20 (T), 21 (C), 22 (G), 28 (G), 29 (indels), 41 (C), 42 (C), 44 (C), 45 (T), 46 (C), 47 (G), 50 (T), 51 (T), 52 (C), 58 (G), 60 (C), 68 (C), 84 (T), 98 (C), 107 (indel), 109 (C), 112 (indels), 114 (G), 116 (C), 124 (G), 125 (A), 127 (C), 130 (C), 131 (T), 132 (C), 136 (A), 137 (G), 140 (T), 141 (T), 144 (A), 153 (indel), 155 (G), 156 (T), 157 (A), 158 (C), 165 (C), 166 (G), 168 (A), 176 (A), 350 (indels), 388 (C), 391 (T), 397 (G), 404 (T), 410 (A), 420 (C), 435 (C), 440 (C), 443 (G), 447 (C), 449 (G), 450 (A), 469 (T), 475 (G), 476 (T), 481 (T), 489 (T), 495 (A), 497 (G), 500 (A), 502 (C); LSU positions 100 (G), 104 (indel), 138 (C), 141 (G), 143 (G), 145 (G), 205 (C), 206 (C), 210 (C), 488 (C), 550 (T), 700 (C), 705 (C), 755 (G), 907 (T).

Culture characteristics.

Colony reaching 39.72 mm diam. on PDA after 7 days in darkness at 25 °C, front side greyish-yellow, reverse side black-brown, margins burr-like (Fig. 24a).

Figure 24. 

Poaceascoma koreanum sp. nov. (CMML 20-44) a front and reverse sides of colony on PDA b–g chlamydospore-like structures on MEA. Scale bars: 10 μm.

Type.

South Korea • South Jeolla Province, Hwasun, isolated from roots of Zoysia japonica, October 2020, H. Liu and H. Sang, holotype CMML 20-44H (permanently preserved in a metabolically inactive state), ex-holotype CMML 20-44, ex-isotype CMML 20-45; CMML 20-46.

Notes.

No conidiogenous structures or sexual morph were observed in strains CMML 20-44, CMML 20-45 and CMML 20-46. On MEA, chlamydospore-like structures (4.5–10.5 μm in width) in hyphae were observed after incubation for two weeks (Fig. 24b–g). In multi-loci phylogeny, based on ITS, LSU, SSU and TEF1, these three strains formed a distinct single clade within the genus Poaceascoma sister to clade comprising strain of P. magnum (ex-type CMML 20-47 and ex-isotype CMML 20-71), P. herbaceum (ex-type GZCC 19-0046), P. helicoides (ex-type MFLUCC 11-0136 and representative strain MFLU 11-0172) and P. lochii (ex-type BRIP 71546) (Fig. 26). Based on nucleotide sequence comparison, P. koreanum (CMML 20-44) differed from ex-type strain of P. lochii (BRIP 71546) in two loci: ITS identities = 456/518 (88.03%), 40 gaps; LSU identities = 882/896 (98.44%). P. koreanum (CMML 20-44) differed from ex-type strain of P. helicoides (MFLUCC 11-0136) in four loci: ITS identities = 381/428 (89.02%), 41 gaps; SSU identities = 915/916 (99.89%); LSU identities = 779/793 (98.23%); TEF1 identities = 896/958 (93.53%). Compared to ex-type of P. herbaceum (GZCC 19-0046), nucleotide sequences were different in these loci: ITS identities = 335/380 (88.16%), 41 gaps; SSU identities = 1021/1025 (99.61%); LSU identities = 890/904 (98.45%); TEF1 identities = 897/958 (93.63%). P. koreanum (CMML 20-44) also differed from ex-holotype strain of P. magnum (CMML 20-47): ITS identities = 433/500 (89.02%), 45 gaps; SSU identities = 1028/1032 (99.61%); LSU identities = 884/900 (98.22%); TEF1 identities = 874/924 (94.59%). All of these species were originally found on herbaceous plants. Specifically, P. lochii was found on leaves of turf-grass Zoysia matrella, P. helicoides and P. herbaceum were saprophytes on dead stem of Digitaria sanguinalis and dead culm of unidentified herbaceous plants, respectively (Phookamsak et al. 2015; Hyde et al. 2017; Tan et al. 2021). Morphologically, P. herbaceum, P. helicoides and P. lochii were described, based on their sexual morph, while only chlamydospore-like structures were observed in strains CMML 20-44, CMML 20-45 and CMML 20-46. Additionally, P. magnum differs from these strains in producing large chlamydospores. Therefore, Poaceascoma koreanum sp. nov. was introduced in this study.

Poaceascoma zoysiiradicicola H. Liu & H. Sang, sp. nov.

MycoBank No: 857266
Fig. 25

Etymology.

Name refers to roots of Zoysia japonica.

Description.

Poaceascoma zoysiiradicicola differs from its closest phylogenetic neighbour P. halophilum (MFLUCC 15-0949) by unique fixed alleles in two loci: LSU positions 2 (T), 48 (T), 49 (T), 52 (indel), 144 (T), 359 (T), 458 (T), 655 (T), 730 (indel), 764 (indel), 866 (A); SSU positions 171 (indel), 969 (indel).

Culture characteristics.

Colony reaching 44.23 mm diam. on PDA after 7 days in darkness at 25 °C, front side reseda green, reverse sides crineous to dark, margin white on both sides (Fig. 25a).

Figure 25. 

Poaceascoma zoysiiradicicola sp. nov. (CMML 20-50) a front and reverse sides of colony on PDA b–g chlamydospore-like structures on MEA. Scale bars: 10 μm.

Type.

Korea • South Jeolla Province, Hwasun, isolated from roots of Zoysia japonica, October 2020, H. Liu and H. Sang, holotype CMML 20-50H (permanently preserved in a metabolically inactive state), ex-holotype CMML 20-50, ex-isotype CMML 20-51.

Notes.

No sporulation was found on synthetic media in this fungus. However, chlamydospore-like structures in hyphae were observed on MEA after two weeks, 4–6.5 μm in width (Fig. 25b–g). In phylogenetic analysis, based on multi-loci of ITS, LSU, SSU and TEF1, strains CMML 20-50 and CMML 20-51 formed a separate clade sister to ex-type strain of P. halophilum (MFLUCC 15-0949) (Fig. 26). Based on nucleotide sequences of LSU and SSU, ex-holotype strain of P. zoysiiradicicola (CMML 20-50) was different with the ex-type strain of P. halophilum (MFLUCC 15-0949): LSU sequence identities = 855/863 (99.07%), gaps = 3; SSU sequence identities = 1038/1038 (100%), gaps = 2. Poaceascoma halophilum was found as a saprophyte on a decaying bamboo stick and its asexual morph is undetermined (Hyde et al. 2017). In terms of culture characteristics, P. halophilum differs from P. zoysiiradicicola in the slower vegetative growth rate on PDA (Hyde et al. 2017). Therefore, P. zoysiiradicicola sp. nov. was introduced in this study to accommodate CMML 20-50 and CMML 20-51 in the genus Poaceascoma.

Figure 26. 

Maximum Likelihood phylogenetic tree based on combined sequences of ITS, LSU, SSU and TEF1 from Poaceascoma species using Maximum Likelihood method. Bootstrap values (BS) and Bayesian posterior probabilities (PP) are given at the nodes (BS/PP). Strains obtained from this study are in bold blue. Ex-type isolates are marked with T. Corynespora torulosa (CBS 136419) was used as the outgroup taxon.

In vitro antifungal activity against Rhizoctonia solani AG2-2(IIIB)

Antifungal activities of the species described above were tested by in vitro dual culture against R. solani AG2-2(IIIB), the casual pathogen of turf-grass brown patch disease. Different antifungal activities were observed amongst these species (Fig. 27a–l). Mycelial growth inhibition rates were 37.97% for D. hwasunensis CMML 20-35, 23.15% for L. jeollanense CMML 20-43, 40.35% for M. zoysiae CMML 20-39, 37.90% for N. dimorphospora CMML 20-40, 21.53% for P. endophyticum CMML 20-49, 21.51% for P. zoysiiradicicola CMML 20-51, 7.95% for P. koreanum CMML 20-46, 35.37% for P. magnum CMML 20-47, 51.68% for S. zoysiae CMML 20-15, 41.07% for S. endophytica CMML 20-37 and 15.12% for P. poae CMML 20-36 (Fig. 27m). Setophoma zoysiae CMML 20-15 showed a significantly higher inhibition to R. solani AG2-2(IIIB) and was therefore selected for further antifungal activity assays.

Figure 27. 

Dual culture of endophytic fungi against turfgrass brown patch pathogen Rhizoctonia solani (AG-2-2 (IIIB)) a control b Dactylaria hwasunensis (CMML 20-35) c Lophiostoma jeollanense (CMML 20-43) d Magnaporthiopsis zoysiae (CMML 20-39) e Niesslia dimorphospora (CMML 20-40) f Poaceascoma endophyticum (CMML 20-49) g P. zoysiiradicicola (CMML 20-51) h P. koreanum (CMML 20-46) i P. magnum (CMML 20-47) j Setophoma zoysiae (CMML 20-15) k Stagonospora endophytica (CMML 20-37) l Pseudorhypophila poae (CMML 20-36) m Inhibition rates of each fungal strains. Error bars indicate the standard errors of the means.

In vitro antifungal activity of Setophoma zoysiae (CMML 20-15) against eight phytopathogens

To test the antifungal spectrum of S. zoysiae (CMML 20-15), eight different agriculturally important phytopathogens were used for dual culture assay. Mycelial growth inhibition rates were 50.65% for R. cerealis (KACC 40154), 48.86% for R. solani (AG2-2(IV) KACC 40132), 54.70% for Clarireedia jacksonii (CMML 20-31), 50.96% for Pythium ultimum (KACC 40705), 60.82% for Sclerotinia sclerotiorum (KACC 40457), 46.57% for Botrytis cinerea (CMML 20-BC04), 21.68% for Fusarium oxysporum (CMML 21-1) and 33.56% for Colletotrichum gloeosporiodes (KACC 40003) (Fig. 28).

Figure 28. 

Dual culture of Setophoma zoysiae (CMML20-15) against eight different plant pathogens a Rhizoctonia solani (AG-2-2(IV) KACC 40132) b R. cerealis (KACC 40154) c Clarireedia jacksonii (CMML 20-31) d Pythium ultimum (KACC 40705) e Sclerotinia sclerotiorum (KACC 40457) f Botrytis cinerea (CMML 20-BC04) g Fusarium oxysporum (CMML 21-1) h Colletotrichum gloeosporiodes (KACC 40003) i mycelial growth inhibition rates of each pathogen. Error bars indicate the standard errors of the means.

Antifungal activities of mycelial crude extracts of Setophoma zoysiae (CMML 20-15)

Mycelial crude extraction of S. zoysiae (CMML 20-15) was conducted using five different solvents (methanol, ethyl acetate, hexane, acetone and butanol). Antifungal activities of the five crude extracts against R. solani (AG2-2(IIIB)) were tested using a paper disc assay. No mycelial growth inhibition was found in methanol, ethyl acetate, hexane and acetone crude extracts, while dramatic growth inhibition was found in butanol extract (Fig. 29).

Figure 29. 

Antifungal activities of five crude extracts (methanol, ethyl acetate, hexane, acetone and butanol) of Setophoma zoysiae (CMML20-15) against Rhizoctonia solani (AG2-2(IIIB)) on PDA media. Paper discs were moistened with 20 μl of the crude extracts. Only solvent was treated in the control groups.

To evaluate the mycelial viability of R. solani (AG2-2(IIIB)), Evans blue and neutral red were used to stain the mycelia from PDA plates with or without butanol extract of S. zoysiae (CMML 20-15). Staining results were examined under a microscope. Mycelia from the control groups (without exposure to butanol extract) were not stained by Evans blue, but were stained red by neutral red. On the contrary, mycelia exposed to butanol extract were stained blue by Evans blue, while remaining unstained by neutral red (Fig. 30). This revealed that cell death of R. solani (AG2-2(IIIB)) occurred under the treatment of butanol extract of S. zoysiae (CMML 20-15).

Figure 30. 

Mycelia of Rhizoctonia solani (AG2-2(IIIB)) with or without exposure to butanol extract of Setophoma zoysiae (CMML20-15) were stained with Evans blue and neutral red. Dead cells stained blue; viable cells, red.

In planta brown patch disease control

Butanol extract of S. zoysiae (CMML 20-15) was re-dissolved in sterile distilled water and used for brown patch control on creeping bentgrass in pots, with fungicide azoxystrobin used for the positive control. Symptoms of severe disease were observed on creeping bentgrass treated with only the pathogen R. solani (AG2-2(IIIB)). Fungicide azoxystrobin showed complete control of brown patch disease and only minor disease symptoms occurred on butanol extract-treated creeping bentgrass. No symptoms were observed in the sterile water-treated control group (Fig. 31).

Figure 31. 

In planta brown patch control of creeping bentgrass by butanol extract of Setophoma zoysiae (CMML20-15), with a comparison to a commercial fungicide azoxystrobin. Control group was treated with sterile water.

Discussion

In this study, both culture-independent and -dependent methods were used to determine endophytic fungal diversity associated with roots of Z. japonica. Novel species were identified based on morphological characterisation and multi-loci phylogenetic analyses. In addition, antifungal activities of the described species were tested against R. solani (AG2-2(IIIB)) with S. zoysiae (CMML20-15) being the best antagonist. Butanol crude extract of this strain was applied to control R. solani (AG2-2(IIIB)) by in vitro mycelial growth inhibition and in planta brown patch control. This is the first comprehensive work revealing fungal community on Z. japonica with an attempt for exploration and application of biological resources.

Through analysis of ITS amplicon reads, abundant OTUs were identified in roots of Z. japonica, dominated by members of the class Sordariomycetes. Fungi in this class have also been reported to dominate epiphytic and endophytic samples of tomato (Dong et al. 2021). The functions of endophytic fungi in this class and their roles in interacting with host plants deserve to be investigated. However, the classification of the most abundant taxa at the family and genus levels have remained unknown, suggesting that a large number of endophytic fungi in Z. japonica are remain unidentified. This supports the fact that endophytic fungi constitute a major part of unexplored fungal diversity (Rajamanikyam et al. 2017).

A variety of endophytic fungi were obtained in roots of Z. japonica by isolation in this study. Taxa in genera Collectotrichum, Fusarium, Curvularia, Lophiostoma, Magnaporthiopsis, Poaceascoma and Stagonospora were detected by both culture-independent and culture-dependent methods. Amongst these taxa, Biappendiculispora (currently Lophiostoma) and Poaceascoma were in the top 20 most abundant genera in ITS amplicon sequencing analysis and Poaceascoma spp. were also detected with high portions (18.52%) during isolation. Therefore, Poaceascoma spp. may have potential functions in the root-inhabiting mycobiome of Z. japonica.

Combining morphological characterisation and phylogenetic analysis represents a reliable strategy for fungal taxonomy. Based on this approach, a newly-recorded species (Niesslia dimorphospora) in Korea and 10 new species (Dactylaria hwasunensis, Lophiostoma jeollanense, Magnaporthiopsis zoysiae, Poaceascoma endophyticum, P. koreanum, P. magnum, P. zoysiiradicicola, Setophoma zoysiae, Stagonospora endophytica and Pseudorhypophila poae) were identified. Some genera such as Magnaporthiopsis, Poaceascoma and Stagonospora have been reported to be associated with grasses. Specifically, Magnaporthiopsis spp., such as M. dharug, M. gadigal, M. gumbaynggirr and M. yugambeh, were isolated from diseased turf-grass species in Australia (Wong et al. 2022). Magnaporthiopsis meyeri-festucae was associated with a summer patch-like disease of fescue turf-grasses in New Jersey (Luo et al. 2017). Magnaporthiopsis zoysiae was isolated from healthy roots of turf-grass Z. japonica in this study, it being genetically different from other Magnaporthiopsis spp. The genus Poaceascoma was established with type species P. helicoides, which is isolated as a saprophyte from dead stems of a grass in Poaceae (Phookamsak et al. 2015). Herein, four novel species P. endophyticum, P. koreanum, P. magnum and P. zoysiiradicicola were isolated as endophytes from turf-grass Z. japonica. Species in Stagonospora are commonly associated with grasses (Thambugala 2017) as pathogens and S. endophytica was supplemented as an endophyte in this study.

In previous studies, most of the endophytic fungi have not sporulated during culture. For example, Lin et al. (2007) reported that 48.9% of the obtained endophytic taxa from Camptotheca acuminata were non-sporulating fungi. Tejesvi et al. (2011) isolated 87 endophytic fungi from asymptomatic leaf tissues of Rhododendron tomentosum and most of the isolates were unable to sporulate. Therefore, it is difficult to identify endophytic non-sporulating fungi, based on morphological characters and sequence-based molecular tools can be used to achieve their classification (Promputtha et al. 2005; Huang et al. 2009; Tejesvi et al. 2011; Selim 2012). In the present study, species L. jeollanense, P. endophyticum, P. koreanum, P. magnum and P. zoysiiradicicola did not sporulate on synthetic media – only chlamydospores were produced. Similarly, sporulation was not observed in endophytic fungi, such as Cyanodermella asteris, Nemania aquilariae, N. yunnanensis, Batnamyces globulariicola, Endopandanicola thailandica, Endomelanconiopsis freycinetiae, Diaporthe pandanicola and Mycoleptodiscus endophyticus, which were proposed as novel species recently (Jahn et al. 2017; Tibpromma et al. 2018, 2021; Noumeur et al. 2020).

Endophytic fungi are also an important source of novel and potential bioactive compounds (Strobel and Daisy 2003; Tomita 2003; Rajamanikyam et al. 2017). Application of endophytic fungi as biocontrol agents has increased considerably. To date, endophytic fungi have been reported to control a variety of plant pathogens, ranging from pathogenic fungi, bacteria, oomycetes to plant-parasitic nematodes (Latz et al. 2018; Huang et al. 2020; Poveda et al. 2020; Hapida et al. 2021; Simamora et al. 2021). Brown patch caused by R. solani is a major disease of turf-grasses worldwide and its control relies heavily on fungicide applications (Daniels and Latin 2013). To reduce the use of fungicides, biocontrol agents have been applied for brown patch control, such as Bacillus velezensis (GH1-13) (Lee et al. 2023), Paenibacillus ehimensis (KWN38), Stenotrophomonas maltophilia (C3) (Yuen and Zhang 2001) and Trichoderma harzianum (Guerrero et al. 2008). In this study, antifungal activities against R. solani (AG2-2(IIIB)) were observed amongst the tested endophytic fungi and S. zoysiae (CMML20-15) could serve as an antagonist for brown patch management. Additional dual culture assay suggested that S. zoysiae (CMML20-15) was potentially able to control the turf-grass large patch pathogen R. solani (AG2-2 (IV)), yellow patch pathogen R. cerealis, dollar spot pathogen C. jacksonii, Pythium blight pathogen P. ultimum and non-turf-grass pathogens S. sclerotiorum, B. cinerea, F. oxysporum and C. gloeosporiodes.

In conclusion, this study provides insights from the mycobiome diversity in Z. japonica to an application of a biocontrol agent in controlling pathogens of turf-grass, which will be valuable for future management of Z. japonica. Identifying the functions of other root-colonizing fungi of Z. japonica and searching for their bioactive compounds warrant future investigation.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Adherence to national and international regulations

All the fungal strains used in this study have been legally obtained, respecting the Convention on Biological Diversity (Rio Convention).

Funding

This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry (IPET) through Agricultural Microbiome R&D Program for Advancing innovative technology Program funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (RS-2024-00395538), Republic of Korea and the BK21 FOUR Program, Graduate Program for Integrative Food, Bioscience and Biotechnology, funded by the National Research Foundation of Korea (NRF).

Author contributions

Haifeng Liu: Methodology, Investigation, Data curation, Writing – Original Draft. Hyeongju Choi: Methodology, Investigation. Narayan Chandra Paul: Conceptualisation, Methodology. Hiran A. Ariyawansa: Conceptualisation, Writing – review and editing. Hyunkyu Sang: Conceptualisation, Writing – review and editing, Supervision, Project administration.

Author ORCIDs

Haifeng Liu https://orcid.org/0000-0002-9733-9240

Hyeongju Choi https://orcid.org/0000-0003-0435-8483

Narayan Chandra Paul https://orcid.org/0000-0001-6568-515X

Hiran A. Ariyawansa https://orcid.org/0000-0001-8526-7721

Hyunkyu Sang https://orcid.org/0000-0002-7459-5217

Data availability

The raw reads of ITS amplicon sequencing generated in this study were deposited in NCBI (BioProject no. PRJNA1165193). Nucleotide sequences of species described in this study were submitted to GenBank with accession numbers shown in Suppl. material 1.

References

  • Abeywickrama P, Qian N, Jayawardena R, Li Y, Zhang W, Guo K, Zhang L, Zhang G, Yan J, Li X, Guo Z, Hyde K, Peng Y, Zhao W (2023) Endophytic fungi in green manure crops; friends or foe? Mycosphere 14: 1–106. https://doi.org/10.5943/mycosphere/14/1/1
  • Andreasen M, Skrede I, Jaklitsch WM, Voglmayr H, Nordén B (2021) Multi-locus phylogenetic analysis of lophiostomatoid fungi motivates a broad concept of Lophiostoma and reveals nine new species. Persoonia 46: 240–271. https://doi.org/10.3767/persoonia.2021.46.09
  • Bharadwaj R, Jagadeesan H, Kumar SR, Ramalingam S (2020) Molecular mechanisms in grass-Epichloë interactions: Towards endophyte driven farming to improve plant fitness and immunity. World Journal of Microbiology and Biotechnology 36: 92. https://doi.org/10.1007/s11274-020-02868-5
  • Chen Z, Jin Y, Yao X, Chen T, Wei X, Li C, White JF, Nan Z (2020) Fungal endophyte improves survival of Lolium perenne in low fertility soils by increasing root growth, metabolic activity and absorption of nutrients. Plant and Soil 452: 185–206. https://doi.org/10.1007/s11104-020-04556-7
  • Crous PW, Wingfield MJ, Roux JJL, Richardson DM, Strasberg D, Shivas RG, Alvarado P, Edwards J, Moreno G, Sharma R, Sonawane MS, Tan YP, Altés A, Barasubiye T, Barnes CW, Blanchette RA, Boertmann D, Bogo A, Carlavilla JR, Cheewangkoon R, Daniel R, De Beer ZW, Yáñez-Morales MDJ, Duong TA, Fernández-Vicente J, Geering ADW, Guest DI, Held BW, Heykoop M, Hubka V, Ismail AM, Kajale SC, Khemmuk W, Kolařík M, Kurli R, Lebeuf R, Lévesque CA, Lombard L, Magista D, Manjón JL, Marincowitz S, Mohedano JM, Nováková A, Oberlies NH, Otto EC, Paguigan ND, Pascoe IG, Pérez-Butrón JL, Perrone G, Rahi P, Raja HA, Rintoul T, Sanhueza RMV, Scarlett K, Shouche YS, Shuttleworth LA, Taylor PWJ, Thorn RG, Vawdrey LL, Solano-Vidal R, Voitk A, Wong PTW, Wood AR, Zamora JC, Groenewald JZ (2015) Fungal Planet description sheets: 371–399. Persoonia 35: 264–327. https://doi.org/10.3767/003158515X690269
  • Crous PW, Wingfield MJ, Burgess TI, Hardy GEStJ, Crane C, Barrett S, Cano-Lira JF, Leroux JJ, Thangavel R, Guarro J, Stchigel AM, Martín MP, Alfredo DS, Barber PA, Barreto RW, Baseia IG, Cano-Canals J, Cheewangkoon R, Ferreira RJ, Gené J, Lechat C, Moreno G, Roets F, Shivas RG, Sousa JO, Tan YP, Wiederhold NP, Abell SE, Accioly T, Albizu JL, Alves JL, Antoniolli ZI, Aplin N, Araújo J, Arzanlou M, Bezerra JDP, Bouchara J-P, Carlavilla JR, Castillo A, Castroagudín VL, Ceresini PC, Claridge GF, Coelho G, Coimbra VRM, Costa LA, Da Cunha KC, Da Silva SS, Daniel R, De Beer ZW, Dueñas M, Edwards J, Enwistle P, Fiuza PO, Fournier J, García D, Gibertoni TB, Giraud S, Guevara-Suarez M, Gusmão LFP, Haituk S, Heykoop M, Hirooka Y, Hofmann TA, Houbraken J, Hughes DP, Kautmanová I, Koppel O, Koukol O, Larsson E, Latha KPD, Lee DH, Lisboa DO, Lisboa WS, López-Villalba Á, Maciel JLN, Manimohan P, Manjón JL, Marincowitz S, Marney TS, Meijer M, Miller AN, Olariaga I, Paiva LM, Piepenbring M, Poveda-Molero JC, Raj KNA, Raja HA, Rougeron A, Salcedo I, Samadi R, Santos TAB, Scarlett K, Seifert KA, Shuttleworth LA, Silva GA, Silva M, Siqueira JPZ, Souza-Motta CM, Stephenson SL (2016) Fungal Planet description sheets: 469–557. Persoonia 37: 218–403. https://doi.org/10.3767/003158516X694499
  • Crous PW, Begoude BAD, Boers J, Braun U, Declercq B, Dijksterhuis J, Elliott TF, Garay-Rodriguez GA, Jurjević Ž, Kruse J, Linde CC, Loyd A, Mound L, Osieck ER, Rivera-Vargas LI, Quimbita AM, Rodas CA, Roux J, Schumacher RK, Starink-Willemse M, Thangavel R, Trappe JM, Van Iperen AL, Van Steenwinkel C, Wells A, Wingfield MJ, Yilmaz N, Groenewald JZ (2022a) New and interesting fungi. Fungal Systematics and Evolution 10: 19–90. https://doi.org/10.3114/fuse.2022.10.02
  • Crous PW, Boers J, Holdom D, Osieck, Steinrucken TV, Tan YP, Vitelli JS, Shivas RG, Barrett M, Boxshall A-G, Broadbridge J, Larsson E, Lebel T, Pinruan U, Sommai S, Alvarado P, Bonito G, Decock CA, De La Peña-Lastra S, Delgado G, Houbraken J, Maciá-Vicente JG, Raja HA, Rigueiro-Rodríguez A, Rodríguez A, Wingfield MJ, Adams SJ, Akulov A, AL-Hidmi T, Antonín V, Arauzo S, Arenas F, Armada F, Aylward J, Bellanger J-M, Berraf-Tebbal A, Bidaud A, Boccardo F, Cabero J, Calledda F, Corriol G, Crane JL, Dearnaley JDW, Dima B, Dovana F, Eichmeier A, Esteve-Raventós F, Fine M, Ganzert L, García D, Torres-Garcia D, Gené J, Gutiérrez A, Iglesias P, Istel Ł, Jangsantear P, Jansen GM, Jeppson M, Karun NC, Karich A, Khamsuntorn P, Kokkonen K, Kolarík M, Kubátová A, Labuda R, Lagashetti AC, Lifshitz N, Linde C, Loizides M, Luangsa-ard JJ, Lueangjaroenkit P, Mahadevakumar S, Mahamedi AE, Malloch DW, Marincowitz S, Mateos A, Moreau P-A, Miller AN, Molia A, Morte A, Navarro-Ródenas A, Nebesářová J, Nigrone E, Nuthan BR, Oberlies NH, Pepori AL, Rämä T, Rapley D, Reschke K, Robicheau BM, Roets F, Roux J, Saavedra M, Sakolrak B, Santini A, Ševčíková H, Singh PN, Singh SK, Somrithipol S, Spetik M, Sridhar KR, Starink-Willemse M, Taylor VA, Van Iperen AL, Vauras J, Walker AK, Wingfield BD, Yarden O, Cooke AW, Manners AG, Pegg KG, Groenewald JZ (2022b) Fungal Planet description sheets: 1383–1435. Persoonia 48: 261–371. https://doi.org/10.3767/persoonia.2022.48.08
  • Cubero OF, Crespo A, Fatehi J, Bridge PD (1999) DNA extraction and PCR amplification method suitable for fresh, herbarium-stored, lichenized, and other fungi. Plant Systematics and Evolution 216: 243–249. https://doi.org/10.1007/BF01084401
  • De Hoog G (1985) Taxonomy of the Dactylaria complex, VI. Key to the genera and checklist of epithets. Studies in Mycology 26: 97–122.
  • Edgar RC (2016) SINTAX: a simple non-Bayesian taxonomy classifier for 16S and ITS sequences. Bioinformatics. Preprint at bioRxiv. https://doi.org/10.1101/074161
  • Ge Y, Norton T, Wang Z-Y (2006) Transgenic zoysiagrass (Zoysia japonica) plants obtained by agrobacterium-mediated transformation. Plant Cell Reports 25: 792–798. https://doi.org/10.1007/s00299-006-0123-8
  • Glass NL, Donaldson GC (1995) Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Applied and Environmental Microbiology 61: 1323–1330. https://doi.org/10.1128/aem.61.4.1323-1330.1995
  • Guerrero C, Vitoriano J, Neto L, Dionísio L (2008) Control of fungi diseases on turfgrass using Trichoderma harzianum. Wseas Transactions on Environment and Development 9: 736–754.
  • Hapida Y, Elfita E, Widjajanti H, Salni S (2021) Biodiversity and antibacterial activity of endophytic fungi isolated from jambu bol (Syzygium malaccense). Biodiversitas Journal of Biological Diversity 22: 5668–5677. https://doi.org/10.13057/biodiv/d221253
  • Harms K, Milic A, Stchigel AM, Stadler M, Surup F, Marin-Felix Y (2021) Three new derivatives of zopfinol from Pseudorhypophila mangenotii gen. et comb. nov. Journal of Fungi 7: 181. https://doi.org/10.3390/jof7030181
  • Hassen H (1929) Etiology of the pink-root disease of onions. Phytopathology 19: 691–704.
  • Huang L-Q, Niu Y-C, Su L, Deng H, Lyu H (2020) The potential of endophytic fungi isolated from cucurbit plants for biocontrol of soilborne fungal diseases of cucumber. Microbiological Research 231: 126369. https://doi.org/10.1016/j.micres.2019.126369
  • Huang W, Cai Y, Surveswaran S, Hyde K, Corke H, Sun M (2009) Molecular phylogenetic identification of endophytic fungi isolated from three Artemisia species. Fungal Diversity: 69–88.
  • Hyde KD, Norphanphoun C, Abreu VP, Bazzicalupo A, Thilini Chethana KW, Clericuzio M, Dayarathne MC, Dissanayake AJ, Ekanayaka AH, He M-Q, Hongsanan S, Huang S-K, Jayasiri SC, Jayawardena RS, Karunarathna A, Konta S, Kušan I, Lee H, Li J, Lin C-G, Liu N-G, Lu Y-Z, Luo Z-L, Manawasinghe IS, Mapook A, Perera RH, Phookamsak R, Phukhamsakda C, Siedlecki I, Soares AM, Tennakoon DS, Tian Q, Tibpromma S, Wanasinghe DN, Xiao Y-P, Yang J, Zeng X-Y, Abdel-Aziz FA, Li W-J, Senanayake IC, Shang Q-J, Daranagama DA, De Silva NI, Thambugala KM, Abdel-Wahab MA, Bahkali AH, Berbee ML, Boonmee S, Bhat DJ, Bulgakov TS, Buyck B, Camporesi E, Castañeda-Ruiz RF, Chomnunti P, Doilom M, Dovana F, Gibertoni TB, Jadan M, Jeewon R, Jones EBG, Kang J-C, Karunarathna SC, Lim YW, Liu J-K, Liu Z-Y, Plautz HL, Lumyong S, Maharachchikumbura SSN, Matočec N, McKenzie EHC, Mešić A, Miller D, Pawłowska J, Pereira OL, Promputtha I, Romero AI, Ryvarden L, Su H-Y, Suetrong S, Tkalčec Z, Vizzini A, Wen T-C, Wisitrassameewong K, Wrzosek M, Xu J-C, Zhao Q, Zhao R-L, Mortimer PE (2017) Fungal diversity notes 603–708: Taxonomic and phylogenetic notes on genera and species. Fungal Diversity 87: 1–235. https://doi.org/10.1007/s13225-017-0391-3
  • Jahn L, Schafhauser T, Pan S, Weber T, Wohlleben W, Fewer D, Sivonen K, Flor L, Van Pée K-H, Caradec T, Jacques P, Huijbers Mieke ME, Van Berkel Willem JH, Ludwig-Müller J (2017) Cyanodermella asteris sp. nov. (Ostropales) from the inflorescence axis of Aster tataricus. Mycotaxon 132: 107–123. https://doi.org/10.5248/132.107
  • Jha P, Kaur T, Chhabra I, Panja A, Paul S, Kumar V, Malik T (2023) Endophytic fungi: hidden treasure chest of antimicrobial metabolites interrelationship of endophytes and metabolites. Frontiers in Microbiology 14: 1227830. https://doi.org/10.3389/fmicb.2023.1227830
  • Kalyaanamoorthy S, Minh BQ, Wong TKF, Von Haeseler A, Jermiin LS (2017) ModelFinder: fast model selection for accurate phylogenetic estimates. Nature Methods 14: 587–589. https://doi.org/10.1038/nmeth.4285
  • Khidir HH, Eudy DM, Porras-Alfaro A, Herrera J, Natvig DO, Sinsabaugh RL (2010) A general suite of fungal endophytes dominate the roots of two dominant grasses in a semiarid grassland. Journal of Arid Environments 74: 35–42. https://doi.org/10.1016/j.jaridenv.2009.07.014
  • Latz MAC, Jensen B, Collinge DB, Jørgensen HJL (2018) Endophytic fungi as biocontrol agents: elucidating mechanisms in disease suppression. Plant Ecology & Diversity 11: 555–567. https://doi.org/10.1080/17550874.2018.1534146
  • Lee G, Choi H, Liu H, Han Y-H, Paul NC, Han GH, Kim H, Kim PI, Seo S-I, Song J, Sang H (2023) Biocontrol of the causal brown patch pathogen Rhizoctonia solani by Bacillus velezensis GH1-13 and development of a bacterial strain specific detection method. Frontiers in Plant Science 13: 1091030. https://doi.org/10.3389/fpls.2022.1091030
  • Lin X, Lu C, Huang Y, Zheng Z, Su W, Shen Y (2007) Endophytic fungi from a pharmaceutical plant, Camptotheca acuminata: isolation, identification and bioactivity. World Journal of Microbiology and Biotechnology 23: 1037–1040. https://doi.org/10.1007/s11274-006-9329-8
  • Loch DS, Ebina M, Choi JS, Han L (2017) Ecological implications of Zoysia species, distribution, and adaptation for management and use of zoysiagrasses. International Turfgrass Society Research Journal 13: 11–25. https://doi.org/10.2134/itsrj2016.10.0857
  • Luo J, Vines PL, Grimshaw A, Hoffman L, Walsh E, Bonos SA, Clarke BB, Murphy JA, Meyer WA, Zhang N (2017) Magnaporthiopsis meyeri-festucae sp. nov., associated with a summer patch-like disease of fine fescue turfgrasses. Mycologia 109: 780–789. https://doi.org/10.1080/00275514.2017.1400306
  • Luo Z-L, Bahkali AH, Liu X-Y, Phookamsak R, Zhao Y-C, Zhou D-Q, Su H-Y, Hyde KD (2016) Poaceascoma aquaticum sp. nov. (Lentitheciaceae), a new species from submerged bamboo in freshwater. Phytotaxa 253: 71. https://doi.org/10.11646/phytotaxa.253.1.5
  • Manzotti A, Bergna A, Burow M, Jørgensen HJL, Cernava T, Berg G, Collinge DB, Jensen B (2020) Insights into the community structure and lifestyle of the fungal root endophytes of tomato by combining amplicon sequencing and isolation approaches with phytohormone profiling. FEMS Microbiology Ecology 96: fiaa052. https://doi.org/10.1093/femsec/fiaa052
  • Marin-Felix Y, Miller AN, Cano-Lira JF, Guarro J, García D, Stadler M, Huhndorf SM, Stchigel AM (2020) Re-evaluation of the order Sordariales: Delimitation of Lasiosphaeriaceae s. str., and introduction of the new families Diplogelasinosporaceae, Naviculisporaceae, and Schizotheciaceae. Microorganisms 8: 1430. https://doi.org/10.3390/microorganisms8091430
  • Matheny PB, Liu YJ, Ammirati JF, Hall BD (2002) Using RPB1 sequences to improve phylogenetic inference among mushrooms (Inocybe, Agaricales). American Journal of Botany 89: 688–698. https://doi.org/10.3732/ajb.89.4.688
  • Meyer WA, Torres MS, White JF (2015) Biology and applications of fungal endophytes in turfgrasses. In: Stier JC, Horgan BP, Bonos SA (Eds) Turfgrass: biology, use, and management. American Society of Agronomy, Madison, WI, USA, 713–731. https://doi.org/10.2134/agronmonogr56.c20
  • Monard C, Gantner S, Stenlid J (2013) Utilizing ITS1 and ITS2 to study environmental fungal diversity using pyrosequencing. FEMS Microbiology Ecology 84: 165–175. https://doi.org/10.1111/1574-6941.12046
  • Nguyen L-T, Schmidt HA, Von Haeseler A, Minh BQ (2015) IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Molecular Biology and Evolution 32: 268–274. https://doi.org/10.1093/molbev/msu300
  • Nilsson RH, Larsson K-H, Taylor AFS, Bengtsson-Palme J, Jeppesen TS, Schigel D, Kennedy P, Picard K, Glöckner FO, Tedersoo L, Saar I, Kõljalg U, Abarenkov K (2019) The UNITE database for molecular identification of fungi: Handling dark taxa and parallel taxonomic classifications. Nucleic Acids Research 47: D259–D264. https://doi.org/10.1093/nar/gky1022
  • Noumeur SR, Teponno RB, Helaly SE, Wang X-W, Harzallah D, Houbraken J, Crous PW, Stadler M (2020) Diketopiperazines from Batnamyces globulariicola, gen. & sp. nov. (Chaetomiaceae), a fungus associated with roots of the medicinal plant Globularia alypum in Algeria. Mycological Progress 19: 589–603. https://doi.org/10.1007/s11557-020-01581-9
  • O’Donnell K, Cigelnik E (1997) Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Molecular Phylogenetics and Evolution 7: 103–116. https://doi.org/10.1006/mpev.1996.0376
  • Oksanen J, Simpson GL, Blanchet FG, Kindt R, Legendre P, Minchin PR, O’Hara RB, Solymos P, Stevens MHH, Szoecs E, Wagner H, Barbour M, Bedward M, Bolker B, Borcard D, Carvalho G, Chirico M, De Caceres M, Durand S, Evangelista HBA, FitzJohn R, Friendly M, Furneaux B, Hannigan G, Hill MO, Lahti L, McGlinn D, Ouellette M-H, Cunha ER, Smith T, Stier A, Ter Braak CJF, Weedon J (2022) vegan: Community ecology package. https://cran.r-project.org/web/packages/vegan/index.html
  • Phookamsak R, Manamgoda DS, Li W-J, Dai D-Q, Singtripop C, Hyde KD (2015) Poaceascoma helicoides gen et sp. nov., a new genus with scolecospores in Lentitheciaceae. Cryptogamie, Mycologie 36: 225–236. https://doi.org/10.7872/crym/v36.iss2.2015.225
  • Porras-Alfaro A, Herrera J, Sinsabaugh RL, Odenbach KJ, Lowrey T, Natvig DO (2008) Novel root fungal consortium associated with a dominant desert grass. Applied and Environmental Microbiology 74: 2805–2813. https://doi.org/10.1128/AEM.02769-07
  • Poveda J, Abril-Urias P, Escobar C (2020) Biological control of plant-parasitic nematodes by filamentous fungi inducers of resistance: Trichoderma, mycorrhizal and endophytic fungi. Frontiers in Microbiology 11: 992. https://doi.org/10.3389/fmicb.2020.00992
  • Promputtha I, Jeewon R, Lumyong S, McKenzie EH, Hyde KD (2005) Ribosomal DNA fingerprinting in the identification of non sporulating endophytes from Magnolia liliifera (Magnoliaceae). Fungal Diversity 20: 167–186.
  • Quaedvlieg W, Verkley GJM, Shin H-D, Barreto RW, Alfenas AC, Swart WJ, Groenewald JZ, Crous PW (2013) Sizing up Septoria. Studies in Mycology 75: 307–390. https://doi.org/10.3114/sim0017
  • Rajani P, Rajasekaran C, Vasanthakumari MM, Olsson SB, Ravikanth G, Uma Shaanker R (2021) Inhibition of plant pathogenic fungi by endophytic Trichoderma spp. through mycoparasitism and volatile organic compounds. Microbiological Research 242: 126595. https://doi.org/10.1016/j.micres.2020.126595
  • Rehner SA, Buckley E (2005) A Beauveria phylogeny inferred from nuclear ITS and EF1-α sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs. Mycologia 97: 84–98. https://doi.org/10.1080/15572536.2006.11832842
  • Ripa FA, Cao W, Tong S, Sun J (2019) Assessment of plant growth promoting and abiotic stress tolerance properties of wheat endophytic fungi. BioMed Research International 2019: 1–12. https://doi.org/10.1155/2019/6105865
  • Ronquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61: 539–542. https://doi.org/10.1093/sysbio/sys029
  • Schmitt I, Crespo A, Divakar PK, Fankhauser JD, Herman-Sackett E, Kalb K, Nelsen MP, Nelson NA, Rivas-Plata E, Shimp AD, Widhelm T, Lumbsch HT (2009) New primers for promising single-copy genes in fungal phylogenetics and systematics. Persoonia 23: 35–40. https://doi.org/10.3767/003158509X470602
  • Simamora AV, Hahuly MV, Henuk JB (2021) Endophytic fungi as potential biocontrol agents of Phytophthora palmivora in the cocoa plant. Biodiversitas Journal of Biological Diversity 22: 2601–2609. https://doi.org/10.13057/biodiv/d220519
  • Soreng RJ, Peterson PM, Romaschenko K, Davidse G, Zuloaga FO, Judziewicz EJ, Filgueiras TS, Davis JI, Morrone O (2015) A worldwide phylogenetic classification of the Poaceae (Gramineae). Journal of Systematics and Evolution 53: 117–137. https://doi.org/10.1111/jse.12150
  • Sridhar KR (2019) Diversity, ecology, and significance of fungal endophytes. In: Jha S (Ed.) Endophytes and secondary metabolites. Reference Series in Phytochemistry. Springer, Cham, 61–100. https://doi.org/10.1007/978-3-319-90484-9_5
  • Stiller JW, Hall BD (1997) The origin of red algae: Implications for plastid evolution. Proceedings of the National Academy of Sciences 94: 4520–4525. https://doi.org/10.1073/pnas.94.9.4520
  • Tan Y, Marney T, Bishop-Hurley S, Bransgrove K, Shivas R (2021) Index Fungorum no. 490.
  • Tanney JB, Di Stefano J, Miller JD, McMullin DR (2023) Natural products from the Picea foliar endophytes Niesslia endophytica sp. nov. and Strasseria geniculata. Mycological Progress 22: 17. https://doi.org/10.1007/s11557-023-01869-6
  • Tejesvi MV, Kajula M, Mattila S, Pirttilä AM (2011) Bioactivity and genetic diversity of endophytic fungi in Rhododendron tomentosum Harmaja. Fungal Diversity 47: 97–107. https://doi.org/10.1007/s13225-010-0087-4
  • Thambugala KM, Hyde KD, Tanaka K, Tian Q, Wanasinghe DN, Ariyawansa HA, Jayasiri SC, Boonmee S, Camporesi E, Hashimoto A, Hirayama K, Schumacher RK, Promputtha I, Liu Z-Y (2015) Towards a natural classification and backbone tree for Lophiostomataceae, Floricolaceae, and Amorosiaceae fam. nov. Fungal Diversity 74: 199–266. https://doi.org/10.1007/s13225-015-0348-3
  • Tibpromma S, Hyde KD, Bhat JD, Mortimer PE, Xu J, Promputtha I, Doilom M, Yang J-B, Tang AMC, Karunarathna SC (2018) Identification of endophytic fungi from leaves of Pandanaceae based on their morphotypes and DNA sequence data from southern Thailand. MycoKeys 33: 25–67. https://doi.org/10.3897/mycokeys.33.23670
  • Tibpromma S, Zhang L, Karunarathna SC, Du T-Y, Phukhamsakda C, Rachakunta M, Suwannarach N, Xu J, Mortimer PE, Wang Y-H (2021) Volatile constituents of endophytic fungi isolated from Aquilaria sinensis with descriptions of two new species of Nemania. Life 11: 363. https://doi.org/10.3390/life11040363
  • Tiwari P, Kang S, Bae H (2023) Plant-endophyte associations: Rich yet under-explored sources of novel bioactive molecules and applications. Microbiological Research 266: 127241. https://doi.org/10.1016/j.micres.2022.127241
  • Tomita F (2003) Endophytes in southeast Asia and Japan: Their taxonomic diversity and potential applications. Fungal Diversity 14: 187–204.
  • Tothill JC, Hacker JB (1983) The grasses of southern Queensland. University of Queensland Press, St. Lucia, Queensland, Australia.
  • Vilgalys R, Hester M (1990) Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4238–4246. https://doi.org/10.1128/jb.172.8.4238-4246.1990
  • Vines PL, Hoffmann FG, Meyer F, Allen TW, Luo J, Zhang N, Tomaso-Peterson M (2020) Magnaporthiopsis cynodontis, a novel turfgrass pathogen with widespread distribution in the United States. Mycologia 112: 52–63. https://doi.org/10.1080/00275514.2019.1676614
  • White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (Eds) PCR protocols: a guide to methods and applications. Academic Press, San Diego, 315–322. https://doi.org/10.1016/B978-0-12-372180-8.50042-1
  • Xia Y, Sahib MR, Amna A, Opiyo SO, Zhao Z, Gao YG (2019) Culturable endophytic fungal communities associated with plants in organic and conventional farming systems and their effects on plant growth. Scientific Reports 9: 1669. https://doi.org/10.1038/s41598-018-38230-x
  • Yang D-H, Sun H-J, Jeong O-C, Jin I-D, Kang H-G, Lee H-Y (2023) Development of ‘Halla Green 12’ cultivar of zoysiagrass, a hybrid of Z. matrella and Z. japonica. Korean Journal of Breeding Science 55: 147–155. https://doi.org/10.9787/KJBS.2023.55.2.147
  • Yuen G, Zhang Z (2001) Control of brown patch disease using the bacterium Stenotrophomonas maltophilia strain C3 and culture fluid. International Turfgrass Society Research Journal 9: 742–747.
  • Zhang N, Zhao S, Shen Q (2011) A six-gene phylogeny reveals the evolution of mode of infection in the rice blast fungus and allied species. Mycologia 103: 1267–1276. https://doi.org/10.3852/11-022

Supplementary materials

Supplementary material 1 

Genbank accession numbers for the strains used for phylogenetic analysis in this study

Haifeng Liu, Hyeongju Choi, Narayan Chandra Paul, Hiran A. Ariyawansa, Hyunkyu Sang

Data type: xlsx

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (31.61 kb)
Supplementary material 2 

Sequence alignments

Haifeng Liu, Hyeongju Choi, Narayan Chandra Paul, Hiran A. Ariyawansa, Hyunkyu Sang

Data type: zip

Explanation note: Sequence alignments (fas.-files in ZIP.archive).

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (60.05 kb)
login to comment