New nematicidal and antimicrobial secondary metabolites from a new species in the new genus, Pseudobambusicola thailandica

Abstract Abstract

During the course of a study on the functional biodiversity of the mycobiota inhabiting rainforests in Thailand, a fungal strain was isolated from a plant sample and shown to represent an undescribed species, as inferred from a combination of morphological and molecular phylogenetic methods. Molecular phylogenetic analyses, based on four DNA loci, revealed a phylogenetic tree with the newly generated sequences clustering in a separate branch, together with members of the Sulcatisporaceae (Pleosporales, Ascomycota). The Thai specimen morphologically resembled Neobambusicola strelitziae in having pycnidial conidiomata with phialidic conidiogenous cells that produce both fusoid-ellipsoid macroconidia and subcylindrical microconidia. However, the new fungus, for which the name Pseudobambusicola thailandica is proposed, differs from N. strelitziae in having conidiomata with well-defined necks, the presence of globose to subglobose thick-walled cells adjacent to conidiomata and the production of chlamydospores in culture. When cultures of P. thailandica, growing on water agar, were confronted with Caenorhabditis elegans nematodes, worms approaching the fungal mycelia were killed. This observation gave rise to a study of its secondary metabolites and six novel and two known compounds were isolated from submerged cultures of P. thailandica. The structures of metabolites 1–6, for which the trivial names thailanones A–F are proposed, were elucidated using a combination of spectral methods, including extensive 1 and 2D NMR analysis and high resolution mass spectrometry. Compounds 4 and 8 showed strong nematicidal and weak antifungal activity, whereas all other tested compounds showed moderate to weak nematicidal activity but no significant effects in the serial dilution assay against various fungi and bacteria. Compounds 1 and 8 also inhibited growth of the pathogenic basidiomycete Phellinus tremulae in a plate diffusion assay.

Introduction

Fungi are regarded as prolific sources of secondary metabolites with prominent and selective biological activities that can serve as a basis for development of new antimicrobials, agrochemical pesticides and other useful compounds (Bills and Gloer 2016, Karwehl and Stadler 2017). In particular, the mycobiota of tropical countries are widely unexplored and can still yield a plethora of novel chemical entities. In recent years, many novel compounds with, for example, antimicrobial (Helaly et al. 2016, 2017, Richter et al. 2016, Kuephadungphan et al. 2017), cytotoxic (Surup et al. 2017) and antioxidative (Kuhnert et al. 2015) effects were isolated in the authors’ laboratory from tropical fungi. Furthermore, fungi represent a rich source of nematicidal compounds because they are both prey and natural antagonists of nematodes. Thus, understanding the chemical basis for fungi nematode interactions offers natural biocontrol strategies (Anke et al. 1995). According to Degenkolb and Vilcinskas (2016), approximately 700 species of nematophagous fungi have been described so far and four ecophysiological categories have been proposed. However, little has been done to screen for metabolites in nematophagous fungi or, for that measure, nematicidal metabolites in other fungi since the first studies of this kind during the 1990s (Stadler et al. 1993a, b, 1994).

Environmentally compatible and low-cost alternatives to chemical control measures for phytoparasitic nematodes are urgently needed and these must not affect vertebrates, crops and other non-target organisms. Highly specific, preferably soil-borne antagonists are best suited for this purpose (Degenkolb and Vilcinskas 2016).

In this context, fungi isolated from nature were examined for morphological features and by ITS sequencing. The strains that turned out to belong to well-studied, ubiquitous mycotoxin-producing genera (in particular Trichocomaceae and Hypocreaeae) were discarded. Those strains that belong to less studied phylogenetic lineages were selected for studies of their antagonistic activities. They were first tested using a water agar assay to detect nematicidal effects and, in parallel, extracts were prepared and checked in an agar plate diffusion assay for antifungal and nematicidal activities. Herein, the authors report the discovery of a new genus and species Pseudobambusicola thailandica and its six novel and two known secondary metabolites, including their isolation, structure elucidation and biological activity.

Materials and methods

Fungal isolation

During a fungal exploration in Thailand in 2015, an unrecognised fungus was found growing on a twig of an unidentified plant. The twig was incubated in a damp chamber and treated according to Castañeda-Ruiz et al. (2016). Single conidial isolates were established from sporulating conidiomata in Petri dishes containing water agar (WA; Difco agar 5 g, tap water 1 l). Colonies were sub-cultured on potato carrot agar (PCA; potatoes 20 g; carrots 20 g; agar 20 g; distilled water 1 l) and oatmeal agar (OA; oatmeal 30 g; agar 18 g; distilled water 1 l) as described previously (Hernández-Restrepo et al. 2017). Herbarium type material and the ex-type strain are maintained at the BIOTEC Bangkok herbarium (BBH) and at the BIOTEC culture collection (BCC; both Pathum Thani, Thailand), respectively.

Morphology

Morphological features were characterised from colonies growing on OA or on synthetic nutrient-poor agar (SNA; Nirenberg 1976) supplemented by fragments of autoclaved pine needles and incubated at 25 °C under continuous near-ultraviolet light to promote sporulation. Colony colours were assessed according to the charts of Rayner (1970). Micromorphological descriptions and measurements for 30 replicates of relevant features were carried out from mature conidiomata and conidia mounted in lactic acid 90%. Photomicrographs were made following Hernández-Restrepo et al. (2017).

DNA isolation, amplification and sequences analyses

Genomic DNA was extracted from fungal colonies growing on MEA using the Wizard® Genomic DNA purification kit (Promega, Madison, USA) following the manufacturer’s protocols. The nuclear rDNA operon spanning the 3’ end of the 18S nrRNA gene, the first internal transcribed spacer (ITS1), the 5.8S nrRNA gene, the second ITS region (ITS2) and approximately 900 bp of the 5’ end of the large subunit of the nrRNA gene (LSU), part of the RNA polymerase II second largest subunit gene (rpb2) and part of the translation elongation factor 1-α gene (tef1) were amplified following Hernández-Restrepo et al. (2016). The programme SeqMan Pro v. 10.0.1 (DNASTAR, Madison, WI, USA) was used to obtain consensus sequences for each DNA region. Blast searches using ITS and LSU sequences were performed and the closest matches and related taxa were retrieved from GenBank and included in the phylogenetic analyses (Table 1, See Suppl. material 1). Alignments were produced with MAFFT v. 7 (Katoh and Standley 2013), checked and refined using MEGA v. 6 (Tamura et al. 2013) and SequenceMatrix (Vaidya et al. 2011). Individual alignments for each locus and the concatenated four-loci dataset were analysed by maximum likelihood (ML) with gamma model of rate heterogeneity using the RAxML HPC BlackBox v. 8.2.8 (Stamatakis 2014) online server of the Cipres Science gateway portal (Miller et al. 2010). The maximum likelihood search option was used to search for the best-scoring tree after bootstrapping. By default, the RAxML BlackBox calculates statistical support for branches by rapid bootstrap analyses of 1000 replicates (Stamatakis 2014). Bootstrap support (bs) values ≥ 70 % were considered significant. Incongruence amongst datasets was tested by visual inspection of all groups with ≥ 70 % bs in partial trees of each locus to search for potentially conflicting groups. A Markov Chain Monte Carlo (MCMC) algorithm was used to generate phylogenetic trees with Bayesian probabilities from the concatenated four-loci dataset using MrBayes v. 3.2.6 (Ronquist et al. 2012). Two analyses of four MCMC chains were run from random trees, trees were sampled every 100 generations and 25 % of them were discarded as the burn-in phase. Posterior probabilities (pp) were determined from the remaining trees. The sequences generated during this study and the alignments used in the phylogenetic analyses were deposited in GenBank and TreeBASE, respectively.

Table 1.

Isolates and GenBank accession numbers used in the phylogenetic analyses.

Taxa	Strain number1	GenBank accession numbers2				References
		ITS	LSU	rpb2	tef1
Alternaria tenuissima	CBS 918.96	–	KC584311	KC584435	KC584693	Woudenberg et al. 2013
Bambusicola didymospora	MFLUCC 10-0557	KU940116	KU863105	KU940163	KU940188	Dai et al. 2017
B. loculata	MFLU 15-0056	KP761732	KP761729	KP761715	KP761724	Dai et al. 2015
B. pustulata	MFLUCC 15-0190	KU940118	KU863107	KU940165	KU940190	Dai et al. 2017
B. splendida	MFLUCC 11-0611	KU940121	KU863110	KU940168	–	Dai et al. 2017
Coniothyrium palmicola	CBS 161.37	JX681086	JX681086	–	–	Verkley et al. 2014
Dendrothyrium longisporum	CBS 824.84	JX496115	JX496228	–	–	Verkley et al. 2014
Dydimella exigua	CBS 183.55	NR135936	EU754155	GU357800	KR184187	De Gruyter et al. 2009, Schoch et al. 2009, Kim et al. 2016
Keissleriella culmifida	KT 2308	–	AB807591	–	AB808570	Tanaka et al. 2015
K. quadriseptata	KT 2292	NR145135	AB807593	–	AB808572	Tanaka et al. 2015
Latorua caligans	CBS 576.65	NR132923	KR873266	–	–	Crous et al. 2015a
Leptosphaeria doliolum	CBS 505.75	JF740205	GQ387576	KY064035	GU349069	De Gruyter et al. 2013, Schoch et al. 2009
Lophiostoma arundinis	AFTOL-ID 1606	–	DQ782384	DQ782386	DQ782387	Schoch et al. 2009
Macrodiplodiopsis desmazieri	CBS 140062	NR132924	KR873272	–	–	Crous et al. 2015a
Magnicamarosporium iriomotense	KT 2822	AB809640	AB807509	–	AB808485	Tanaka et al. 2015
Massarina phragmiticola	CBS 110446	–	DQ813510	–	–	Kodsueb et al. 2007
Montagnula bellevaliae	MFLUCC 14-0924	KT443906	KT443902	–	–	Hongsanan et al. 2015
M. scabiosae	MFLUCC 14-0954	KT443907	KT443903	–	–	Hongsanan et al. 2015
Murilentithecium clematidis	MFLUCC 14-0562	KM408757	KM408759	KM454447	KM454445	Wanasinghe et al. 2014
Neobambusicola strelitziae	CBS 138869	NR 137945	KP004495	–	MG976037	Crous et al. 2014, this study
Palmiascoma gregariascomum	MFLUCC 11-0175	KP744452	KP744495	KP998466	–	Liu et al. 2015
Parabambusicola bambusina	H 4321	–	AB807536	–	AB808511	Tanaka et al. 2015
Paraconiothyrium brasiliense	CBS 122851	JX496036	JX496149	–	–	Verkley et al. 2014
Phoma herbarum	CBS 615.75	NR135967	EU754186	KP330420	KR184186	Aveskamp et al. 2009, Chen et al. 2015
Pleurophoma ossicola	CPC 24985	KR476737	KR476770	–	–	Crous et al. 2015b
Polyschema congolensis	CBS 542.73	–	EF204502	EF204486	–	Shenoy et al. 2010
P. terricola	CBS 301.65	–	EF204504	EF204487	–	Shenoy et al. 2010
Pseudobambusicola thailandica sp. nov.	BCC 79462	MG926559	MG926560	MG926561	MG926562	This study
Pseudoleptosphaeria etheridgei	CBS 125980	NR111620	JF740291	–	–	De Gruyter et al. 2013
Pseudoxylomyces elegans	KT 2887	–	AB807598	–	AB808576	Tanaka et al. 2015
Setoseptoria arundinacea	KT 552	–	AB807574	–	AB808550	Tanaka et al. 2015
Stemphylium vesicarium	CBS 191.86	KC584239	JX681120	KC584471	KC584731	Woudenberg et al. 2013
Sulcatispora acerina	KT 2982	LC014597	LC014610	–	LC014615	Tanaka et al. 2015
S. berchemiae	KT 1607	AB809635	AB807534	–	AB808509	Tanaka et al. 2015
Trematosphaeria pertusa	CBS 122368	NR132040	FJ201990	FJ795476	KF015701	Zhang et al. 2009

¹ BCC: BIOTEC Culture Collection, Thailand; CBS: Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; CPC: Culture collection of Pedro Crous, housed at CBS; KT and H: Culture collection of K. Tanaka and K. Hirayama, housed at the National Institute of Agrobiological Science, Japan (MAFF); MFLU: Mae Fah Laung University Herbarium, Chiang Rai, Thailand; MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand.

² ITS: internal transcribed spacer regions 1 & 2 including 5.8S nrRNA gene; LSU: large subunit of the nrRNA gene, rpb2: partial RNA polymerase II second largest subunit gene; tef1: partial translation elongation factor 1-α gene. Sequences generated in the present study are in bold.

Chromatography and spectral methods

1D and 2D nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance III 700 spectrometer with a 5 mm TXI cryoprobe (¹H 700 MHz, ¹³C 175 MHz) and a Bruker Avance III 500 (¹H 500 MHz, ¹³C 125 MHz) spectrometer, UV spectra were recorded with a Shimadzu UV-2450 UV−Vis spectrophotometer and optical rotations were measured on a Perkin-Elmer 241 polarimeter. Analytical HPLC was carried out on an Agilent 1200 Series, equipped with degasser, binary pump SL, autosampler and connected to a diode array detection/light scattering detector Corona Ultra RS. A Waters C18 Acquity UPLC BEH column (2.1 × 50 mm, 1.7 μm) was used as stationary phase. The mobile phase consisted of H₂O + 0.1% formic acid (solvent A) and acetonitrile + 0.1% formic acid (solvent B) with the following gradient: 0–0.5 min 5% B, 0.5–20 min 100% B, 20–30 min 100% B; injection volume was 2 µl, flow rate 600 µl/min.

HPLC-ESI-MS spectra were recorded on an ion trap mass spectrometer [scan range 100–2000 m/z, capillary voltage 4000 V, dry temperature 250 °C] (amaZon speed, Bruker) and HR-ESIMS spectra on a time-of-flight (TOF) MS [scan range 250–25000 m/z, capillary voltage 4500 V, dry temperature 200 °C] (MaXis, Bruker). In parallel, UV/Vis spectra in the range of 200–600 nm were recorded.

Chemicals and solvents were obtained from AppliChem GmbH (Darmstadt, Germany), Avantor Performance Materials (Deventer, Netherlands), Carl Roth GmbH & Co. KG (Karlsruhe, Germany) and Merck KGaA (Darmstadt, Germany) in analytical and HPLC grade.

Fermentation and extraction

A seed culture was prepared as follows: five mycelial plugs (0.5 × 0.5 cm²) were cut from actively growing colonies maintained on YM 6.3 agar (malt extract 10 g/l, D-glucose 4 g/l, yeast extract 4 g/l, agar 20 g/l, pH 6.3 before autoclaving) and placed into a 500 mL Erlenmeyer flask containing 200 mL Q6½ medium (D-glucose 2.5 g/l, glycerol 10 g/l, cotton seed flour 5 g/l, pH 6.3) and incubated on a rotary shaker for 96 hours at 24 °C and 140 rpm. 20 mL of the seed culture were added into 10 × 1000 ml sterile Erlenmeyer flasks with 500 ml of Q6 ½ medium (5 l total) and incubated on a rotary shaker (288 hours, 24 °C, 140 rpm).

Biomass and supernatant were separated by means of centrifugation and filtration. The mycelia were extracted twice with acetone (2 l), the extract was evaporated in vacuo and the remaining aqueous phase extracted with equal amounts of ethyl acetate three times. One percent (1 %) of Amberlite XAD-16N was given to the culture broth and stirred for 1 h. After filtration, the XAD resin was extracted as described above. 220 mg and 88 mg of mycelial and supernatant crude extracts were obtained, respectively.

Isolation of the compounds 1–8

The supernatant crude extract was fractionated on preparative HPLC (Gilson GX270 Series HPLC system). The reversed phase C18 column (Nucleodur 150/40, 10 µm, 110 Å; with a precolumn VP 100/10; Macherey-Nagel) was used as a stationary phase and the mobile phase was composed of deionised water + TFA 0.05 % (Milli-Q, Millipore, Schwalbach, Germany; solvent A) and acetonitrile (ACN) + TFA 0.05 % (solvent B). The fractionation was accomplished with the following gradient: 15 % of B isocratic for 5 min, followed by a linear increase to 80 % B over 30 min, afterwards increasing to 100% B in 5 min and thereafter isocratic conditions at 100 % for 5 min. In total, 7 compounds were obtained from the supernatant crude extract: Compound 1 (thailanone A; 1 mg) was obtained at the retention time t_R = 6 min, compound 2 (thailanone B; 1 mg) at t_R = 4.3 min, compound 3 (thailanone C; 1.3 mg) at t_R = 6.4 min, compound 4 (thailanone D; 1 mg) at t_R = 8.1 min; compound 5 (thailanone E; 4.2 mg) at t_R = 8.2, compound 6 (thailanone F; 1.6 mg) at t_R = 8.6 min) and compound 7; monocerin (7.8 mg) at t_R = 9.1 min. The mycelial crude extract was chromatographed in a similar manner as described above, yielding 77.8 mg of deoxyphomalone (8, t_R = 11.2 min) but none of the other compounds.

Evaluation of antimicrobial activities

Minimum inhibitory concentrations (MIC) of compounds 1–8 were determined in serial dilution assays against Bacillus subtillis DSM10, Mucor plumbeus MUCL 49355 and Candida tenuis MUCL 29892 as described previously by Chepkirui et al. (2016). The assays were carried out in 96-well microtiter plates in YMG (yeast-malt-glucose) medium for filamentous fungi and yeasts and MH (Müller-Hinton) medium for the bacterium. For all tested compounds, the starting concentration was 100 µg/mL and final 0.78 µg/mL.

Water agar plate assay

The fungal cultures were tested in the water agar plate assay against Caenorhabditis elegans nematodes (wild type strain, see Ashrafi et al. 2017), in a similar manner as previously described by Stadler et al. (1994). After 3–7 days, nematicidal effects became visible by many dead and immotile nematodes in the vicinity of the mycelia. Fungal colonies exhibiting toxic effects were selected for submerged cultivation and production/isolation of nematicidal compounds.

Microtiter plate assay for nematicidal activities

The nematicidal activity against C. elegans of all isolated compounds was determined by a slightly modified method (Stadler et al. 1994, Kuephadungphan et al. 2017 and Ashrafi et al. 2017). C. elegans was inoculated monoxenically on nematode agar (soy peptone 2 g/l, NaCl 1 g/l, agar-agar 20 g/l) and, after autoclaving, the following ingredients were added as sterile filtered solutions: cholesterol (1 mg/mL dissolved in EtOH) 0.5 ml, 1M CaCl₂ 1 mll, 1M MgSO₄ 1 ml, 40 mM potassium phosphate buffer 12.5 ml; pH 6.8) with living Escherichia coli DSM498 (1 ml of a suspension containing approximately 10 cells/ml, pre-inoculated for 12 h at 37 °C) and the plates were incubated at 21 °C for 4–5 days. Thereafter, nematodes were washed down from the plates with M9 buffer (3 g KH₂PO₄, 6 g Na₂HPO₄, 5 g NaCl and, after autoclaving, the addition of 1 ml 1 M MgSO₄). Finally, a nematode suspension of approximately 500 nematodes/ml in M9 buffer was prepared and used in the microtiter plate assay.

The assay was performed in 24-well microtiter plates at four concentrations (100, 50, 25 and 12.5 µg/ml) for each compound. Ivermectin was used as a positive control, while methanol was used as a negative control. The plates were incubated at 20 °C in the dark and nematicidal activity was recorded after 18 h of incubation and expressed as LD₉₀ (i.e. concentration causing over 90 % immobility of the nematodes).

Antifungal activity assay against Phellinus tremulae

Growth inhibition of Phellinus tremulae CBS 123.40 for compounds 1–8 was tested according to the modified protocol published by Ayer and Jimenez (1994). The assay was performed in 24-well microtiter plates where 1 mL of YM agar was added in each well and thereafter the compounds were dissolved in methanol (100, 50, 25 and 12.5 µg/ml) and added to the wells. Shortly after the media solidified, 0.5 × 0.5 mm² agar plugs of actively growing colonies of Ph. tremulae CBS 123.40, grown on a YM 6.3 agar plate, were placed in each well of the microtiter plate. Nystatin and methanol were used as positive and negative controls, respectively, together with control wells without additives. Inhibition of the radial growth of the colonies of Ph. tremulae CBS 123.40 relative to the control was recorded as a positive result. The radial growth was measured after 3, 5, 7 and 9 d.

Phytotoxic activity assay

Phytotoxic activities were carried out by germination and seedling growth bioassay against Setaria italica and Lepidum sativum according to the protocol from Anke et al. (1989). The amount of 100 µg/paper disc of compound was tested; as a positive control herbicide methyl vilogen dichloride hydrate was used. The negative controls were the seeds only and the solvent alone (the one used for dissolving the compounds).

Results and discussion

Molecular phylogenetic analysis

The combined dataset consisted of 35 taxa with 3126 characters of which 396 bp corresponded to ITS, 853 bp to LSU, 904 bp to rpb2 and 973 bp to tef1. The alignment had 100% representation for LSU, 74% for ITS, 46% for rpb2 and 57% for tef1. The phylogenetic tree (Fig. 1) shows two fully supported main clades, corresponding to the sub-orders Massarineae and Pleosporineae (Pleosporales, Dothideomycetes). In the Massarineae, eleven clades representing families are shown, i.e., Bambusicolaceae (96%, 1 pp), Coniotyriaceae (100%, 1 pp), Didymosphaeriaceae, Latoruaceae (100%, 1 pp), Lentitheciaceae (97%, 1 pp), Macrodiplodiopsidaceae, Massarinaceae (100%, 1 pp), Montagnulaceae (100%, 1 pp), Parabambusicolaceae, Sulcatisporaceae (100%, 1 pp) and Trematosphaeriaceae and an additional subclade comprising Pseudoxylomyces elegans. In the phylogenetic tree (Fig. 1), the sequence data of the new species indicate a systematic position in an independent branch in the Sulcatisporaceae close to Magnicamarosporium eriomotense without any support.

Figure 1.

Phylogenetic tree (RAxML) inferred from the DNA sequence data of four loci (ITS, LSU, tef1 and rpb2) of Pseudobambusicola thailandica and related species in Pleosporales (Dothideomycetes). The new taxon is indicated in bold. Taxa reported to produce deoxyphomalone are indicated by an underlined. Maximum likelihood bootstrap values ≥ 70 % and Bayesian posterior probabilities ≥ 0.95 are shown at the nodes and the scale bar indicates the number of expected mutations per site. Clades with 100 BML and 1 PP are indicated by thickened lines . The tree was rooted to Lophiostoma arundinis (AFTOL-ID 1606). ^T = ex-type strain; ^ET = epitype strain.

Taxonomy

Pseudobambusicola

Hern.-Restr. & Crous gen. nov.

MB824299

Etymology.

The name reflects its morphological similarity of the type species to the asexual morphs of Bambusicola and Neobambusicola.

Type species.

Pseudobambusicola thailandica Hern.-Restr. & Crous.

Diagnosis.

Differs from Neobambusicola in having conidiomata with a neck, the presence of globose to subglobose thick-walled cells adjacent to the conidiomata and the production of chlamydospores in culture.

Mycelium composed of hyaline to pale brown, septate, smooth to slightly verruculose, hyphae. Conidiomata pycnidial, semi- or entirely immersed in the agar, solitary or aggregated, erumpent, globose with a neck, opening via central ostiole, dark brown, surrounded by dark brown, smooth to slightly verruculose hyphae, at the base globose to subglobose, thick-walled cells often present. Conidiophores reduced to conidiogenous cells. Conidiogenous cells phialidic with periclinal thickening at the conidiogenous locus, subcylindrical to ampulliform, hyaline, smooth. Conidia exposed in white, mucous drops at the ostioles of the pycnidia, composed by macro- and microconidia. Macroconidia produced in white, mucous heads, solitary, fusoid-ellipsoid, apex bluntly to subobtusely rounded, tapering to a distinctly truncate base, prominently guttulate, hyaline, smooth, 0–3-septate. Microconidia produced in the same pycnidia as macroconidia, solitary, oblong to cuneiform, non-guttulate to slightly guttulate, hyaline, smooth, aseptate. Chlamydospores brown, terminal at the tips of vegetative hyphae, in chains. Sexual morph not observed.

Pseudobambusicola thailandica

Hern.-Restr. & Crous sp. nov.

MB824300

Etymology.

The epithet refers to Thailand, where this species was collected.

Type.

THAILAND. Lop Buri Province: Chai Badan, Wang Kan Lueang Arboretum, Wang Kan Lueang Waterfall, on twig (unidentified), 14 Jul 2015, M. Hernández-Restrepo, MHR 1534 (holotype: BBH 42022!, culture ex-type BCC 79462!).

Description of fungal structures on SNA.

Mycelium composed by hyaline to pale brown, septate, smooth to slightly verruculose, hyphae, 1–2.5 µm wide. Conidiomata pycnidial, semi- or entirely immersed in the agar, solitary or aggregated, erumpent, globose, sometimes with a neck, opening via central ostiole, dark brown, 63–360 µm diam., sometimes with a cylindrical neck 50–125 × 40–50 µm, opening via central ostiole; at the base of the conidiomata are often present globose to subglobose cells, thick-walled, 5–9 µm wide; conidiomata surrounded by dark brown, smooth to slightly verruculose hyphae, 2–2.5 µm wide. Conidiophores reduced to conidiogenous cells. Conidiogenous cells phialidic with periclinal thickening, subcylindrical to ampulliform, hyaline, smooth, 6.5–7 × 2.5–4 µm. Conidia exposed in white, mucous drops at the ostiole of pycnidia, composed by macro- and microconidia. Macroconidia produced in white, mucous heads, solitary, fusoid-ellipsoid, apex bluntly to subobtusely rounded, tapering to a distinctly truncate base, mostly straight, but sometimes slightly curved, prominently guttulate, hyaline, smooth, 0–3-septate, 10–20 × 2–4(–6) µm. Microconidia produced in the same pycnidia with macroconidia, solitary, oblong to cuneiform, non-guttulate to slightly guttulate, hyaline, smooth, aseptate, 2–4(–5.5) × 1–2 µm, apex rounded, base truncate. Chlamydospores brown, terminal, in chains, 16–38 × 5–6 µm. Sexual morph not observed.

Culture characteristics.

Colonies on OA at 25 °C reaching 24 mm diam. in 2 weeks, elevated, with dense cottony mycelium at the centre, mouse grey, margin whitish, effuse to fimbriate; reverse dark mouse grey.

Notes.

Pseudobambusicola is introduced here for a pycnidial coelomycete producing two kinds of conidia. Morphologically, it is similar to the species of Bambusicola and Neobambusicola. However, asexual morphs in Bambusicola are characterised by brown or pale brown conidia and annellidic rather than phialidic conidiogenous cells and hyaline conidia as in Pseudobambusicola (Dai et al. 2012, 2017). Neobambusicola is a monotypic genus erected for N. strelitziae, first described from South Africa growing on necrotic leaf tissue associated with Phyllachora strelitziae (Phyllachoraceae, Phyllachorales, Sordariomycetes) (Crous et al. 2014). Both genera are similar in having pycnidial conidiomata and phialidic conidiogenous cells that produce fusoid-ellipsoid macro- and subcylindrical microconidia. However, in the new genus, the conidiomata are surrounded by dark brown, smooth to slightly verruculose hyphae and, in mature conidiomata, a cylindrical neck is often present; furthermore, chlamydospores can be present in culture. Although both genera belong to the Sulcatisporaceae (Pleosporales, Dothideomycetes), they are placed in different clades, Neobambusicola is more closely related to Sulcatispora (100 %, 1 pp), while Pseudobambusicola was placed in a distinct branch with Magnicamarisporium (Fig. 1). Additionally, based on LSU, ITS and tef1 sequences, P. thailandica is 97 % (KP004495) and 83 % (KP004467) and 93 % similar to N. strelitziae, respectively.

Figure 2.

Pseudobambusicola thailandica (BCC 79462) on SNA. A Colony overview B–C Pycnidia D–G Conidiogenous cells H globose to subglobose cells, thick-walled, at the base of the conidiomata I Microconidia J Macroconidia K Chlamydospores. Scale bars: 200 µm (B), 100 µm (C), 10 µm (D, H, I–K), 5 µm (E–G).

Water agar plate assay

Out of 66 fungal strains investigated, 18 exhibited antagonistic activity towards nematodes in the water agar assay. Of those, 3 strains produced compounds with nematicidal activity in submerged culture, while in 5 strains, antimicrobial activity was observed. Extracts from P. thailandica (BCC 79462) submerged fermentation displayed strong activity towards nematodes and were subjected to extensive chromatographic studies as described in the Experimental part.

Structure elucidation of compounds 1–8

Fractionation of the crude extracts obtained from submerged cultures of P. thailandica (BCC 79462) resulted in the identification of six previously undescribed polyketides for which the authors propose the trivial names thailanones A–F (1–6) and two known compounds, monocerin and deoxyphomalone, 7 and 8 (see chemical structures in Fig. 3). The NMR spectroscopic data are compiled in Tables 2 and 3 and the spectra and chromatograms are compiled in the Suppl. material 1.

Figure 3.

Chemical structures of thailanones A–F (1–6), monocerin (7) and deoxyphomalone (8).

Table 2.

NMR spectroscopic data for compounds 1–3 in D₆-acetone (¹H NMR at 700 MHz; ¹³C at 500 MHz).

	1			2			3
No.	13C	DEPT	1H/HSQC	13C	1H/HSQC		DEPT
1	202.6	C		202.6		83.9	C
2	120.2	C		119.0		194.1	C
3	187.9	C		191.2		120.3	C
4	38.9	CH2	2.61 (s),3.29 (s)	43.8	2.41 (s)2.88 (s)	170.1	C
5	86.3	C		84.4		58.3	CH	3.46 (s)
6	15.5	CH2	2.11 (q), J= 7.53 Hz	15.2	2.11 (q), J= 7.53 Hz	16.7	CH2	2.16 (q), J=7.53 Hz
7	12.7	CH3	0.93 (t), 7.53 Hz	12.8	0.93 (t), 7.53 Hz	13.2	CH3	0.78 (t), J=7.53 Hz
8	210.7	C		210.1		57.4	CH3	3.84 (s)
9	40.1	CH2	2.61 (m), 2.74(m)	38.9	2.53 (dt), J=7.10, 17.96 Hz2.68 (m), J=7.10, 17.96 Hz
10	17.8	CH2	1.55 (m)	17.6	1.55 (m)
11	14.0	CH3	0.88 (t), 7.42 Hz	13.9	0.86 (t), 7.31 Hz
12	58.3	CH3	4.05 (s)

Table 3.

NMR spectroscopic data for compounds 4–6 in D₆-acetone (¹H NMR at 700 MHz; ¹³C at 500 MHz).

		4		5			6
No.	13C	DEPT	1H/HSQC	13C	DEPT	1H/HSQC	13C	DEPT	1H/HSQC
1	196.36	C	–	104.2	C		164.3	C
2	106.4	C	–	164.7	C		88.7	CH	5.42(s)
3	190.6	C	–	116.1	C		171.9	C
4	96.0	CH	5.56 (s)	160.7	C		112.3	C
5	177.9	C	–	111.1	CH	6.36(s)	25.0	CH2	2.35 (t), J=7.74 Hz
6	79.1	C	–	146.2	C		32.8	CH2	1.40 (p), J=7.31Hz
7	203.2	C	–	174.7	C		23.6	CH2	1.34 (m),
8	41.5	CH2	2.92 (m)	16.8	CH2	2.64 (q), J=7.53 Hz	14.7	CH3	0.91 (t), J=7.31 Hz
9	19.34	CH2	1.65 (m)	13.7	CH3	1.08 (t), J=7.53 Hz	159.3	C
10	14.25	CH3	0.96 (t), J=7.31Hz	39.3	CH2	2.86 (t), 7.31Hz	17.7	CH3	2.19 (s)
11	36.12	CH2	1.79 (dq), J=7.53, 13.34 1.92 (dq), J=7.53, 13.34	26.0	CH2	1.59 (sext), J=7.31 Hz
12	8.2	CH3	0.79 (t), J=7.53	14.6	CH3	0.93 (t), J=7.31
13	57.7	CH3	3.94 (s)
		OH	4.44 (s)

Compound 1 (thailanone A) was isolated as a white solid from the supernatant with the molecular formula C₁₂H₁₈O₄ and four degrees of unsaturation established from the HRMS data. ¹³C and DEPT NMR data revealed the presence of 12 carbons in the molecule: three methyl groups, four methylene groups and five quaternary carbons (Table 2). ¹H NMR spectra on the other hand revealed the presence of two methyl triplets at δ 0.88 (H-11) and δ 0.93 (H-7) together with a methoxy group singlet resonating at δ 4.05 (H-12).

HMBC correlations of H-4 to C-1/C-2/C-3-C-5/C-6, H-6 to C-1/C-2/C-2/C-7 and H-9 to C-5/C-8/C-10/C-11 indicated the presence of an isohumulone moiety differing in the ring substitution (Fig. 4). Furthermore, HMBC correlations between H-11 to C-10/C-9 and H-7 to C-2/C-6 were observed. These correlations were further supported by the COSY correlations observed between H-10 and H-9/H-11 and H-6 and H-7. The methoxy proton H-12 showed a HMBC correlation to C-3. Therefore, the structure of compound 1 was established as 5-butanoyl-2-ethyl-5-hydroxy-3-methoxycyclopent-2-en-1-one.

Figure 4.

COSY, HMBC and ROESY correlations of 1.

Compound 2 (thailanone B) was obtained from the supernatant as a white solid. From the HR mass spectrum, its molecular formula was deduced as C₁₁H₁₆O₄ with four degrees of unsaturation. Analysis of the ¹H NMR and ¹³C NMR spectra of 2 suggested a closely related structure to that of 1 with the difference being the absence of the methoxy group at C-3. Further, the HMBC and COSY correlations observed were similar to those observed for 1. Hence, the structure was elucidated as (5S)-5-butanoyl-2-ethyl-3,5-dihidroxycyclopent-2-en-1-one.

The white solid compound 3 (thailanone C) with the molecular formula C₈H₁₀O₄ and 4 degrees of unsaturation deduced from HR mass spectrum was isolated from the supernatant. The 1D and 2D NMR data of 3 suggested that the molecule has a closely related structure to 1 with one of the side chains missing. Analysis of the ¹H NMR spectrum indicated the presence of a triplet at δ 0.78 (H-7) and a singlet at δ 3.84 (H-8) for methyl and methoxy groups, respectively. A COSY correlation was observed between H-6/H-7. Further, H-7 exhibited HMBC correlations to C-3/C-6, while H-6 was correlating to C-2/C3/C-4/C-7 in the HMBC spectra. H-5 on the other hand showed HMBC correlations to C-1/C-2/C-3/C-4. The epoxide ring was assigned based on the chemical shifts of C-1 (δ 86.3) and C-5 (δ 58.3) and also the established molecular formula. The methoxy group showed HMBC a correlation to C-4 (δ 170.1). The structure of 3 was established as 3-ethyl-1-hydroxy-4-methoxy-6-oxabicyclo[3.1.0]hex-3-en-2-one.

Compound 4 (thailanone D) was isolated as white solid. The molecular formula C₁₃H₁₈O₅ was deduced from the HRMS data. ¹³C and DEPT NMR data indicated the presence of two methyl groups, a methoxy group, three methylene groups and five quaternary carbons in the molecule (Table 3). The ¹H NMR spectra revealed 2 methyl triplets at δ 0.79 (C-12) and δ 0.96 (C-10). In addition, a methoxy singlet was observed at δ 3.94 (C-13). Networks of COSY correlations were observed between H-9 and H-8/H-10 and H-11 to H-12. In the HMBC spectra, H-4 showed correlations to C-2/C-3/C-5/C-6. Proton H-8 was correlating to C-2/C-7/C-9 /C-10, while the methyl protons H-10 were correlating to C-8/C-9 in the HMBC spectra. Further, H-11 showed HMBC correlations to C-1/C-5/C-6/C-12. The hydroxy group singlet at δ 4.44 showed HMBC correlations to C-1/C-5/C-6/C-11. Cross peaks in the ROESY spectra between the methoxy proton H-13 (δ 3.94) and the aromatic proton H-4 (δ 5.56) were observed. No ROESY correlations were observed between the hydroxy group proton (δ 4.44) and H-11/H-12/H-13. Hence the relative stereochemistry at C-6 can be assigned as S. The structure of compound 4 was elucidated as (6S)-2-butanoyl-6-ethyl-3,6-dihidroxy-5-methoxycyclohexa-2,4-dien-1-one.

The white solid compound 5 (thailanone E) showed the molecular formula C₁₂H₁₆O₄ as deduced from HRMS data. The 1D and 2D NMR data of 5 suggested a closely related structure to 4 with the difference being in the ring substitution: The C-7 to C-10 chain and the carbon resonating δ 79.1 in 4 were missing. Analysis of the COSY spectra revealed correlations of H-8 to H-9 and H-11 to H-10/H-12. HMBC correlations of H-5 to C-1/C-3/C-4/C-10, H-8 to C-2/C-3/C-4/C-9, H-10 to C-1/C-5/C-6/C-11/C-12 and H-12 to C-10/C-11 were observed. Hence, the structure of the compound 5 was elucidated as 3-ethyl-2,4-dihydroxy-6-propylbenzoic acid.

Compound 6 (thailanone F) was obtained from the supernatant as a white solid with the molecular formula C₁₁H₁₈O₄ established from HRMS data. Analysis of the ¹H NMR data revealed a methyl group triplet at δ 0.91 (H-8), a methyl group singlet at δ 2.19 (H-10) and a methoxy singlet at 3.87 (H-11). HMBC correlations of H-2 to C-1/C-3/C-4 and H-10 to C-4/C-9 were recorded. Furthermore, HMBC correlations between H-5 and C-3/C-4/C-6/C-7/C-9, H-6 and C-4/C-5/C-7/C-8, H-7 and C-5/C-6/C-8 and H-8 and C-6/C-7 were observed. These correlations were further supported by the COSY correlations of H-6 to H-5/H-7 and H-7 to H-6/H-8. Cross peaks between H-2 (5.42) and methoxy protons H-11 (δ 3.87) were not observed in the ROESY spectra, indicating that the olefinic bond at position two had E configuration. The olefinic bond between C-4 (δ 112.3) and C-9 (δ 159.3) was assigned E configuration, since H-5 (δ 2.35) and H-10 (δ 2.19) also did not correlate in the ROESY spectra. The structure of the compound 6 was established as (2Z, 4E)-4-(1-hydroxyethylidene)-3-methoxyoct-2-enoic acid.

Monocerin (7) and deoxyphomalone (8) were identified by comparing their NMR and HRMS data with those reported in literature (Aldridge et al. 1970, Ayer and Jimenez 1994, respectively). Monocerin was reported before as a potent herbicide and insecticide against Johnson grass and woolly aphids, respectively (Grove et al. 1979, Robeson et al. 1982), while deoxyphomalone has been reported from other pleosporalean fungi like Alternaria. To the best of the authors’ knowledge, it has not been reported previously from a species of the Sulcatisporaceae.

Biological activity

The results of the biological assays that were performed to detect antibacterial, antifungal and nematicidal activities are summarised in Table 4. Compound 6 was moderately active against M. plumbeus with MIC of 25 µg/ml, while deoxyphomalone (8) exhibited moderate activities against B. subtilis and M. plumbeus with MIC values of 12.5 and 25 µg/ml, respectively. Compounds 1–6 and 8 were also the only compounds with significant activities against M. plumbeus. The results by Ayer and Jimenez (1994), regarding the antifungal activity of phomalone and its deoxy derivative, were also repeated in the serial dilution assay. Compounds 2–7 failed to significantly inhibit growth of Ph. tremulae, whereas the new phomalone derivative 1 showed moderate inhibition and was more weakly active than the known compound 8. No phytotoxic effects in plant germination and seedling growth bioassay with S. italica and L. sativum at 100 μg/paper disk were observed for any of tested compounds.

Table 4.

Biological activities of compounds 1–8.

Compounds	Antimicrobial activity MIC (µg/mL)		Nematicidal activity LD90 (µg/mL)	Antifungal activity (% growth inhibition at ≤ 12.5 µg/mL)
	Bacillus subtillis DSM10	Mucor plumbeus MUCL 49355	Caenorhabditis elegans	Phellinus tremulae CBS 123.40
Thailanone A (1)	≤ 50	–	≤ 50	50
Thailanone B (2)	–	–	≤ 25	28.6
Thailanone C (3)	–	–	≤ 25	31.4
Thailanone D (4)	–	≤ 25	≤ 12.5	28.6
Thailanone E (5)	–	–	≤ 50	25.
Thailanone F (6)	–	–	≤ 25	41.4
Monocerin (7)	–	–	–	28.6
Deoxyphomalone (8)	≤ 12.5	≤ 25	≤ 12.5	50
Standards
Nystatin #	–	≤ 0.782	–	100
Ciprofloxacin ††	≤ 0.78	–	–	–
Ivermectin ‡‡	–	–	≤ 12.5	–
Methanol	–	–	–	–

No activity against Candida tenuis, Setaria italica and Lepidum sativum was observed for any of tested compounds up to concentrations of 100 µg/mL. # Nystatin-antifungal reference; †† Ciprofloxacin-antibacterial reference; ‡‡ Ivermectin-nematicidal reference.

Compound 7 was reported as a potent herbicide and insecticide against Johnson grass and woolly aphids, respectively (Grove et al. 1979, Robeson et al. 1982). It was first isolated from a fungus described as Phoma etheridgei (Hutchison et al. 1994) and recently, 7 was also isolated from Alternaria tenuissima (Pleosporaceae) and the bioactivity was tested against E. coli (Anyanwu and Sorensen 2015). It has previously been reported to be active against the pathogenic basidiomycete Phellinus tremulae, which infects different species of poplar (Ayer and Jimenez 1994). This fungus causes extensive damage to hardwoods in North America and, in Canada, Ph. tremulae seriously reduces the economic value of Populus tremuloides (Trifonov et al. 1992). This prompted the authors to re-evaluate the compound in an antifungal assay against Ph. tremulae, in which all purified metabolites from P. thailandica were tested.

Moreover, the phytotoxic activity of terrein and congeners on plant growth and induction of lesions on fruit surfaces were previously investigated by Zaehle et al. (2014). Terrein, a major metabolite of Aspergillus terreus, resembles 1 and 7 in its chemical structure. The authors therefore performed a phytotoxicity assay, but they did not find significant effects of these compounds on germination and shoot/root elongation.

Conclusion

In the course of this investigation of the fungal specimens collected in the rainforest of Thailand, several nematode-antagonistic strains were detected. The use of nematodes as test organisms can detect bioactivity from the compounds that are not detected by whole-cell-based screening for antimicrobial activities. As an outcome of the antihelmintic screening, six new compounds (thailanones 1–6) and two known compounds, deoxyphomalone (7) and monocerin (8) were isolated and further evaluated regarding their antifungal activity. Even though these results are just preliminary and the biological activities of the new compounds are rather moderate, they are very likely to play an important chemo-ecological role in the natural habitat of the fungal producer organisms, e.g. to protect against nematode predation. The authors have not yet tried to detect the metabolites on water agar in the presence of nematodes because of the experimental limitations that would first need to be overcome. The moderate activity of the new compounds (as compared to, for example, the standard ivermectin, which is at least ten times more active) probably precludes their adoption as a nematicidal agent that could serve as a candidate for an antihelmintic drug or an agrochemical nematicide. On the other hand, the fungus might turn out to be a candidate for a biocontrol agent to act as an antagonist of pathogenic nematodes and fungi.

Citation

Rupcic Z, Chepkirui C, Hernández-Restrepo M, Crous PW, Luangsa-ard JJ, Stadler M (2018) New nematicidal and antimicrobial secondary metabolites from a new species in the new genus, Pseudobambusicola thailandica. MycoKeys 33: 1–23. https://doi.org/10.3897/mycokeys.33.23341

Funding Statement

European Union’s Horizon 2020 research and innovation programme (RISE) under the Marie Skłodowska-Curie grant agreement No 645701, Project acronym “GoMyTri” Kenya National Council for Science and Technology (NACOSTI)and German Academic Exchange Service (DAAD)