Research Article |
Corresponding author: Marc Stadler ( marc.stadler@t-online.de ) Academic editor: Imke Schmitt
© 2018 Zeljka Rupcic, Clara Chepkirui, Margarita Hernández-Restrepo, Pedro W. Crous, Janet Jennifer Luangsa-ard, Marc Stadler.
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:
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
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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.
Antifungal agent, deoxyphomalone, monocerin, nematode-antagonism, nematicide, phylogeny
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 (
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 (
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.
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
Morphological features were characterised from colonies growing on OA or on synthetic nutrient-poor agar (SNA;
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
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 |
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Bambusicola didymospora | MFLUCC 10-0557 | KU940116 | KU863105 | KU940163 | KU940188 |
|
B. loculata | MFLU 15-0056 | KP761732 | KP761729 | KP761715 | KP761724 |
|
B. pustulata | MFLUCC 15-0190 | KU940118 | KU863107 | KU940165 | KU940190 |
|
B. splendida | MFLUCC 11-0611 | KU940121 | KU863110 | KU940168 | – |
|
Coniothyrium palmicola | CBS 161.37 | JX681086 | JX681086 | – | – |
|
Dendrothyrium longisporum | CBS 824.84 | JX496115 | JX496228 | – | – |
|
Dydimella exigua | CBS 183.55 | NR135936 | EU754155 | GU357800 | KR184187 |
|
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 | – | – |
|
Leptosphaeria doliolum | CBS 505.75 | JF740205 | GQ387576 | KY064035 | GU349069 | De Gruyter et al. 2013, |
Lophiostoma arundinis | AFTOL-ID 1606 | – | DQ782384 | DQ782386 | DQ782387 |
|
Macrodiplodiopsis desmazieri | CBS 140062 | NR132924 | KR873272 | – | – |
|
Magnicamarosporium iriomotense | KT 2822 | AB809640 | AB807509 | – | AB808485 | Tanaka et al. 2015 |
Massarina phragmiticola | CBS 110446 | – | DQ813510 | – | – |
|
Montagnula bellevaliae | MFLUCC 14-0924 | KT443906 | KT443902 | – | – |
|
M. scabiosae | MFLUCC 14-0954 | KT443907 | KT443903 | – | – |
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Murilentithecium clematidis | MFLUCC 14-0562 | KM408757 | KM408759 | KM454447 | KM454445 |
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Neobambusicola strelitziae | CBS 138869 | NR 137945 | KP004495 | – | MG976037 |
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Palmiascoma gregariascomum | MFLUCC 11-0175 | KP744452 | KP744495 | KP998466 | – |
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Parabambusicola bambusina | H 4321 | – | AB807536 | – | AB808511 | Tanaka et al. 2015 |
Paraconiothyrium brasiliense | CBS 122851 | JX496036 | JX496149 | – | – |
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Phoma herbarum | CBS 615.75 | NR135967 | EU754186 | KP330420 | KR184186 |
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Pleurophoma ossicola | CPC 24985 | KR476737 | KR476770 | – | – |
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Polyschema congolensis | CBS 542.73 | – | EF204502 | EF204486 | – |
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P. terricola | CBS 301.65 | – | EF204504 | EF204487 | – |
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Pseudobambusicola thailandica sp. nov. |
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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 |
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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 |
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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.
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.
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 tR = 6 min, compound 2 (thailanone B; 1 mg) at tR = 4.3 min, compound 3 (thailanone C; 1.3 mg) at tR = 6.4 min, compound 4 (thailanone D; 1 mg) at tR = 8.1 min; compound 5 (thailanone E; 4.2 mg) at tR = 8.2, compound 6 (thailanone F; 1.6 mg) at tR = 8.6 min) and compound 7; monocerin (7.8 mg) at tR = 9.1 min. The mycelial crude extract was chromatographed in a similar manner as described above, yielding 77.8 mg of deoxyphomalone (8, tR = 11.2 min) but none of the other compounds.
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
The fungal cultures were tested in the water agar plate assay against Caenorhabditis elegans nematodes (wild type strain, see
The nematicidal activity against C. elegans of all isolated compounds was determined by a slightly modified method (
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).
Growth inhibition of Phellinus tremulae CBS 123.40 for compounds 1–8 was tested according to the modified protocol published by
Phytotoxic activities were carried out by germination and seedling growth bioassay against Setaria italica and Lepidum sativum according to the protocol from
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.
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.
The name reflects its morphological similarity of the type species to the asexual morphs of Bambusicola and Neobambusicola.
Pseudobambusicola thailandica Hern.-Restr. & Crous.
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.
The epithet refers to Thailand, where this species was collected.
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:
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.
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.
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 (
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 (
Fractionation of the crude extracts obtained from submerged cultures of P. thailandica (
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
NMR spectroscopic data for compounds 1–3 in D6-acetone (1H NMR at 700 MHz; 13C at 500 MHz).
1 | 2 | 3 | ||||||
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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) |
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.
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
NMR spectroscopic data for compounds 4–6 in D6-acetone (1H NMR at 700 MHz; 13C at 500 MHz).
4 | 5 | 6 | |||||||
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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) |
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,
The results of the biological assays that were performed to detect antibacterial, antifungal and nematicidal activities are summarised in Table
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 | – | – | – | – |
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 (
Moreover, the phytotoxic activity of terrein and congeners on plant growth and induction of lesions on fruit surfaces were previously investigated by
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.
This project has received funding from the European Union’s Horizon 2020 research and innovation programme (RISE) under the Marie Skłodowska-Curie grant agreement No 645701, Project acronym “GoMyTri” (lead beneficiaries JJL, PWC and MS). Financial support by the German Academic Exchange Service (DAAD) and the Kenya National Council for Science and Technology (NACOSTI), who provided a personal PhD stipend for CC is gratefully acknowledged. We are grateful to Cäcilia Bergmann, Vanessa Stiller and Anke Skiba for expert technical assistance and to Christel Kakoschke for recording NMR spectra.