Strong antagonism of an endophyte of Fraxinus excelsior towards the ash dieback pathogen, Hymenoscyphus fraxineus, is mediated by the antifungal secondary metabolite PF1140

ABSTRACT

Ash dieback, caused by the fungal pathogen Hymenoscyphus fraxineus (Helotiales, Ascomycota), is threatening the existence of the European ash, Fraxineus excelsior. During our search for biological control agents for this devastating disease, endophytic fungi were isolated from healthy plant tissues and co-cultivated with H. fraxineus to assess their antagonistic potential. Among the strains screened, Penicillium cf. manginii DSM 104493 most strongly inhibited the pathogen. Initially, DSM 104493 showed promise in planta as a biocontrol agent. Inoculation of DSM 104493 into axenically cultured ash seedlings greatly decreased the development of disease symptoms in seedlings infected with H. fraxineus. The fungus was thus cultivated on a larger scale in order to obtain sufficient material to identify active metabolites that accounted for the antibiosis observed in dual culture. We isolated PF1140 (1) and identified it as the main active compound in the course of a bioassay-guided isolation strategy. Furthermore, its derivative 2, the mycotoxin citreoviridin (3), three tetramic acids of the vancouverone type (4–6), and penidiamide (7) were isolated by preparative chromatography. The structures were elucidated mainly by NMR spectroscopy and high-resolution mass spectrometry (HRMS), of which compounds 2 and 6 represent novel natural products. Of the compounds tested, not only PF1140 (1) strongly inhibited H. fraxineus in an agar diffusion assay but also showed phytotoxic effects in a leaf puncture assay. Unfortunately, both the latent virulent attributes of DSM 104493 observed subsequent to these experiments in planta and the production of mycotoxins exclude strain Penicillium cf. manginii DSM 104493 from further development as a safe biocontrol agent.

IMPORTANCE

Environmentally friendly measures are urgently needed to control the causative agent of ash dieback, Hymenoscyphus fraxineus. Herein, we show that the endophyte DSM 104493 exhibits protective effects in vitro and in planta. We traced the activity of DSM 104493 to the antifungal natural product PF1140, which unfortunately also showed phytotoxic effects. Our results have important implications for understanding plant-fungal interactions mediated by secondary metabolites, not only in the context of ash dieback but also generally in plant-microbial interactions.

INTRODUCTION

The ash dieback disease, caused by the introduced Asian pathogen Hymenoscyphus fraxineus (Helotiales, Ascomycota), has spread throughout Europe in the course of the past 30 years and has become a serious threat to several species of ash (Fraxinus spp.) and ash-related ecosystems (1). Among other measures taken to control the disease, several research groups have evaluated antagonistic endophytes of Fraxinus species as biocontrol agents (2–6). However, most of the strains were not investigated for their function in planta. To the best of our knowledge, only Halecker et al. (7) and Barta et al. (8) inoculated Fraxineus excelsior with endophytes to determine their potential to mitigate disease development.

In our previous study of an endophytic isolate of Hypoxylon rubiginosum (Xylariales, Ascomycota) derived from F. excelsior, we showed that the strain not only colonized axenically cultured seedlings asymptomatically but also that it produced large quantities of antifungal phomopsidin in dual culture with H. fraxineus (7). A follow-up study showed that several species of the H. rubiginosum complex, which are likewise often associated with Fraxinus, also produce the same compound in dual culture (9).

To the best of our knowledge, no other research groups have elucidated the structures of the antagonistic metabolites that endophytes of F. excelsior produce. Even though many other studies reported antibiosis of the endophytes directed against the pathogen, no attempt has been made to determine the bioactive compounds and identify them using state-of-the-art analytics. Nor have there been any other comparable studies that have investigated the in planta capacity of an endophyte to prevent the development of disease symptoms.

During our search for additional endophytic antagonists of the ash dieback pathogen, we came across a fungal strain that showed particularly strong inhibition of H. fraxineus in our initial screening both in dual culture and in planta. The present paper is dedicated to describing its antagonism against H. fraxineus in dual culture, its apparent inhibition of H. fraxineus in planta, and its major secondary metabolites, at least one of which seems to be responsible for the phytotoxic activity of the strain.

RESULTS

Taxonomic assessment of DSM 104493

For the preliminary molecular identification of DSM 104493 (Fig. S1), the complete sequences of the ITS region of the rRNA gene cluster and the partial sequence of the β-tubulin-encoding gene were submitted to Nucleotide BLAST in the NCBI GenBank database (https://blast.ncbi.nlm.nih.gov), as recommended by Houbraken et al. (10). The analysis of ITS sequences (GenBank accession OR996288) suggested that the strain belongs to the genus Penicillium with a 99% sequence similarity to the type strain of Penicillium manginii (CBS 253.31, GenBank accession MH855205). However, the locality of this isolate is not known (10). The β-tubulin sequence (GenBank accession PP182370) was also 98% similar to P. manginii strain CBS 126233 (GenBank accession JN606645) isolated from soil under Cyathea tree ferns on the Rio Jaba Trail near Quebrada Culebra, Wilson Botanical Garden/La Cruces Biological Station, Costa Rica. This suggests that the isolate belongs to the Penicillium section Citrina (10) and is closely related to P. manginii; however, the precise identification requires the analysis of more strains.

To align DNA barcoding results with the phenotypic attributes of fungi from the Penicillium section Citrina, we tested exolite profiles, production of sclerotia, and conidia. Exolites for the species P. manginii include citrinin, citreoviridin, citreoviridinol A1 and A2, epicitreoviridinol, and, in some isolates, phenicin. DSM 104493 strain produced citreoviridin and phenicin (Fig. S2). Also, the morphology of our strain corroborated our molecular diagnostics and extrolite approach. DSM 104493 did not sporulate on any of the tested media: Czapek yeast extract agar (CYA), yeast extract sucrose agar (YES), dichloran 18% glycerol agar (DG18), malt extract agar (MEA), creatine sucrose agar (CREA), and oatmeal agar (OA), as known for some isolates of the Citrina section (10). Identification was also narrowed down by the production of sclerotia, which all but one of the species that produce sclerotia within the Penicillium westlingii clade, to which P. manginii belongs, do.

In consideration of the molecular identification and the phenotypic characteristics, we conclude that the tested isolate is closely related to P. manginii sensu NCBI Taxonomy and MycoBank (note, Index Fungorum/Species Fungorum current name is Penicillium atrosanguineum, www.speciesfungorum.org). However, the precise determination of the taxonomic position of this isolate requires further analysis, which was beyond the scope of this investigation. Therefore, we assign the strain P. cf. manginii DSM 104493.

Inhibition of H. fraxineus in dual culture and in planta

In the course of our screening for endophytic fungi with antagonistic activities against the plant pathogenic fungus H. fraxineus, dual culture with various strains was conducted on agar media to assess the strains’ antagonistic potential (7, 9). DSM 104493 stood out with a growth inhibition of 64%, which has been the greatest value of inhibition thus far observed (7, 9, 11). Following initial inoculation into axenically cultured ash seedlings in 2016, seedlings infected with DSM 104493 had no more disease symptoms than the control (Table S1).

Based on the results in dual cultures and in planta, DSM 104493 was further tested for its ability to control H. fraxineus in planta in axenically cultured ash seedlings. Before repetition of the experiment, tests were run to determine the most virulent of the eight isolates of H. fraxineus, all of which had been isolated from F. excelsior. Results showed that they varied greatly in their virulence toward the ash seedlings, with the evaluation of disease symptoms ranging from 1.7 to 4.3 (evaluation of 1 = asymptomatic, that of 5 = dead; Fig. 1A). Based on an evaluation of 4.3, the most virulent isolate, H. fraxineus NWE 1/2/H1 was selected for dual inoculation experiments with DSM 104493.

Fig 1.

(A) Disease symptoms of axenically cultured ash seedlings inoculated with isolates of H. fraxineus to determine their virulence. (B) Disease symptoms of ash seedlings inoculated with the most virulent strain H. fraxineus NWE 1/2/H1 and DSM 104493, inoculated simultaneously and inoculated 7 days after inoculation with H. fraxineus NWE 1/2/H1, respectively. Evaluation of disease symptoms on a scale of 1 = no disease symptoms to 5 = dead, based on necroses, chloroses, wilting, and loss of leaves.

H. fraxineus NWE1/2/H1 and DSM 104493 were inoculated into ash seedlings in two separate experiments: (i) simultaneously with H. fraxineus and (ii) H. fraxineus 7 days before inoculation with the endophyte. The latter experiment was conducted to give H. fraxineus, which has a slower growth rate than DSM 104493, a head start. The results of the two experiments were similar. Inoculation with H. fraxineus NWE1/2/H1 alone led to disease symptoms of 4.3 (Fig. 1A). However, inoculation with DSM 104493 with H. fraxineus NWE1/2/H1 only resulted in a disease evaluation between 1.5 and 2.0 (Fig. 1B), demonstrating that DSM 104493 greatly reduced the disease symptoms that inoculation with H. fraxineus alone would have resulted in (Fig. 1). Whereas the inoculated endophyte was isolated from the tissue segments following surface sterilization, H. fraxineus was not.

Unfortunately, in contrast to the very promising results attained in dual culture and in the above-reported inoculation experiments with DSM 104493 in 2017, in a subsequent experiment (2021), which ran for 3 not 2 months, disease symptoms did develop. As in 2017, no disease symptoms were present 8 weeks after inoculation. However, they did develop in the inoculated seedlings 3 months after inoculation. The plants were either dead (evaluation 5) or almost dead (evaluation 4; Fig. S3; Table S1). All control seedlings were asymptomatic. Nevertheless, we considered the activity of P. cf. manginii DSM 104493 sufficiently interesting for the analysis of its secondary metabolites.

Structure elucidation of secondary metabolites

We chose a bioactivity-guided isolation approach to pinpoint the observed activity of individual natural products. The antifungal compounds could be extracted with ethyl acetate from the crude extract, indicating that indeed a small molecule is responsible for the activity. The crude extract was subsequently fractionated by reversed-phase high-performance liquid chromatography (HPLC), and the obtained fractions were tested against Mucor hiemalis (Mucorales, Mycoromycota) as the indicator organism. The active fraction was analyzed by HPLC/MS; it contained a single compound 1 with m/z 278.1744. By analyzing 1D and 2D NMR data, compound 1 was identified as the known antifungal compound, PF1140 (12).

Large-scale cultivation resulted in the isolation of sufficient quantities for the structure elucidation of additional metabolites (Fig. 2). Major pigment 3 was identified as citreoviridin, whose identity was confirmed by UV/vis and HRMS data (13). The ¹H and ¹³C NMR spectra of compound 2 were highly similar to those of compound 1 (Fig. S30). However, additional signals were detected for a para-hydroxy benzyl moiety, which was connected to C–5 based on the HMBC correlations from 2′/6′–H to C–5 and 6–H to C–1′ (Fig. S30). Consequently, compound 2 was identified as the 5-para-hydroxyphenyl derivative of PF1140 (1). The structure of compound 6 was elucidated as the dehydroxy derivative of compound 5. Metabolites 4, 5, and 7 were identified as vancouverone A, vancouverone B, and penidiamide based on their MS and NMR data (See also Fig. S7 to S29 for MS data and NMR spectra.).

Fig 2.

Secondary metabolites isolated from DSM 104493: PF-1140 (1), its new 5-p-phenyl derivative (2), citreoviridin (3), vancouverones A–C (4–6), and penidiamide (7).

Biological activities of secondary metabolites

The antifungal properties of compounds 1–7 were investigated in an agar disc diffusion assay against H. fraxineus DSM 117123. Of the seven compounds, only compound 1 showed strong activity against H. fraxineus (Fig. 3), while the rest showed no activity. Additionally, the antimicrobial activities of compounds 1, 5, and 6 were assessed against our standard panel of test organisms (Table S2). PF-1140 (1) displayed a broad range of activity against most of the tested strains and also inhibited our test organism for anti-fungal activities, Mucor hiemalis DSM 2656. The strongest activity of this compound was observed against Bacillus subtilis DSM 10 and Staphylococcus aureus DSM 346, both were inhibited with a MIC value of 4.2 µg/mL. Weak antimicrobial activities were observed for compounds vancouverone B (5) and the new derivative 6, which we named vancouverone C, with an MIC value of 66.6 µg/mL. Vancouverone C (6) was active against Gram-positive bacteria (B. subtilis DSM 10 and S. aureus DSM 346) and the Gram-negative bacterium Escherichia coli DSM 1116, while vancouverone B was only active against B. subtilis. According to cytotoxicity assays, compounds 1, 5, and 6 exhibited significant activities against the tested cell lines (Table 1). PF-1140 (1) generally showed the strongest activities against the tested cell lines, with the highest effects against A431 with half-maximal inhibitory concentrations (IC₅₀) of 0.13 µM.

Fig 3.

Agar disc (6 mm diameter) diffusion assay of compound PF1140 against H. fraxineus DSM 117123, grown on 9-cm petri dishes on agar-based PD medium with H. fraxineus. N, positive control nystatin; M, negative control methanol; and PF, PF1140.

TABLE 1.

Cytotoxic activities of compounds 1, 5, and 6^a

Cell line	IC50 (µM)
	1	5	6	Ref.
Endocervical adenocarcinoma KB3.1	0.35	16	21	4.5 × 10−5
Mouse fibroblast L929	0.47	15	19	4.3 × 10−4
Human lung carcinoma A549	0.43	18	25	5.3 × 10−5
Squamous cell carcinoma A431	0.13	6.7	10	6.5 × 10−5
Human prostate cancer PC-3	9.0	59	35	2.8 × 10−4
Human breast adenocarcinoma MCF-7	0.61	5.9	8.6	8.3 × 10−5
Human ovarian carcinoma SKOV-3	0.47	19	23	1.8 × 10−4

^a Ref.: epothilone B.

The phytotoxic activities of PF1140 (1), citreoviridine (3), and vancouverones A–C (4–6) were assessed with a leaf puncture assay (Fig. S4). In this assay, we applied solutions of 60 µg of the test compounds on European ash (F. excelsior) leaves, causing brownish lesions in all cases. Using the GNU image manipulation program, symptoms resulting from the application of the toxins were quantified (Fig. S4 and S5). While vancouverones A (4) and B (5) had remarkable toxicity with large lesions on ash leaves, PF1140 (1) was obviously the least phytotoxic of the compounds tested (Fig. S6).

DISCUSSION

Dual culture on agar plates is a standard assay for assessing the biocontrol potential of endophytes against H. fraxineus. However, this technique might not be a good predictor of a biocontrol agents’ protective capacity since it is uncertain how interactions take place in planta. Thus, it is necessary to study the interaction of endophytes with the ash plants, i.e., after inoculation. Although several studies have shown antagonistic effects of fungal endophytes against H. fraxineus under in vitro conditions, their effects in planta have so far only been studied by Halecker et al. (7) and Barta et al. (8). Halecker et al. (7) also selected an isolate for the elucidation of the structures of its active metabolites on the basis of the results in dual inoculation experiments, i.e., H. fraxineus with a selection of endophytes and following inoculation into axenically cultured ash seedlings. Of the tested endophytes, only H. rubiginosum caused no disease symptoms in planta. Barta et al. (8) conducted trunk inoculations with four endophytic isolates, which had been selected from 75 isolates showing strong inhibition of H. fraxineus in dual culture. The total length of necrotic lesions formed by H. fraxineus infection was reduced in the ash trunks co-inoculated with endophytes. However, the effect was not significant, nor was the mode of action determined.

Taken together with data from our previous study comparing endophytic isolates in co-culture with H. fraxineus, DSM 104493 was determined to be the strongest inhibitor of the ash dieback pathogen. In contrast to other endophytes, it was not inhibited by H. fraxineus in co-culture (7), which is also relevant when selecting endophytes for biocontrol. Most importantly, the strain was able to initially inhibit/reduce symptoms of H. fraxineus infection in planta, i.e., in axenically cultured seedlings. Unfortunately, DSM 104493 itself showed signs of phytotoxicity in later experiments, which were run not for 8 weeks post-inoculation as in the experiments presented here but for 3 months (Table S1).

Although we did not study the production of secondary metabolites as a final proof in planta, it is plausible that the observed bioactivities of DSM 104493 against H. fraxineus as well as those found in the phytotoxicity test using ash leaves can be correlated with the bioactivities of individual natural products. We identified PF1140 (1) as the bioactive, antifungal compound of DSM 104493. The key metabolite PF1140 belongs to the group of 4-oxy-3-alkyl-2-pyridone alkaloids (14), which is known to have strong antifungal activity. Initially described in a Japanese patent (15), the compound was recently isolated from a New Zealand marine-derived Penicillium species (16). The biosynthesis of compound 1 explains the co-occurrence of metabolites 2 and 4–6 as precursors of compound 1 (17). With fusaricide/epipyridone, an analogous pair of a 4-oxy-3-alkyl-2-pyridone together with its p-hydroxyphenyl-modified derivative lacking the N-OH group is known. The absence of activity of compound 2 compared to compound 1 suggests that the N-OH functionality plays a key role in the biological activities of this group of metabolites (16).

The antifungal as well as phytotoxic activities of compound 1 suggest the compound has a Janus-faced nature: on the one hand, compound 1 might be the key factor responsible for DSM 104493’s inhibition of the growth of H. fraxineus in planta. On the other hand, compound 1 and mainly its derivatives 4 and 5 could be responsible for the phytotoxic effects of DSM 104493 observed at a concentration of 1 mg/mL. The role of citreoviridin (3), which has been identified along with the phospholipase A₂ inhibitor penidiamide (7) as causing beri beri disease, remains elusive. Finding that DSM 104493 produces not only antifungal but also phytotoxic metabolites is not surprising. Endophytes often interact antagonistically with their plant hosts using virulence factors, e.g., the phytotoxic metabolites, exoenzymes, and phytohormones that are required to infect and colonize the host. These are necessary to counter the plant defense reactions and, thus, maintain an equilibrium between plant defense and fungal virulence, thus enabling asymptomatic colonization (4).

Overall, the data presented in our study exemplify that fungal endophytes may well be exerting a protective effect against H. fraxineus in planta. The antifungal effect of DSM 104493 can be attributed mainly to the production of the antifungal compound PF1140 (1). The fact that H. fraxineus was not isolated from the seedlings inoculated with DSM 104493 together with H. fraxineus suggests that the metabolite inhibited the growth of H. fraxineus in planta.

Aside from expanding our knowledge on this plant disease, our results provide an interesting example as to how secondary metabolites may shape particular interactions between host plant, endophytes, and pathogens in nature.

MATERIALS AND METHODS

Fungal material

The H. fraxineus strains were all isolated from F. excelsior. DSM 104493 was isolated from the roots of a small F. excelsior sapling, collected and isolated from the Elm Forest (52.204514, 10.719210°) in central Germany in October 2015. H. fraxineus DSM 117123 was isolated in 2021 from Rhüden, Lower Saxony, by M. Ridley from a diseased branch. H. fraxineus DSM 106868 was isolated in 2009 from Verditz, Kempten, by T. Kirisits and S. Mottinger-Kroupa from a necrotic shoot; H. fraxineus isolates from the culture collection of the Institute of Forest Entomology, Forest Pathology and Forest Protection (IFFF), Vienna, can be obtained directly from the institute. H. fraxineus IFFF NWE/1/2/H1, CBS 123137, was isolated in 2008 in Vienna by T. Kirisits and S. Mottinger-Kroupa from discolored wood of a dead shoot; H. fraxineus IFFF N/5/4/A, CBS 122192, was isolated in 2007 at the Altausee, Steiermark, by E. Halmschlager and S. Mottinger-Kroupa from the bast of a dead shoot; H. fraxineus IFFF N1/3/Holz was isolated in 2007 from Edt, Upper Austria, by E. Halmschlager and S. Mottinger-Kroupa from discolored wood of a small stem; H. fraxineus IFFF ST/FE/4 was isolated in 2014 from Stinatz, Burgenland, by T. Kirisits from a necrotic stem of a young plant; H. fraxineus IFFF ST/FE/11 was isolated in 2015 from Stinatz, Burgenland, by T. Kirisits from a necrotic stem of a young plant; H. fraxineus IFFF ST/FE/17 was isolated in 2015 from Stinatz, Burgenland, by T. Kirisits from a necrotic stem of a young plant; H. fraxineus IFF LOF5 was isolated in 2009 by T. Kirisits and M. Matlakova from a necrotic shoot.

Taxonomic assessment of DSM 104493

The DNA was isolated with the help of MasterPureTM Yeast DNA purification kit, and the nucleic acid concentration was controlled with agarose gel electrophoresis (5 mL DNA per sample was applied to 1% agarose gel, the electrophoresis was performed for 40 min at 120 V, and then the DNA was visualized in the gel with GelStarTM staining technology and UV light. Visible bands were considered to have suitable DNA concentration for PCR). The PCR was performed with an initial denaturation step for 2 min at 96°C, followed by 34–40 cycles of denaturation (60 s at 96°C), primer annealing (60 s, 64°C β-tubulin, 54.5°C ITS/LSU), and elongation (60 s at 72°C). Final extension step was for 7 min at 72°C. The quality of PCR products was checked in 1% agarose gel, stained with GelStarTM technology, using Thermo ScientificTM Gene RulerTM 1 kb DNA Ladder. The PCR products of sufficient quality were purified with the PCRpure kit (Analytik Jena) and used for sequencing.

Bt2a (Forward) (18) GGTAACCAAA TCGGTGCTGC TTTC

Bt2b (Reverse) (18) ACCCTCAGTG TAGTGACCCT TGGC

ITS ITS1 (Forward) (19) TCCGTAGGTG AACCTGCGG

LSU LR5 (reverse) (20) TCCTGAGGGA AACTTCG

Purified PCR amplification products were sequenced at DSMZ (Braunschweig, Germany) on Applied Biosystems (ABI) Sanger sequencing machines (Model 3500xL, 24-capillary), using the same primers as used for the PCR at a concentration of 0.5 pmol/µL. Exception: NL4 Primer (sequence 5′–3′: GGTTCGTGTTTCAAGACGG) was used for sequencing of the LSU gene for PCR products, amplified with LR5 Primer. The obtained sequences were corrected according to their chromatograms using Sequencher software (genecodes.com). Forward and reverse sequences were assembled to a consensus sequence; the ends displaying bad quality were trimmed.

The following media were used to induce sporulation: CYA, YES, DG18, MEA (Oxoid), CREA, and OA. All media were prepared as described by Samson et al. (21).

For the extrolite analysis, DSM 104493 was grown on CYA medium for 7 days at 25°C. Thereafter, agar plates were cut into small pieces and extracted with the extraction solvent [ethyl acetate/dichloromethane/methanol (3:2:1, vol/vol/vol) with 1% (vol/vol) formic acid] and subsequently ultrasonicated for 50 min. The solvents were dried in vacuo at 40°C. The extracts were analyzed by high-performance liquid chromatography using diode array UV-VIS detection and HR-ESIMS (high-resolution electrospray ionization mass spectrometry), spectra were recorded with an Agilent 1200 Infinity Series HPLC−UV system [Agilent Technologies, Santa Clara, CA, USA; column 2.1 × 50 mm, 1.7 µm, C18 Acquity UPLC BEH (Waters), UV/vis detection 200–640 nm] connected to a MaXis ESI-TOF mass spectrometer (Bruker) (scan range 100–2,500 m/z, capillary voltage 4,500 V, and dry temperature 200°C). Identification of the extrolite phenicin was performed by comparison of the UV-visible spectra and HR-ESIMS spectra with the literature.

Endophyte inhibition of H. fraxineus in planta

Tests for potential virulence of DSM 104493 and H. fraxineus in planta were conducted using axenically cultured ash seedlings (F. excelsior). To establish in vitro cultures of ash seedlings, a modified method of Junker et al. (22) and Raquin et al. (23) was employed using ash tree seeds from 2003 (Niedersächsische Landesforsten, Oerrel, from Ostholstein, Friederikenhof, Germany, D-01 001 1 0059 03). After developing leaves and roots, the seedlings were transplanted into baby food jars containing H₁₀ medium (22). Each seedling was inoculated with 0.5 cm² of a mycelial culture at the base of the seedling’s stem. Cultivation was on a window facing north for 3 months with evaluation after 2 and/or 3 months. Virulence was evaluated based on necroses, chloroses, and loss of leaves on a subjective score of 1 (asymptomatic) to 5 (dead) (7). At the end of the experiments, to ascertain that the fungus had colonized the seedlings, the seedlings were surface sterilized and separated into shoots and roots, which in turn were cut into segments and plated on biomalt agar medium with five to seven segments per shoot or root and plant. The number of emerging colonies/segments and seedlings was tallied (22).

DSM 104493 was inoculated into seedlings in 2017 with cultivation for 8 weeks and in 2021 with cultivation for 3 months. H. fraxineus NWE/1/2/H1, N/5/4/A, ST/FE/4, N1/3/Holz, ST/FE/11, ST/FE/17, VER/2, and LOF5 were inoculated into seedlings in 2017 (cultivation for 8 weeks) to select the most virulent isolate.

Dual inoculation of ash seedlings with H. fraxineus and DSM 104493

Based on the results of tests to determine the virulence of H. fraxineus, strain NWE/1/2/H1 was selected as the most virulent strain and employed in the dual inoculation experiments. In the first experiment, H. fraxineus and DSM 104493 were inoculated simultaneously. Since H. fraxineus grows slower than DSM 104493, in the second experiment, H. fraxineus was inoculated 1 week before DSM 104493. In both experiments, there were three parallels. Cultivation was as above for 8 weeks.

Instrumentation for spectral data

HPLC-DAD/MS measurements were performed using an amaZon speed electron transfer dissociation ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany) and measured in positive and negative ion modes simultaneously. An HPLC C18 Acquity UPLC BEH column (Waters, Milford, MA, USA) was used with this system: solvent A: H₂O; solvent B: acetonitrile (MeCN) supplemented with 0.1% formic acid; gradient conditions: 5% B for 0.5 min, increasing to 100% B in 20 min, maintaining isocratic conditions at 100% B for 10 min; flow rate: 0.6 mL/min; and UV/vis detection: 200–600 nm.

HR-ESIMS spectra were recorded with an Agilent 1200 Infinity Series HPLC−UV system [Agilent Technologies, Santa Clara, CA, USA; column 2.1 × 50 mm, 1.7 µm, C18 Acquity UPLC BEH (Waters); solvent A: H₂O + 0.1% formic acid; solvent B: MeCN + 0.1% formic acid; gradient: 5% B for 0.5 min increasing to 100% B in 19.5 min and then maintaining 100% B for 5 min; flow rate: 0.6 mL/min; and UV/vis detection: 200–640 nm] connected to a MaXis ESI-TOF mass spectrometer (Bruker) (scan range: 100–2,500 m/z, capillary voltage: 4,500 V, and dry temperature 200°C).

NMR spectra were recorded on a Bruker Avance III HD 700 MHz spectrometer (¹H 700 MHz, ¹³C 175 MHz), equipped with a 5 mm TXI cryoprobe, and a Bruker Avance III 500 MHz spectrometer with a BBFOplus SmartProbe (¹H 500 MHz and ¹³C 125 MHz).

Chemicals and solvents were obtained from AppliChem GmbH (Darmstadt, Germany), Avantor Performance Materials (Deventor, The Netherlands), Carl Roth GmbH & Co. KG (Karlsruhe, Germany), and Merck KGaA (Darmstadt, Germany).

Fermentation and extraction

DSM 104493 was cultured on solid rice medium (25 g rice and 100 mL base liquid: yeast extract 1 g/L, sodium tartrate 0.5 g/L, and KH₂PO₄ 0.5 g/L) in 500 mL Erlenmeyer flasks. After autoclaving the flasks at 121°C for 20 min and cooling to room temperature, five 50 mm²-sized pieces of well-grown mycelium of DSM 104493, which had been previously prepared and grown on yeast-malt agar plates for about 7 days at 23°C, were each transferred to a 500 mL Erlenmeyer flask under sterile conditions. The fermentation was retained under static conditions at room temperature until fungal growth completely covered the surface of the rice medium, which took about 30 days. The fungal cultures were fragmented mechanically and extracted with 3 × 200 mL of EtOAc, then put in an ultrasonic bath for 30 min, followed by evaporation to dryness to yield a yellow crude extract.

Isolation of the compounds

First, the crude extract was adsorbed onto an RP-18 cartridge, and MeOH was used for elution. Isolation procedures were carried out with reversed-phase HPLC (Gilson, PLC 2020, Middleton, USA). VP Nucleodur C18 ec column (Macherey-Nagel, 150 × 40 mm, 10 µm) was used as the stationary phase. UV detection was carried out at 210, 254, and 356 nm; the elution gradient comprised deionized water (Millipore, Milli-Q) with 0.05% trifluoroacetic acid (TFA) and acetonitrile (MeCN) with 0.05% TFA. The elution gradient used was 15%–100% MeCN for 73 min followed by isocratic conditions with 100% MeCN for 10 min. Twenty-three fractions were collected according to the observed peaks. Fraction 15 contained compound 2 (0.5 mg) eluted after 9.9 min, fraction 19 contained compound 4 (0.4 mg) eluted after 13.8 min, fraction 16 contained compound 5 (7 mg) eluted after 10.6 min, fraction 18 contained compound 6 (1.1 mg) eluted after 12.4 min, and fraction 9 contained compound 7 (0.9 mg) eluted after 7.1 min.

For further purification, a C18 Nucleodur column of dimensions 250 × 21 mm, 7 µm was used as a stationary phase. Fraction 13 was purified by reversed-phase HPLC with an elution gradient of 40%–50% solvent B in 33 min to afford 5.8 mg citreoviridin (85% purity) (t_R: 8.9 min). Fraction 17 was also purified by reverse-phase HPLC, elution gradient 45%–60% solvent B for 43 min to give 13.5 mg of the compound 1 (t_R: 10.4 min).

Physico-chemical characteristics of the compounds

PF1140 (1): colorless oil; ¹H NMR (CH₃OH-d₄, 700 MHz): δ_H 8.55 (s, 6–N1OH), 7.55 (d, J = 7.6 Hz, 6–H), 5.89 (d, J = 7.6 Hz, 5–H), 4.70 (q, J = 6.5 Hz, 13–H₃), 2.23 (d, J = 10.8 Hz, 7–H), 1.79 (br d, J = 13.8 Hz, 11–H_a), 1.71 (br d, J = 13.8 Hz, 9–H_a), 1.64 (m, 10–H), 1.64 (m, 12–H), 1.26 (d, J = 6.5 Hz, 14–H₃), 1.03 (dd, J = 13.8, 13.2 Hz, 9–H_b), 0.92 (d, J = 6.7 Hz, 17–H₃), 0.89 (d, J = 6.5 Hz, 16–H₃), 0.86 (m, 11–H_b), 0.70 (s, 15–H₃) ppm; ¹³C NMR (CH₃OH-d₄, 175 MHz): δ_C 162.3, 161.6, 134.4, 111.6, 99.8, 75.6, 46.7, 45.8, 45.7, 39.1, 35.1, 28.2, 23.4, 22.1, 21.2, and 15.1 ppm; HR-ESIMS: m/z 278.1744 [M + H]⁺ (calculated for C₁₆H₂₄NO₃ 278.1751); data are consistent with those of de Silva et al. (12, 16).

Compound 2: light yellow amorphous solid; [α]²⁰_D = −80 (c = 0.001, MeOH); UV (MeOH, c = 0.02 mg/mL) λmax (log ε) 250 (4.1), 211 (4.2) nm; ¹H NMR (CH₃OH-d₄, 700 MHz): δ_H 7.22 (br d, J = 8.7 Hz, 2′–H/6′–H), 7.13 (s, 6–H), 6.78 (br d, J = 8.7 Hz, 3′–H/5′–H), 4.78 (q, J = 6.5 Hz, 13–H₃), 2.26 (d, J = 10.8 Hz, 7–H), 1.79 (br d, J = 13.8 Hz, 9–H_a), 1.73 (br d, J = 13.8 Hz, 11–H_a), 1.66 (m, 10–H), 1.66 (m, 12–H), 1.24 (d, J = 6.5 Hz, 14–H₃), 1.05 (dd, J = 13.8, 13.0 Hz, 9–H_b), 0.95 (d, J = 6.7 Hz, 17–H₃), 0.90 (d, J = 6.5 Hz, 16–H₃), 0.88 (m, 8–H), 0.75 (d, J = 6.5 Hz, 15–H₃) ppm; ¹³C NMR (CH₃OH-d₄, 175 MHz): δ_C 165.7 (C, C–4), 162.0 (C, C–2), 157.7 (C, C–4′), 131.5 (CH, C–6), 131.1 (CH, C–2′/C–6′), 126.4 (C, C–1′), 116.5 (C, C–5), 115.7 (CH, C–2′/C–5′), 110.6 (C, C–3), 45.8 (CH, C–8), 45.4 (CH₂, C–9), 45.4 (CH₂, C–11), 38.6 (CH, C–12), 34.4 (CH, C–8), 27.8 (CH, C–10), 23.0 (CH₃, C–16), 21.9 (CH₃, C–15), 20.8 (CH₃, C–17), and 14.6 (CH₃, C–14) ppm, signals of CH₂–9 and CH₂–11 might be interchanged; ¹³C chemical shifts were extracted from HMBC data; HR-ESIMS: m/z 354.2068 [M + H]⁺ (calculated for C₂₂H₂₈NO₃, 354.2064).

Citreoviridine (3): yellow oil; HR-ESIMS: m/z 403.2113 [M + H]⁺ (calculated for C₂₃H₃₁O₆ 403.2115) (13).

Vancouverone A (4): yellow amorphous solid; ¹H NMR (acetone-d₆, 700 MHz): δ_H 7.36 (5′–H/9′–H), 6.84 (6′–H/8′–H), 5.14 (9–H), 4.21 (4–H), 2.02 (7–H_a), 1.74 (5–H_a), 1.67 (7–H_b), 1.60 (6–H),1.54 (10–H₃), 1.51 (13–H₃), 0.95 (11–H₃), 0.88 (5–H_b), 0.78 (12–H₃), ¹³C NMR (acetone-d₆, 175 MHz): δ_C 129.9 (CH, C–5′/C–9′), 119.0 (CH, C–9), 115.6 (CH, C–5′/C–9′), 48.2 (CH₂–7), 41.5 (CH₂–5), 37.1 (CH–4), 28.8 (CH–6), 19.5 (CH₃–12), 18.8 (CH₃–11), 14.9 (CH₃–13), 12.7 (CH₃–10); chemical shifts were extracted from HSQC data; HRESIMS: m/z 370.2015 [M + H]⁺ (calculated for C₂₂H₂₈NO₄, 370.2013). Data are consistent with those of Yokoyama et al. (24).

Vancouverone B (5): yellow amorphous solid; ¹H NMR (methanol-d₄, 700 MHz): δ_H 7.12 (5′–H/9′–H), 6.66 (6′–H/8′–H), 5.14 (9–H), 4.99 (3′–H), 4.23 (3′–OH), 3.65 (4–H), 1.84 (7–H_a), 1.77 (7–H_b), 1.68 (5–H_a), 1.56 (10–H₃), 1.50 (13–H₃), 1.36 (6–H), 0.95 (5–H_b), 0.94 (11–H₃), 0.77 (12–H₃), ¹³C NMR (methanol-d₄, 175 MHz): δ_C 129.5 (CH, C–5′/C–9′), 120.8 (CH, C–9), 115.4 (CH, C–5′/C–9′), 74.7 (CH, C–3′), 49.1 (CH₂–7), 41.1 (CH₂–5), 35.1 (CH–4), 29.7 (CH–6), 20.0 (CH₃–12), 18.6 (CH₃–11), 15.4 (CH₃–13), 13.3 (CH₃–10); chemical shifts were extracted from HSQC data; HRESIMS: m/z 388.2121 (calculated for C₂₂H₃₀NO₅, 388.2118); data are consistent with those of Yokoyama et al. (24).

Vancouverone C (6): yellow amorphous solid; [α]²⁰_D = −110 (c = 0.1, MeOH); UV (MeOH, c = 0.02 mg/mL) λmax (log ε) 350 (2.9), 280 (3.9), 200 (4.2) nm; ¹H NMR (methanol-d₄, 700 MHz): δ_H 7.00 (br d, J = 8.4 Hz, 5′–H/9′–H), 6.64 (br d, J = 8.4 Hz, 6′–H/8’–H), 5.15 (q, J = 6.7 Hz, 9–H), 3.89 (m, 2′–H), 3.88 (m, 4–H), 2.99 (dd, J = 13.9, 4.0 Hz, 3′–H_a), 2.79 (m, 3′–H_b), 1.96 (dd, J = 12.6, 6.0 Hz, 7′–H_a), 1.72 (m, 7′–H_b), 1.70 (m, 5′–H_a), 1.56 (d, J = 6.7 Hz, 10–H₃), 1.52 (s, 13–H₃), 1.48 (m, 6–H), 0.99 (d, J = 6.7 Hz, 11–H₃), 0.91 (dd, J = 12.6, 6.8 Hz, 5–H_b), 0.76 (d, J = 6.6 Hz, 12–H₃), ¹³C NMR (methanol-d₄, 175 MHz): δ_C 131.6 (CH, C–5′/C–9′), 120.4 (CH, C–9), 115.8 (CH, C–5′/C–9′), 62.8 (CH, C–2′), 49.2 (CH₂–7), 41.8 (CH₂–5), 40.2 (CH–4), 37.9 (CH₂–3′), 29.8 (CH–6), 20.0 (CH₃–12), 18.8 (CH₃–11), 15.2 (CH₃–13), and 13.3 (CH₃–10); chemical shifts were extracted from HSQC data; HR-ESIMS: m/z 372.2167 [M + H]⁺ (calculated for C₂₂H₃₀NO₄, 372.2169).

Penidiamide (7): colorless oil; ¹H NMR (pyridine-d₅, 700 MHz): δ_H 12.16 (br s, 9–NH), 11.13 (br d, J = 9.9 Hz, 1–NH), 9.52 (m, 2′–NH), 8.21 (dd, J = 14.8, 9.9 Hz, 1–H), 8.09 (d, J = 8.0 Hz, 5–H), 8.03 (d, J = 7.8 Hz, 7″–H), 7.57 (m, 8–H), 7.48 (d, J = 2.3 Hz, 10–H), 7.28 (m, 7–H), 7.25 (m, 5″–H), 7.22 (m, 6–H), 7.00 (br d, J = 8.2 Hz, 4″–H), 6.82 (br d, J = 14.8 Hz, 2–H), 6.64 (m, 2–H), 4.60 (m, 2′–H₂); ¹³C NMR (pyridine-d₅, 175 MHz): δ_C 171.3 (C, C–1″), 168.3 (C, C–1′), 151.5 (C, C–3″), 138.6 (C, C–9), 133.0 (CH, C–5″), 129.4 (CH, C–7″), 126.8 (C, C–4), 124.5 (CH, C–10), 122.7 (CH, C–7), 121.8 (CH, C–1), 120.6 (CH, C–5), 120.5 (CH, C–6), 117.7 (CH, C–4″), 116.3 (C, C–2″), 116.0 (CH, C–6″), 113.8 (C, C–3), 112.8 (CH, C–8), 107.5 (CH, C–2), and 44.3 (CH₂, C–2′) ppm. HR-ESIMS: m/z 335.1500 [M + H]⁺ (calculated for C₁₉H₁₉N₄O₂ 335.1503); data are consistent with those of Witter et al. (25).

Antimicrobial activity assay

The minimum inhibitory concentrations of compounds 1, 5, and 6 were determined in a serial dilution assay as described previously (26) and were carried out in 96-well microtiter plates (There were not ample quantities of compounds 2, 3, 4, and 7 for antimicrobial and cytotoxicity testing.). Various test organisms were used: Bacteria: Bacillus subtilis (DSM 10), Staphylococcus aureus (DSM 346), Acinetobacter baumannii (DSM 30008), Chromobacterium violaceum (DSM 30191), Escherichia coli (DSM 1116), Pseudomonas aeruginosa (PA14), Mycolicibacterium smegmatis (ATCC 700084); Fungi: Candida albicans (DSM1665), Schizosaccharomyces pombe (DSM 70572), Mucor hiemalis (DSM 2656), Pichia anomala (DSM 6766), and Rhodotorula glutinis (DSM 10134). In these assays, oxytetracycline, gentamycin, ciprofloxacin, and kanamycin were used as the positive controls against bacterial pathogens, while nystatin was used as the positive control against fungi.

Cytotoxicity assay

In vitro cytotoxicity of compounds 1, 5, and 6 was assessed using the 3-(4,5-dimethylthiayol-2-yl)−2,5-diphenyltetrazolium bromide test in 96-well microtiter plates as described previously (26) against several mammalian cell lines (endocervical adenocarcinoma KB3.1, mouse fibroblast L929, human lung carcinoma A549, squamous cell carcinoma A431, human prostate cancer PC-3, human breast adenocarcinoma MCF-7, and ovarian carcinoma SKOV-3). The compounds were first tested using two sensitive cell lines L929 and KB3.1, and subsequently, the active compounds were tested with the other five cell lines. Epothilone B was used as the positive control.

Antifungal activity assay

The test was performed in 9 cm diameter petri dishes as described previously (7), using two-layered agar-based potato-dextrose medium (potato extract dextrose agar; Carl Roth GmbH & Co. KG, Karlsruhe, Germany): a solid lower layer (20 mL of medium containing 2% agar, pH: 5.6) and a soft upper layer (6 mL of medium containing 1% agar, pH: 5.6). The latter was inoculated with 2 mL of the homogenized liquid pre-culture of H. fraxineus DSM 117123 before being poured onto the dried lower agar layer. The compounds were dissolved in MeOH at a concentration of 5 mg/mL, and 10 µL of the solutions was pipetted onto sterile filter discs (diameter 6 mm). Three sterile filter paper discs per plate were gently placed on the dried potato-dextrose agar upper layer. The fungicide nystatin was applied as a positive control (20 µg/paper disc) and methanol as a negative control (10 µL/paper disc). The antifungal activity of the isolated compounds was evaluated compared to nystatin after 7 days of incubation at 21°C.

Phytotoxicity assay

The phytotoxicity of the isolated compounds was determined by the leaf puncture assay (27, 28). The compounds were applied to similar leaves from the same F. excelsior plant. The compounds were dissolved in MeOH in distilled water (final concentration of MeOH, 4%) at a concentration of 1 mg/mL. After the leaves were punctured with a needle, 20 µL of test samples were applied to their adaxial sides. As a negative control, 20 µL of MeOH in distilled water (final concentration of MeOH, 4%) was applied to the leaves, and 1 mg/mL macrocidin A (29) was applied as a positive control. Each treatment was repeated three times, after which the leaves were placed on moistened paper filters in 9 cm petri dishes to prevent the droplets from drying. After 7 days, leaves were evaluated for symptoms. The software GNU Image Manipulation Program was used to measure the symptoms.

DATA AVAILABILITY

Colony morphology on CYA and exolite production of DSM 104493 are shown in Fig. S1 and S2, respectively. ITS and β-tubulin sequence data are available through the NCBI Nucleotide database (GenBank accession: OR996288 for ITS and GenBank accession PP182370 for β-tubulin). Processed MS and NMR spectra of compounds 1–7 are depicted in the supplemental material; raw data files are available from the corresponding author, F.S., upon request.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/aem.00665-24.

Supplemental material. aem.00665-24-s0001.pdf.

Figures S1 to S30; Tables S1 and S2.

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