Research Article |
Corresponding author: Marc Stadler ( marc.stadler@t-online.de ) Academic editor: Thorsten Lumbsch
© 2020 Mohammad Javad Pourmoghaddam, Christopher Lambert, Frank Surup, Seyed Akbar Khodaparast, Irmgard Krisai-Greilhuber, Hermann Voglmayr, 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:
Pourmoghaddam MJ, Lambert C, Surup F, Khodaparast SA, Krisai-Greilhuber I, Voglmayr H, Stadler M (2020) Discovery of a new species of the Hypoxylon rubiginosum complex from Iran and antagonistic activities of Hypoxylon spp. against the Ash Dieback pathogen, Hymenoscyphus fraxineus, in dual culture. MycoKeys 66: 105-133. https://doi.org/10.3897/mycokeys.66.50946
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During a survey of xylarialean fungi in Northern Iran, several specimens that showed affinities to the Hypoxylon rubiginosum complex were collected and cultured. A comparison of their morphological characters, combined with a chemotaxonomic study based on high performance liquid chromatography, coupled with diode array detection and mass spectrometry (HPLC-DAD/MS) and a multi-locus phylogeny based on ITS, LSU, rbp2 and tub2 DNA sequences, revealed a new species here described as Hypoxylon guilanense. In addition, Hypoxylon rubiginosum sensu stricto was also encountered. Concurrently, an endophytic isolate of the latter species showed strong antagonistic activities against the Ash Dieback pathogen, Hymenoscyphus fraxineus, in a dual culture assay in our laboratory. Therefore, we decided to test the new Iranian fungi for antagonistic activities against the pathogen, along with several cultures of other Hypoxylon species that are related to H. rubiginosum. Our results suggest that the antagonistic effects of Hypoxylon spp. against Hym. fraxineus are widespread and that they are due to the production of antifungal phomopsidin derivatives in the presence of the pathogen.
Ascomycota, Chemotaxonomy, Chemical ecology, Hypoxylaceae, Natural Products, Taxonomy, one new species
Hypoxylon Bull., 1791 is one of the largest genera of the Xylariales and comprises more than 200 species, which are mainly associated with angiosperm trees as saprotrophs and endophytes and are predominant in all forest ecosystems of the world (
It traditionally belonged to the Xylariaceae until a recent phylogenetic study has resulted in a re-arrangement of the genera of stromatic Xylariales and the resurrection of the family Hypoxylaceae (
While the type species of Hypoxylon, H. fragiforme, belongs to a relatively small clade in the phylogeny of
Hypoxylon rubiginosum and related taxa have been studied rather well on their stromatal secondary metabolites and, in many cases, morphologically similar species may contain entirely different pigments and other compounds (
The Ash Dieback disease caused by the introduced apothecial ascomycete Hym. fraxineus (Leotiomycetes) has become one of the greatest problems in European forestry and the majority of common ash trees have succumbed to the fungal pathogen. We have recently studied the secondary metabolism of Hym. fraxineus (previously also known under the synonyms, Hym. pseudoalbidus or Chalara fraxinea) and its non-pathogenic domestic relative, Hym. albidus, for secondary metabolite production (
Concurrently, we were about to study the taxonomy of new collections of Hypoxylon species originating from Iran that also belong to the Hypoxylon rubiginosum complex. Since mycelial cultures of these fungi had just become available, it appeared practical to combine the description of their taxonomy with an evaluation of their antagonistic potential to combat Hym. fraxineus. We have also included a number of other Hypoxylon species that colonise Fraxinus in Europe. The current study therefore provides new evidence on both, the taxonomy and chemical ecology of Hypoxylon.
Samples were collected from Guilan and Mazandaran provinces (Northern Iran) during 2015–2017. Parts of corticated branches and trunks bearing Hypoxylaceae stromata were transferred to the laboratory. Details of the specimens used for morphological investigations are listed in the Taxonomy section under the respective descriptions. Specimens have been deposited in the fungarium of the Department of Plant Protection, Faculty of Agricultural Science, University of Guilan, Guilan, Iran (GUM). Living cultures have been deposited in MUCL (Louvain, Belgium).
Microscopic characters of the teleomorph were observed in distilled water and 10% potassium hydroxide (KOH). Melzer’s reagent was used for staining of the apical ascus apparatus. The numbers of perithecia, ascospores, asci, conidia and conidiophores that were measured for size in the descriptions are 10, 30, 10, 30 and 5, respectively. Specimens were cultured from single ascospore isolates, using 2 % malt extract agar (MEA). For examination of culture macro-morphology, the strains were grown on Difco Oatmeal Agar (OA), following the protocols by
DNA extraction of fresh cultures and amplification of the ITS (nuc rDNA internal transcribed spacer region containing ITS1-5.8S-ITS2), LSU (5' 1200 bp of the large subunit nuc 28S rDNA), rpb2 (partial second largest subunit of the DNA-directed RNA polymerase II) and tub2 (partial β-tubulin) loci were performed as described by
The newly generated sequences were aligned with selected sequences from
Maximum Parsimony (MP) analyses were performed with PAUP v. 4.0a165 (
Maximum Likelihood (ML) analyses were performed with RAxML (
Isolates and accession numbers of sequences used in the phylogenetic analyses. Type specimens are labelled with HT (holotype) ET (epitype) and PT (paratype). Isolates/sequences in bold were isolated/sequenced in the present study.
Species | Strain number | Origin | Status | GenBank accession numbers | Reference | |||
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ITS | LSU | rpb2 | tub2 | |||||
Annulohypoxylon annulatum | CBS 140775 | Texas | ET | KY610418 | KY610418 | KY624263 | KX376353 |
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Annulohypoxylon moriforme | CBS 123579 | Martinique | KX376321 | KY610425 | KY624289 | KX271261 |
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Annulohypoxylon truncatum | CBS 140778 | Texas | ET | KY610419 | KY610419 | KY624277 | KX376352 |
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Biscogniauxia nummularia | MUCL 51395 | France | ET | KY610382 | KY610427 | KY624236 | KX271241 |
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Daldinia caldariorum | MUCL 49211 | France | AM749934 | KY610433 | KY624242 | KC977282 |
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Daldinia concentrica | CBS 113277 | Germany | AY616683 | KY610434 | KY624243 | KC977274 |
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Daldinia dennisii | CBS 114741 | Australia | HT | JX658477 | KY610435 | KY624244 | KC977262 |
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Daldinia petriniae | MUCL 49214 | Austria | ET | AM749937 | KY610439 | KY624248 | KC977261 |
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Daldinia placentiformis | MUCL 47603 | Mexico | AM749921 | KY610440 | KY624249 | KC977278 |
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Daldinia theissenii | CBS 113044 | Argentina | PT | KY610388 | KY610441 | KY624251 | KX271247 |
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Daldinia vernicosa | CBS 119316 | Germany | ET | KY610395 | KY610442 | KY624252 | KC977260 |
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Entonaema liquescens | ATCC 46302 | USA | KY610389 | KY610443 | KY624253 | KX271248 |
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Graphostroma platystomum | CBS 270.87 | France | JX658535 | DQ836906 | KY624296 | HG934108 |
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Hypomontagnella barbarensis | STMA 14081 | Argentina | HT | MK131720 | MK131718 | MK135891 | MK135893 |
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Hypomontagnella monticulosa | MUCL 54604 | French Guiana | ET | KY610404 | KY610487 | KY624305 | KX271273 |
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Hypomontagnella submonticulosa | CBS 115280 | France | KC968923 | KY610457 | KY624226 | KC977267 |
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Hypoxylon carneum | MUCL 54177 | France | KY610400 | KY610480 | KY624297 | KX271270 |
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Hypoxylon cercidicola | CBS 119009 | France | KC968908 | KY610444 | KY624254 | KC977263 |
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Hypoxylon crocopeplum | CBS 119004 | France | KC968907 | KY610445 | KY624255 | KC977268 |
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Hypoxylon fendleri | MUCL 54792 | French Guiana | KF234421 | KY610481 | KY624298 | KF300547 |
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Hypoxylon fragiforme | MUCL 51264 | Germany | ET | KC477229 | KM186295 | KM186296 | KX271282 |
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Hypoxylon fuscum | CBS 113049 | France | ET | KY610401 | KY610482 | KY624299 | KX271271 |
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Hypoxylon griseobrunneum | CBS 331.73 | India | HT | KY610402 | KY610483 | KY624300 | KC977303 |
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Hypoxylon guilanense | MUCL 57726 | Iran | HT | MT214997 | MT214992 | MT212235 | MT212239 | This study |
Hypoxylon haematostroma | MUCL 53301 | Martinique | ET | KC968911 | KY610484 | KY624301 | KC977291 |
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Hypoxylon howeanum | MUCL 47599 | Germany | AM749928 | KY610448 | KY624258 | KC977277 |
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Hypoxylon hypomiltum | MUCL 51845 | Guadeloupe | KY610403 | KY610449 | KY624302 | KX271249 |
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Hypoxylon investiens | CBS 118183 | Malaysia | KC968925 | KY610450 | KY624259 | KC977270 |
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Hypoxylon lateripigmentum | MUCL 53304 | Martinique | HT | KC968933 | KY610486 | KY624304 | KC977290 |
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Hypoxylon lenormandii | CBS 119003 | Ecuador | KC968943 | KY610452 | KY624261 | KC977273 |
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Hypoxylon musceum | MUCL 53765 | Guadeloupe | KC968926 | KY610488 | KY624306 | KC977280 |
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Hypoxylon olivaceopigmentum | DSM 107924 | USA | HT | MK287530 | MK287542 | MK287555 | MK287568 |
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Hypoxylon papillatum | ATCC 58729 | USA | HT | KC968919 | KY610454 | KY624223 | KC977258 |
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Hypoxylon perforatum | CBS 115281 | France | KY610391 | KY610455 | KY624224 | KX271250 |
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Hypoxylon petriniae | CBS 114746 | France | HT | KY610405 | KY610491 | KY624279 | KX271274 |
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Hypoxylon pilgerianum | STMA 13455 | Martinique | KY610412 | KY610412 | KY624308 | KY624315 |
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Hypoxylon porphyreum | CBS 119022 | France | KC968921 | KY610456 | KY624225 | KC977264 |
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Hypoxylon pulicicidum | CBS 122622 | Martinique | HT | JX183075 | KY610492 | KY624280 | JX183072 |
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Hypoxylon rickii | MUCL 53309 | Martinique | ET | KC968932 | KY610416 | KY624281 | KC977288 |
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Hypoxylon rubiginosum | MUCL 52887 | Germany | ET | KC477232 | KY610469 | KY624266 | KY624311 |
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Hypoxylon rubiginosum | MUCL 57727 | Iran | MT214998 | MT214993 | MT212236 | MT212240 | This study | |
Hypoxylon aff. rubiginosum | MUCL 57724 | Iran | MT214999 | MT214994 | MT212237 | MT212241 | This study | |
Hypoxylon aff. rubiginosum | MUCL 57725 | Iran | MT215000 | MT214995 | MT212238 | MT212242 | This study | |
Hypoxylon samuelsii | MUCL 51843 | Guadeloupe | ET | KC968916 | KY610466 | KY624269 | KC977286 |
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Hypoxylon texense | DSM 107933 | USA | HT | MK287536 | MK287548 | MK287561 | MK287574 |
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Hypoxylon ticinense | CBS 115271 | France | JQ009317 | KY610471 | KY624272 | AY951757 |
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Hypoxylon trugodes | MUCL 54794 | Sri Lanka | ET | KF234422 | KY610493 | KY624282 | KF300548 |
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Hypoxylon vogesiacum | CBS 115273 | France | KC968920 | KY610417 | KY624283 | KX271275 |
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Jackrogersella cohaerens | CBS 119126 | Germany | KY610396 | KY610497 | KY624270 | KY624314 |
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Jackrogersella minutella | CBS 119015 | Portugal | KY610381 | KY610424 | KY624235 | KX271240 |
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Jackrogersella multiformis | CBS 119016 | Germany | ET | KC477234 | KY610473 | KY624290 | KX271262 |
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Pyrenopolyporus hunteri | MUCL 52673 | Ivory Coast | ET | KY610421 | KY610472 | KY624309 | KU159530 |
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Pyrenopolyporus laminosus | MUCL 53305 | Martinique | HT | KC968934 | KY610485 | KY624303 | KC977292 |
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Pyrenopolyporus nicaraguensis | CBS 117739 | Burkina_Faso | AM749922 | KY610489 | KY624307 | KC977272 |
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Rhopalostroma angolense | CBS 126414 | Ivory Coast | KY610420 | KY610459 | KY624228 | KX271277 |
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Ruwenzoria pseudoannulata | MUCL 51394 | D. R. Congo | HT | KY610406 | KY610494 | KY624286 | KX271278 |
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Thamnomyces dendroideus | CBS 123578 | French Guiana | HT | FN428831 | KY610467 | KY624232 | KY624313 |
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Xylaria arbuscula | CBS 126415 | Germany | KY610394 | KY610463 | KY624287 | KX271257 |
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Xylaria hypoxylon | CBS 122620 | Sweden | ET | KY610407 | KY610495 | KY624231 | KX271279 |
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Stromata of Hypoxylon specimens were extracted as described by
Dual cultures of Hypoxylon spp. and Hym. fraxineus (STMA 18166) were co-incubated on barley-malt agar by inoculation at opposite sites on 9 cm Petri dishes (cf.
Of the 4369 nucleotide characters of the combined matrix, 1618 are parsimony informative (298 of ITS, 156 of LSU, 487 of rpb2 and 677 of tub2). Fig.
Phylogram of the best ML trees (lnL = −63870.651550) revealed by RAxML from an analysis of the combined ITS–LSU–rpb2–tub2 matrix of selected Xylariales. Strains in bold were sequenced in the current study. ML and MP bootstrap support above 50% are given at the first and second positions, respectively, above or below the branches.
Iran, Guilan Province, Rasht County, Saravan forest, 37°04'26"N, 49°38'13"E, 183 m elev., on fallen branch of Quercus castaneifolia, 9 Apr 2015, M.J. Pourmoghaddam. (GUM 989; ex-holotype culture MUCL 57726).
Guilanense, refers to its origin in Guilan province, Iran.
Stromata superficial, hemispherical to pulvinate, up to 2 cm long × 0.1–0.7 cm wide, with conspicuous perithecial mounds, surface Sienna (8), Umber (9) to Buff (45); Scarlet (5) to Orange (7) granules beneath the surface and between the perithecia, with Orange (7) KOH-extractable pigments. Perithecia spherical to obovoid, 0.33–0.66 high × 0.3–0.55 mm wide. Ostioles umbilicate, inconspicuous. Asci not seen. Ascospores smooth, unicellular, brown to dark brown, ellipsoid, inequilateral with narrowly rounded ends, 12–15 × 5–6 µm, with straight germ slit spore-length on convex side; perispore dehiscent in 10% KOH, conspicuous coil-like ornamentation in SEM; epispore smooth.
Colonies on OA covering a 9 cm Petri dish in 4 wk, at first white, becoming Buff (45), cottony, slightly zonate with diffuse margins; finally, becoming Honey (64). Anamorph not produced in culture.
Orsellinic acid, rubiginosin A and an unknown isomer thereof, as well as mitorubrinol acetate as prevailing stromatal components; cultures produce yet unidentified compounds on barley-malt agar.
The description of this taxon is based on a single specimen, which shows the salient features of the teleomorph and can be discriminated easily from all previously described species of the H. rubiginosum complex. The stromata of the holotype specimen differ from H. texense (i.e. the closest relative in the phylogeny), in having stromata with hemispherical to pulvinate shape, Orange (7) KOH-extractable pigments and larger ascospores [12–15 × 5–6 vs. 9.1–10.8 (–11.5) × (4.0–) 4.5–5.4 (–5.7) μm with straight germ slit.
Hypoxylon guilanense can also be easily differentiated from H. rubiginosum sensu stricto and H. petriniae in the peculiar stromatal shape and it also has larger ascospores. H. cercidicola differs from H. guilanense in having erumpent stromata with discoid shape and smaller ascospores [(9–) 9.5–12 × 5–6 μm)] with straight to slightly sigmoid germ slit. Table
Hypoxylon guilanense (Holotype GUM 989) A stromatal habit B close-up view of stromatal surface, with stromatal pigments in 10% KOH C, H, I ascospores in water, with germ-slits D, E ascospores in 10% KOH with dehiscent perispore F, G ascospore under SEM J, K culture on 9 cm OA plates after 1 and 3 wk of incubation (left to right). Scale bars: 2.5 mm (A), 1 mm (B); 10 µm (C–E); 2 µm (F, G); 10 µm (H, I).
Diagnostic characters of Hypoxylon rubiginosum sensu stricto and closely related species.
Taxon | Stromatal shape | Stromatal surface | KOH-extractable pigments | Ascospores (µm) | Germ slit | Host | Known distribution | Anamorph | Secondary metabolites* |
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Hypoxylon canariense | effused to effused-pulvinate | Fulvous, Dark Brick, Dark Vinaceous | Orange to Sienna | 9.5–11.5 × 4.5–5 | straight | Erica, Ocotea, Laurus, Persea | Spain (Canary Islands) | virgariella-like | Rubiginosins A–C, mitorubrinol acetate |
Hypoxylon carneum | Effused-pulvinate | Dark purple, Dark vinaceous | Livid violet, absent in old stromata | (7.5–)8–11.5 × 4.5–5 | straight | Various angiosperm hosts including Fraxinus | probably cosmopolitan but rare | sporothrix-like | Carneic acids A and B, BNT |
Hypoxylon cercidicola | discoid | Dark brick to Sepia | Orange | (9–)9.5–12 × 5–6 | straight to slightly sigmoid | Fraxinus | Europe and North America | unknown | Mitorubrin, rubiginosin A and C |
Hypoxylon guilanense | hemispherical to pulvinate | Sienna, Umber to Buff | Orange | 12–15 × 5–6 | straight | Quercus | Iran | unknown | Rubiginosin A, mitorubrinol acetate |
Hypoxylon lusitanicum | effused | Brown Vinaceous | Sienna | 11–13.5 × 5–7 | straight | Rhamnus | Portugal | unknown | Rubiginosins A and C, rutilin A |
Hypoxylon petriniae | irregularly effused | Lilac, Vinaceous to Brown Vinaceous | Orange to Rust | 8–11.5(–13) × 4.8–6 | straight | Fraxinus (mostly); Acer, Salicaceae | Western and Central Europe | virgariella-like | Rubiginosin A, BNT |
Hypoxylon retpela | effused-pulvinate | Livid Vinaceous, Brown Vinaceous, | Orange or Scarlet | (9–)9.5–12 × 4.5–5 | straight or slightly sigmoid | unknown | Southeast and East Asia, New Guinea | nodulisporium-like | Mitorubrinol acetate, unknown rubiginosins |
Hypoxylon rubiginosum | effused-pulvinate | Dark Brick, Brown Vinaceous | Orange | 9–13 × 4–5.5 | straight | Various angiosperm hosts including Fraxinus | Europe, North America | nodulisporium-like | Mitorubrin, rubiginosin A–C, rubiginosic acid, daldinin C |
Hypoxylon aff. rubiginosum (GUM 1587) | pulvinate to effused-pulvinate | Luteous, Orange to Ochraceous | Orange | 8–10 (–11) × (3–) 4–4.5 (–5) | straight to slightly sigmoid | Quercus | Iran | virgariella-like | like H. rubiginosum |
Hypoxylon aff. rubiginosum (GUM 1588) | pulvinate | Orange to Apricot | Orange | 10–15 × 5–6.5 | straight to slightly sigmoid | unknown | Iran | not observed- | like H. rubiginosum |
Hypoxylon salicicola | effused | Dark rust to Sepia, Brown Vinaceous | Fulvous to Rust | 7.2–9.6 × 3–4.2 | straight | Salix, rarely on Fraxinus and Prunus | Northern Europe, USA | nodulisporium-like | Mitorubrinol acetate |
Hypoxylon texense | effused to effused-pulvinate | Livid Vinaceous to Brown Vinaceous | Rust to Dark Brick | 9.1–10.8(–11.5) × (4.0–)4.5–5.4(–5.7) | straight or slightly sigmoid | unknown | USA | nodulisporium to virgariella-like | Rubiginosin A, mitorubrinol acetate, unknown rubiginosins |
Hypoxylon urriesii | effused | Dark Brick | Orange | 11–14.5 × 5–6 | straight or slightly sigmoid | unknown | Spain (Canary Islands) | unknown | Mitorubrinol acetate, rubiginosin A |
Stromata superficial, effused-pulvinate, up to 8 cm long × 0.3–0.2 cm wide; with inconspicuous to conspicuous perithecial mounds, surface Red (2) to Brick (59); Scarlet (5) to Orange (7) granules beneath the surface and between the perithecia, with Orange (7) to Scarlet (5) KOH-extractable pigments. Perithecia spherical to obovoid, 0.2–0.5 high × 0.15–0.45 mm wide. Ostioles umbilicate, inconspicuous. Asci 8-spored, cylindrical, with amyloid, discoid apical apparatus, 0.5–1 µm high × 1.5–2.5 µm wide, stipe up to 180 µm long and spore-bearing portion 40–80 × 6.5–10 µm. Ascospores smooth, unicellular, brown to dark brown, ellipsoid, inequilateral with narrowly rounded ends, 9–12 (–13) × 4–6 µm, with straight germ slit spore-length on convex side; perispore dehiscent in 10% KOH; epispore smooth.
Colonies on OA covering a 9 cm Petri dish in 3 wk, at first white, becoming Smoke Grey (105), felty, azonate with diffuse margins; finally becoming Pale Luteous (11) to Straw (46). Asexual morph not produced in culture.
Rubiginosin A and an unknown compound of the mitorubrin / rubiginosin azaphilone family prevalent; cultures produce phomopsidin and unidentified compounds on barley-malt agar.
Iran, Guilan Province, Siahkal County, Deilaman forest, 36°57'25"N, 49°51'54"E, 1100 m elev., on fallen branch of Quercus castaneifolia, 6 Oct 2017 (GUM 1586; culture MUCL 57727); Guilan Province, Shaft County, 36°59'08"N, 49°18'43"E, 594 m elev., on fallen trunk of Pterocarya fraxinifolia, 15 Sep 2015 (GUM 1583); Guilan Province, Langaroud County, Liseroud forest, 37°7'44"N, 50°8'41"E, 28 m elev., on fallen branch of Quercus castaneifolia, 10 Sep 2016 (GUM 1584); Guilan Province, Talesh County, Gisoum forest, 37°37'30"N, 48°58'15"E, 477 m elev., on fallen branch of Populus sp., 20 Oct 2016 (GUM 1585). All specimens collected by M.J. Pourmoghaddam.
H. rubiginosum sensu stricto is a very common fungus in the temperate Northern hemisphere (
Hypoxylon rubiginosum (GUM 1586) A, B stromatal habit C close-up view of stromatal surface D close-up view of stromatal surface, with stromatal pigments in 10% KOH E ascospores in 10% KOH with dehiscent perispore F mature and immature asci in water G immature ascus in water H mature ascus in water I ascus in Melzer’s reagent J ascospores in water K ascus tip in Melzer’s reagent. Scale bars: 2 cm (A); 1 cm (B); 4 mm (C); 2 mm (D); 10 µm (E); 20 µm (F–I), 10 µm (J, K).
Below, we describe two collections that may eventually be recognised to represent new species. They appear phylogenetically different from the type specimen, as well as from Iranian records of H. rubiginosum, but share salient features with the latter species. It is explained in the Notes why we hesitate to describe them as new taxa in this complicated species complex.
Stromata superficial, pulvinate to effused-pulvinate, up to 5 cm long × 0.6–2 cm wide, with inconspicuous to conspicuous perithecial mounds; surface Luteous (12), Orange (7) to Ochreous (44); Orange (7) granules beneath the surface, Orange (7) and Leaden Black (126) granules between the perithecia, with KOH-extractable pigments Orange (7). Perithecia obovoid, compressed-obovoid to spherical, 0.27–0.50 high × 0.23–0.35 mm wide. Ostioles umbilicate, inconspicuous, usually overlain with conspicuous white substance. Asci 8-spored, cylindrical, with amyloid, discoid apical apparatus, 0.5–1 µm high × 2–2.5 µm wide, stipe up to 180 µm long; spore-bearing portion 80–100 × 5.5–7 µm. Ascospores smooth, unicellular, brown to dark brown, ellipsoid, inequilateral with narrowly rounded ends, 8–10 (–11) × (3–) 4–4.5 (–5) µm, with straight to slightly sigmoid germ slit spore-length on convex side; perispore dehiscent in 10% KOH, conspicuous coil-like ornamentation in SEM; epispore smooth.
Hypoxylon aff. rubiginosum (GUM 1587) A, B stromatal habit C close-up view of stromatal surface, with stromatal pigments in 10% KOH D stroma in section showing perithecia and ostioles E mature and immature asci in water F ascus in water G ascus in Melzer’s reagent H ascus tip in Melzer’s reagent I ascospores in 10% KOH with dehiscent perispore J ascospore in water, with germ-slit K ascospore under SEM. Scale bars: 5 mm (A, B); 1 mm (C); 0.5 mm (D); 20 µm (E–G); 10 µm (H–J); 2 µm (K).
Colonies on OA covering a 9 cm Petri dish in 3 wk, at first white, becoming Luteous (12) from outwards, cottony, slightly zonate with diffuse margins; finally, attaining a variety of different colours. Conidiogenous structure branching virgariella-like as defined by
Iran, Guilan Province, Astaneh-Ashrafieh County, Safra-Basteh forest, 37°20'19"N, 49°58'26"E, 14 m elev., on fallen branch of Quercus castaneifolia, 4 Oct 2016, M.J. Pourmoghaddam (GUM 1587; culture MUCL 57724).
This specimen resembles H. rubiginosum in many respects. However, it has slightly smaller ascospores [8–10 (–11) × (3–) 4–4.5 (–5) vs. 9–13 × 4–5.5 µm] and the germ slit of the ascospores is often slightly sigmoid. The most significant differences were noted in the anamorphic structures with virgariella-like branching patterns. This anamorph actually resembles that of H. petriniae. However, this species is normally associated with Fraxinus and differs from Hypoxylon aff. rubiginosum GUM 1587 in having Lilac (54), Vinaceous (57) to Brown Vinaceous (84) stromatal surface colours (owing to the presence of BNT, which was not found in the Iranian specimen). It also differs in having more elongate to irregularly effused stromata with black margins and its ascospores are larger (8–11.5 (–13) × 4.8–6 μm) and have a straight germ slit.
Culture and anamorphic structures of Hypoxylon aff. rubiginosum (GUM 1587) on OA A, B surface of colony after 1 and 8 wk of incubation (respectively, left to right) C–G general view of anamorph structure with virgariella-like branching patterns H, I conidiogenous cells and immature conidia J mature conidia. Scale bars: 20 µm (C–G); 10 µm (H–J).
Stromata superficial, pulvinate, up to 1 cm long × 0.2–0.5 cm wide, with inconspicuous to conspicuous perithecial mounds; surface Orange (7) to Apricot (42); Orange (7) granules beneath the surface and Laeden Black (126) granules between the perithecia, with Orange (7) KOH-extractable pigments. Perithecia obovoid to compressed-obovoid, 0.35–0.65 high × 0.3–0.45 mm wide. Ostioles umbilicate, inconspicuous. Asci with amyloid, discoid apical apparatus, 1–1.5 µm high × 2–3 µm wide, stipe up to 160 µm and spore-bearing portion 70–100 × 6–8 µm long. Ascospores smooth, unicellular, brown to dark brown, ellipsoid, inequilateral with narrowly-rounded ends, 10–15 × 5–6.5 µm, with straight to slightly sigmoid germ slit spore-length on convex side; perispore dehiscent in 10% KOH; epispore smooth.
Colonies on OA covering a 9 cm Petri dish in 3 wk, at first white, becoming whitish, cottony, azonate with entire margins; remaining mainly uncoloured with Pale Luteous tinges. Anamorph not produced in culture.
Iran, Mazandaran Province, Tonekabon County, Do-hezar forest, 36°42'30"N, 50°49'43"E, 456 m elev., on dead branches (host unknown), 28 Oct 2016, M.J. Pourmoghaddam (GUM 1588; culture MUCL 57725).
This specimen is morphologically similar to Hypoxylon aff. rubiginosum GUM 1587, but it can be distinguished by its larger ascospores [10–15 × 5–6.5 vs. 8–10 (–11) × (3–) 4–4.5 (–5) μm]. H. rubiginosum sensu stricto differs from this specimen in having smaller ascospores [(8–) 9–12 × 4–5.5 vs. 10–15 × 5–6.5 μm]. In addition, the stromatal secondary metabolite profile is similar to that of H. rubiginosum with two unknown azaphilone compounds of the mitorubrin / rubiginosin family (UC 2, retention time = 8.7 min, 442 Dalton and UC 3, RT = 10.6 min, 884 Da) and rubiginosin A. H. guilanense differs from Hypoxylon aff. rubiginosum GUM 1588 in having stromata with hemispherical to pulvinate shape and difference in average ascospores sizes (12–15 × 5–6 vs. 10–15 × 5–6.5 μm) with straight germ slit. H. texense differs from Hypoxylon aff. rubiginosum GUM 1588 in having Rust (39) to Dark Brick (86) KOH-extractable pigments and much smaller ascospores [9.1–10.8 (–11.5) × (4.0–) 4.5–5.4 (–5.7) vs. 10–15 × 5–6.5 μm].
Hypoxylon aff. rubiginosum (GUM 1588) A stromatal habit B close-up view of stromatal surface, with stromatal pigments in 10% KOH C section of stroma showing perithecia and ostioles D ascus in Melzer’s reagent E ascospores in 10% KOH with dehiscent perispore. Scale bars: 2.5 mm (A); 0.5 mm (B, C); 20 µm (D); 10 µm (E).
Amongst the four studied Iranian Hypoxylon spp., five major metabolites could be identified. Beneath common secondary metabolites of the H. rubiginosum complex like orsellinic acid (1,
In total, 32 different Hypoxylon strains were screened for production of phomopsidin (5,
Identified secondary metabolites in axenic cultures on barley-malt medium of the surveyed strains. Strains in bold have been used concurrently against STMA 18166 (Hymenoscyphus fraxineus) in an antagonism assay. Identified compounds: 5: phomopsidin; 6: 10-hydroxyphomopsidin; 8: rickiol A; 9: orthosporin 10: daldinone B; 11: 1,8-dimethoxynaphtahlene; 13: 5-methyl-mellein. Identified stromal azaphilone groups detected in culture: MI = Mitorubrin type; NA = Naphthalene type; DA =Daldinin type. For chemical structures, see Fig.
Organism | Strain | Culture metabolites | Stromal metabolites | ||||
---|---|---|---|---|---|---|---|
5 | 6 | Others | MI | NA | DA | ||
Hypoxylon guilanense | MUCL 57726 | – | – | – | – | – | – |
Hypoxylon aff. rubiginosum | MUCL 57724 | + | + | – | + | – | – |
Hypoxylon rubiginosum | MUCL 57727 | + | – | – | – | – | – |
Hypoxylon aff. rubiginosum | MUCL 57725 | + | + | – | – | – | – |
Hypoxylon perforatum | MUCL 57728 | – | – | 10 | – | – | – |
Hypoxylon perforatum | CBS 119011 | – | – | 10 | – | – | – |
Hypoxylon perforatum | MUCL 47187 | + | + | – | – | – | – |
Hypoxylon perforatum | MUCL 54798 | – | – | 10 | – | – | – |
Hypoxylon perforatum | STMA 13041 | + | + | – | – | – | – |
Hypoxylon perforatum | STMA 14051 | – | – | 10 | – | – | – |
Hypoxylon perforatum | CBS 140779 | – | – | 10 | – | – | – |
Hypoxylon petriniae | MUCL 53756 | + | + | – | – | – | – |
Hypoxylon petriniae | STMA 12020 | – | – | – | – | – | – |
Hypoxylon petriniae | STMA 13303 | – | – | – | – | – | – |
Hypoxylon petriniae | STMA 13313 | – | – | 10 | – | – | – |
Hypoxylon rubiginosum | MUCL 2354 | – | – | – | – | – | – |
Hypoxylon rubiginosum | MUCL 47152 | + | + | 9, 10 | – | + | – |
Hypoxylon rubiginosum | MUCL 47970 | + | + | 9, 10 | – | + | – |
Hypoxylon rubiginosum | MUCL 47150 | + | – | – | + | – | – |
Hypoxylon rubiginosum | MUCL 52672 | + | + | – | + | – | – |
Hypoxylon rubiginosum | MUCL 54624 | – | – | 8 | – | – | – |
Hypoxylon rubiginosum | MUCL 2709 | – | – | – | – | – | – |
Hypoxylon rubiginosum | MUCL 34183 | + | + | 13 | – | – | – |
Hypoxylon rubiginosum | MUCL 47147 | + | – | – | + | – | – |
Hypoxylon rubiginosum | STMA 04040 | + | + | – | + | – | – |
Hypoxylon rubiginosum | STMA 07027 | + | + | – | – | – | – |
Hypoxylon rubiginosum | STMA 13346 | + | + | – | – | – | – |
Hypoxylon rubiginosum | STMA 17058 | + | + | – | – | – | – |
Hypoxylon cercidicola | MUCL 54180 | + | – | 13 | – | – | – |
Hypoxylon fuscum | STMA 13090 | – | – | 11, 13 | – | + | + |
Hypoxylon texense | DSM 107933 | – | – | – | + | – | – |
Hypoxylon crocopeplum | CBS 119004 | – | – | – | + | – | – |
Hypoxylon carneum | MUCL 54177 | – | – | 10 | – | – | – |
Illustration of antagonist test by dual culture technique of Hypoxlon spp. and Hymenoscyphus fraxineus on barley-malt agar in 9-cm diam. plates A dual culture of H. rubiginosum (MUCL 47152) against Hym. fraxineus (STMA 18166) after 1 wk of incubation B dual culture of H. rubiginosum (MUCL 47152) against Hym. fraxineus (STMA 18166) after 2 wk of incubation C dual culture of H. rubiginosum (MUCL 47152) against Hym. fraxineus (STMA 18166) after 3 wk of incubation D dual culture of H. rubiginosum (MUCL 47152) against Hym. fraxineus (STMA 18166) after 4 wk of incubation E–H (Hypoxylon aff. rubiginosum MUCL 57724) against Hym. fraxineus after 1, 2, 3, 4 wk I–L H. texense (DSM 107933) against Hym. fraxineus after 1, 2, 3, 4 wk M–P H. guilanense (MUCL 57726) against Hym. fraxineus after 1, 2, 3, 4 wk.
Chemical structures of discussed secondary metabolites. Orsellinic acid (1); mitorubrinol acetate (2); rubiginosin A (3); mitorubrinol (4); phomopsidin (5); 10-hydroxyphomopsidin (6); mitorubrin (7); rickiol A (8); orthosporin (9); daldinone B (10); 1,8-dimethoxynaphthalene (11); daldinin F (12); 5-methyl mellein (13); viridiol (14).
HPLC-UV profiles at 210 nm derived from stromal extracts of strains H. rubiginosum (GUM 1586), H. guilanense (from holotype) and Hypoxylon aff. rubiginosum GUM 1587 and GUM 1588. UV/Vis spectra are shown for orsellinic acid (1), mitorubrinol acetate (2), rubiginosin A (3), an unknown rubiginosin A – like derivative (URg) and rubiginosin – like derivatives (UC 2 and UC 3). ESI mass spectra are shown for compounds URg and 2.
Strikingly, during evaluation and comparison of the HPLC UV/Vis chromatograms with our internal database, the mitorubrin derivatives 2, 4 and 7 were identified by direct comparison of chromatograms derived from extracts of stromata and cultures of the ex-type strain and the holotype of H. texense (
Identified secondary metabolites in dual culture (barley-malt medium with Hymenoscyphus fraxineus) of the surveyed strains listed in Table
Organism | Strain | Culture metabolites | Stromal metabolites | |||
---|---|---|---|---|---|---|
5 | 6 | Others | MI | NA | ||
Hypoxylon cercidicola | MUCL 54180 | + | – | 13 | – | – |
Hypoxylon fuscum | STMA 13090 | – | – | 11, 13 | – | + |
Hypoxylon texense | DSM 107933 | – | – | – | + | – |
Hypoxylon crocopeplum | CBS 119004 | – | – | – | + | – |
Hypoxylon perforatum | MUCL 47187 | + | – | – | + | – |
Hypoxylon petriniae | MUCL 53756 | + | – | – | – | – |
Hypoxylon aff. rubiginosum | MUCL 57724 | + | + | – | + | – |
Hypoxylon rubiginosum | MUCL 47152 | + | – | 9, 10 | – | + |
Hypoxylon rubiginosum | MUCL 47970 | + | – | 9, 10 | – | + |
Hypoxylon guilanense | MUCL 57726 | – | – | – | – | – |
Hypoxylon carneum | MUCL 54177 | – | – | 10 | – | – |
HPLC-UV profiles at 210 nm derived from barley-malt agar (A–C, E) and stromal (E) extracts and compound standard (F). UV/Vis spectra are shown for identified compounds in mono- and dual culture (C) experiments of STMA 18166 (Hym. fraxineus, A) and DSM 107933 (H. texense, B; UC 2, 4 – unknown compounds); stromal metabolites (4 – mitorubrinol; URg – unknown rubiginosin A derivative; 3 – rubiginosin A; 2 – mitorubrinol acetate; 7 – mitorubrin; UC2 – Unknown compound 2 of GLM-F116101 (H. texense, D), and ... ESI mass spectra of 8 in positive and negative modes... of 8 8 (rickiol A, F) identified in the mono culture extract of MUCL 54624 (H. rubiginosum, E).
HPLC-UV chromatograms at 210 nm from mono cultural barley-malt agar extracts of MUCL 47152 (H. rubiginosum), STMA 18166 (Hym. fraxineus), STMA 13090 (H. fuscum) and one dual culture experiment thereof. UV/Vis spectra are shown for phomopsidin (5), 10-hydroxyphomopsidin (6), orthosporin (9), daldinone B (10), 1,8-dimethoxynaphthalene (11), daldinin F (12), 5–methylmellein (13), viridiol (14) and an unidentifiable compound (UC 6) after comparison of data with internal databases. The UV signal of UC 6 was enhanced in the dual culture extract.
The present study dealt with the identification of Hypoxylon species from Northern Iran based on morphological, chemotaxonomic and phylogenetic data, focusing on the H. rubiginosum complex. The specimens encountered appeared morphologically and chemotaxonomically related to H. rubiginosum sensu stricto, as revealed from their morphology and secondary metabolite profiles. While the majority of specimens were assigned to typical H. rubiginosum, we have encountered a new taxon that significantly deviates from the complex in both stromatal and ascospore morphology and appears most closely related to a species that was so far only reported from the southern USA (
Our phylogenetic analyses confirmed previous results (
A main objective of this work was to assess the antagonistic potential of the newly isolated cultures and some strains of related species against an important pathogen, following the recent discovery that an endophytic isolate of H. rubiginosum from a resistant ash tree inhibited the growth of the alien pathogen, Hym. fraxineus (
Last but not least, the current study also revealed some interesting aspects for potential follow-up projects. For instance, the examination of H. fuscum (a species that has never been isolated from Fraxinus, but is actually associated with Corylus and other Betulaceae) in the antagonism assay, revealed the production of several hitherto unknown compounds whose production was significantly enhanced in the presence of Hym. fraxineus. This observation suggests that it will be worthwhile to further study the secondary metabolism of Hypoxylon species in other scenarios using the dual culture approach. The first step would be to scale-up the production of the unknown molecules, isolating enough for structure elucidation and biological studies. This should not be expected to be a trivial task, but it appears doable using the methodology that is presently available.
The production of known and yet unidentified azaphilones (i.e. a compound class that is normally found in high concentrations in the stromata of various Hypoxylaceae, but was rarely observed in their mycelial cultures) in H. rubiginosum and allies, is another interesting observation relating to the differential expression of biosynthetic genes encoding secondary metabolites. It should be rewarding to evaluate the regulation mechanisms that lead to the production of the pigments, aided by genomic and transcriptomic studies.
This study was partially financed by the Ministry of Science, Research and Technology (MSRT) of Iran. Mohammad Javad wants to express his appreciation to all colleagues in the labs of Irmgard Krisai-Greilhuber (Vienna) and Marc Stadler (Braunschweig) for their support, especially Kathrin Wittstein and Silke Reinecke and to all colleagues in the University of Guilan. The authors want to express their gratitude to Manfred Rohde for recording the SEM pictures.
Discovery of a new species of the Hypoxylon rubiginosum complex from Iran and antagonistic activities of Hypoxylon spp. against the Ash Dieback pathogen, Hymenoscyphus fraxineus, in dual culture
Data type: Multimedia.