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. 2020 Jan 26:96:1-16.
doi: 10.1016/j.simyco.2020.01.001. eCollection 2020 Jun.

Identification of Rosellinia species as producers of cyclodepsipeptide PF1022 A and resurrection of the genus Dematophora as inferred from polythetic taxonomy

K Wittstein  1   2 , A Cordsmeier  1   3 , C Lambert  1   2 , L Wendt  1   2 , E B Sir  4 , J Weber  1   2 , N Wurzler  1   2 , L E Petrini  5 , M Stadler  1   2
Affiliations

Identification of Rosellinia species as producers of cyclodepsipeptide PF1022 A and resurrection of the genus Dematophora as inferred from polythetic taxonomy

K Wittstein et al. Stud Mycol. .

Abstract

Rosellinia (Xylariaceae) is a large, cosmopolitan genus comprising over 130 species that have been defined based mainly on the morphology of their sexual morphs. The genus comprises both lignicolous and saprotrophic species that are frequently isolated as endophytes from healthy host plants, and important plant pathogens. In order to evaluate the utility of molecular phylogeny and secondary metabolite profiling to achieve a better basis for their classification, a set of strains was selected for a multi-locus phylogeny inferred from a combination of the sequences of the internal transcribed spacer region (ITS), the large subunit (LSU) of the nuclear rDNA, beta-tubulin (TUB2) and the second largest subunit of the RNA polymerase II (RPB2). Concurrently, various strains were surveyed for production of secondary metabolites. Metabolite profiling relied on methods with high performance liquid chromatography with diode array and mass spectrometric detection (HPLC-DAD/MS) as well as preparative isolation of the major components after re-fermentation followed by structure elucidation using nuclear magnetic resonance (NMR) spectroscopy and high resolution mass spectrometry (HR-MS). Two new and nine known isopimarane diterpenoids were identified during our mycochemical studies of two selected Dematophora strains and the metabolites were tested for biological activity. In addition, the nematicidal cyclodepsipeptide PF1022 A was purified and identified from a culture of Rosellinia corticium, which is the first time that this endophyte-derived drug precursor has been identified unambiguously from an ascospore-derived isolate of a Rosellinia species. While the results of this first HPLC profiling were largely inconclusive regarding the utility of secondary metabolites as genus-specific chemotaxonomic markers, the phylogeny clearly showed that species featuring a dematophora-like asexual morph were included in a well-defined clade, for which the genus Dematophora is resurrected. Dematophora now comprises all previously known important plant pathogens in the genus such as D. arcuata, D. bunodes, D. necatrix and D. pepo, while Rosellinia s. str. comprises those species that are known to have a geniculosporium-like or nodulisporium-like asexual morph, or where the asexual morph remains unknown. The extensive morphological studies of L.E. Petrini served as a basis to transfer several further species from Rosellinia to Dematophora, based on the morphology of their asexual morphs. However, most species of Rosellinia and allies still need to be recollected in fresh state, cultured, and studied for their morphology and their phylogenetic affinities before the infrageneric relationships can be clarified.

Keywords: Dematophora; Dematophora acutispora (Theiss.) C. Lambert, K. Wittstein & M. Stadler; Dematophora arcuata (Petch) C. Lambert, K. Wittstein & M. Stadler; Dematophora asperata (Massee ex Wakef.) Lambert, K. Wittstein & M. Stadler; Dematophora beccariana (Ces.) C. Lambert, K. Wittstein & M, Stadler; Dematophora boedijnii (L.E. Petrini) C. Lambert, K. Wittstein & M. Stadler; Dematophora bothrina (Berk. & Broome) C. Lambert, K. Wittstein & M. Stadler; Dematophora bunodes (Berk. & Broome) C. Lambert, K. Wittstein & M. Stadler; Dematophora buxi (Fabre) C. Lambert, K. Wittstein & M. Stadler; Dematophora compacta (Takemoto) C. Lambert, K. Wittstein & M. Stadler; Dematophora francisiae (L.E. Petrini) C. Lambert, K. Wittstein & M. Stadler; Dematophora freycinetiae (L.E. Petrini) C. Lambert, K. Wittstein & M. Stadler; Dematophora gigantea (Ellis & Everh.) C. Lambert, K. Wittstein & M. Stadler; Dematophora grantii (L.E. Petrini) C. Lambert, K. Wittstein & M. Stadler; Dematophora hsiehiae (L.E. Petrini) C. Lambert, K. Wittstein & M. Stadler; Dematophora hughesii (L.E. Petrini) C. Lambert, K. Wittstein & M. Stadler; Dematophora javaensis (L.E. Petrini) C. Lambert, K. Wittstein & M. Stadler; Dematophora macdonaldii (Bres.) C. Lambert, K. Wittstein & M. Stadler; Dematophora obregonii (L.E. Petrini) C. Lambert, K. Wittstein & M. Stadler; Dematophora obtusiostiolata (L.E. Petrini) C. Lambert, K. Wittstein & M. Stadler; Dematophora paraguayensis (Starbäck) C. Lambert, K. Wittstein & M. Stadler; Dematophora pepo (Pat.) C. Lambert, K. Wittstein & M. Stadler; Dematophora puiggarii (Pat.) C. Lambert, K. Wittstein & M. Stadler; Dematophora pyramidalis (Lar.N. Vassiljeva) C. Lambert, K. Wittstein & M. Stadler; Dematophora samuelsii (L.E. Petrini) C. Lambert, K. Wittstein & M. Stadler; Dematophora siggersii (L.E. Petrini) C. Lambert, K. Wittstein & M. Stadler; Genus resurrection; Isopimarane diterpenoids; PF1022A; Polythetic taxonomy; Rosellinia; Xylariaceae.

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Figures

Fig. 1
Fig. 1
Inferred phylogenetic tree of selected Xylariaceae, Hypoxylaceae and Graphostromataceae calculated by PhyML with 1 000 bootstrap replicates from a multigene alignment of the ITS-LSU ribosomal DNA region and the TUB2 and RPB2 regions. Bootstrap values above 50% are displayed at their respective branches. Sequence information originating from type strains are highlighted in bold.
Fig. 2
Fig. 2
New isopimarane diterpenoids, dematophoranes A and B (1-2), isolated from Dematophora bunodes (CBS123585).
Fig. 3
Fig. 3
Relevant 1H,1H-COSY- (red arrows) and 1H-13C-HMBC (green arrows) correlations of dematophorane A (1).
Fig. 4
Fig. 4
Isolated secondary metabolites (3-12), known from different species of the orders Xylariales and Hypocreales.
Fig. 5
Fig. 5
HPLC-UV chromatograms (210 nm) of crude extracts from Dematophora bunodes (CBS123584), D. bunodes (CBS123597), D. pepo (CBS123592), Rosellinia corticium (STMA13324), R. aquila (STMA15208), R. marcucciana (MUCL51704), and Astrocystis mirabilis (ATTC66432) cultivated in liquid CM medium (200 mL). The detected isopimarane diterpenoids (1–3, 5–6, 8–11) are marked.
Fig. 6
Fig. 6
HPLC-UV chromatograms (210 nm) of crude extracts containing PF1022A (top to bottom: pure PF1022A, Rosellinia corticium (STMA13324), R. corticium (STMA15209), Astrocystis mirabilis (ATTC66432).
Fig. 7
Fig. 7
Sexual and asexual morph structures of different Rosellinia species. A, L, T. R. breensis (Sir & Hladki 841-LIL). B, S. R. hyalospora (Sir & Hladki 463-LIL). C. R. megalospora (Sir & Hladki 972-LIL). D, E, J, M, P. R. longispora (Sir & Hladki 939-LIL). F–I, K. R. rickii (Sir & Hladki 062-LIL). N, O. R. canzacotoana (Sir & Hladki 198-LIL). Q, R. Rosellinia sp. (Sir & Hladki 377-LIL). A, B, D. Stromata in substrate. C. Stromata emerging from the subiculum (arrow). E. Cross section of stromata. F. Stromata and conidiophores (arrows). G–I. Conidiogenous structure in 3% KOH. J, K. Ascus in 3% KOH. L–N. Ascus apical plugs in Melzer's reagent. O, P, S, T. Ascospores showing germ slit in 3% KOH (arrows). Q, R. Ascospores showing cellular appendages in 3% KOH (arrows). Scale bars: A = 2 mm; B–F = 1 mm; G, H, L–T = 10 μm; I, J = 50 μm; K = 20 μm.
Fig. 8
Fig. 8
Asexual and sexual morph structures of different Dematophora species. A, B, F, O. D. necatrix (Hladki 4004-LIL). C-E, G, H, L–N, P. D. paraguayensis (Sir & Hladki 1098-LIL). I–K. D. arcuata (Sir & Hladki 1098-LIL). A, B. Stromata on substrate. D, C. Stromata and subiculum (arrows). E. Stromata and synnemata (arrows) on substrate. F. Short stipitate stroma in cross section. G. Synnema on substrate. H, I. Synnemata in 3 % KOH solution. J. Details of conidiogenous region. K. Detail of conidiogenous cell. L. Asci in Melzer’s reagent. M. Ascus apical plugs in 3 % KOH. N. Ascus apical plug in Melzer's reagent. O, P. Ascospores showing short and central germ slit in 3 % KOH (arrows). Scale bars: A = 5 mm; B, C = 2 mm; D, E = 1 mm; F, G = 500 μm; H, I = 200 μm; J, L = 50 μm; K, M, N, O, P = 10 μm.
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