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

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.


INTRODUCTION
The genus Rosellinia was erected by De Notaris (1844) based on R. aquila as type species and later synonymised with various other genera of pyrenomycetes, including Dematophora (Hartig 1883), which had been typified by D. necatrix. The sexual morphs of Rosellinia are characterised by uniperithecioid stromata usually growing from a subiculum. The ascospore morphology, as well as the conidiogenous structures and other characteristics of the asexual morphs are rather variable, even though most of the conidiogenous structures of Rosellinia spp. known can be assigned to the Geniculosporium type. In fact, only some species of the "R. thelena group" for which no molecular data are available, have been reported to form nodulisporium-like conidiophores according to the definition of Ju & Rogers (1996). Many species are only known from one or a few collections and have never been cultured or included in molecular phylogenetic analyses. Recent studies of stromatic Xylariales by Hsieh et al. (2010) and Wendt et al. (2018) included some taxa of Rosellinia and have clearly showed that the genus has affinities to the genus Xylaria and other genera of Xylariaceae sensu stricto, in particular Entoleuca, Euepixylon and Nemania. The Hypoxylon lenormandii complex, which contains some species that were previously included in Rosellinia for their superficially similar stromatal morphology (Kuhnert et al. 2015), has been shown to belong to the hypoxyloid clade of the Xylariales, which have stromatal pigments and a nodulisporium-like asexual morphs. From polythetic studies employing multi locus molecular phylogenies, this complex now resides in the Hypoxylaceae (Daranagama et al. 2018, Wendt et al. 2018, while Rosellinia is retained in the Xylariaceae, owing to its phylogenetic affinities to Xylaria. In general, the recent segregation of the stromatic Xylariales proposed in the latter papers demonstrated that ascospore morphology is often incongruent with the lineages defined by multi locus phylogeny than conidiogenous structures or secondary metabolite profiles. The current study embarks from the comprehensive monograph by Petrini (2013), which compiled all relevant information on the taxonomy of the genus. This work was based on decades of meticulous morphological studies (Petrini 1992, 2003, Petrini & Petrini 2005. The monograph classified the more than 140 currently accepted species into seven informal "Groups" rather than into formal subgeneric taxa. Notably, Petrini (2013) predicted that the genus would need to be further subdivided as data on the phylogeny of its species accumulated. Out of the seven Groups, two, viz. the R. buxi and the R. necatrix groups, are characterised by dematophora-like asexual morphs, which are also often found in nature because of the rather conspicuous synnemata. In the phylogeny of Wendt et al. (2018), Rosellinia buxi and R. necatrix formed a sister clade to a clade containing the type species, R. aquila, R. corticium and Entoleuca mammata, making Rosellinia sensu Petrini (2013) paraphyletic. This observation motived a detailed study of the phylogeny and the secondary metabolites of these fungi in an attempt to further unravel their phylogenetic affinities. The results and conclusions are presented in the current paper.

Fungal material and chemicals
All scientific names of fungi are given without authorities and years of publications, in accordance with MycoBank (www. mycobank.org), with the exception of the species discussed in the taxonomic part of this manuscript. Reference cultures are deposited at the Westerdijk Fungal Biodiversity Institute (formerly known as CBS, Utrecht, The Netherlands), BCCM/MUCL Agrofood & Environmental Fungal Collection (Louvain, Belgium) and the STMA culture collection of the HZI (Braunschweig, Germany). Unless indicated otherwise solvents were obtained in analytical grade from J.T. Baker (Deventer, Netherlands) or Merck (Darmstadt, Germany) and media ingredients from Carl Roth (Karlsruhe, Germany) or Sigma Aldrich (St. Louis, US). For glucose monitoring test stripes Medi-Test glucose by Macherey-Nagel (Düren, Germany) were used. In order to study their molecular and chemical diversity 14 strains of the genus Rosellinia and closely related species were selected. For molecular phylogenetic analyses additional GenBank sequences were selected. The origin of the fungal strains and their corresponding GenBank accessions are listed in Table 1.

Taxon selection
The general strategy of taxon selection followed the recent study by Wendt et al. (2018) and a previous comprehensive study by Hsieh et al. (2010), mainly using representatives of the Xylariaceae s. str. species of Rosellinia and presumably closely related genera like Coniolariella, Entoleuca, Euepixylon and Nemania made up the bulk of the taxa. The phylogeny thus covers all relevant taxa that have ever been connected with the genus Rosellinia and of which multi locus sequence data are extant in the public domain. In case sequences of the type species or the ex-(epi-)type strains were not available, data derived from vouchers of a related species in the same genus were chosen. As outgroups, members of the Graphostromataceae (Graphostroma platystomum) and the Hypoxylaceae (Hypoxylon fragiforme) were chosen (Fig. 1G, H).

Molecular phylogenetic analysis
DNA extraction, primer selection, resulting sequence analysis and processing as well as the alignment calculations were carried out as described previously (Wendt et al. 2018, Lambert et al. 2019 with Geneious® v. 7.1.9 (http://www.geneious.com, Kearse et al. 2012). Sequences were aligned via MAFFT v. 7.017 with the G-INS-I algorithm set to default in respect to gap opening and extension penalties. Regions of phylogenetic information were filtered by processing via the Castresana Lab Gblocks Server with stringency set to low (allows smaller final blocks and gap positions within final blocks, Talavera & Castresana 2007). The resulting alignments were concatenated with Geneious and the resulting multigene alignment submitted to the PhyML Online Server (http://www.atgcmontpellier.fr/phyml/) with the PhyML v. 3.0 algorithm for phylogenetic relationship inference and preliminary determination of the best fitting substitution model via smart model selection (SMS, see Guindon et al. 2010, Lefort et al. 2017. Automatic model selection was carried out by Bayesian Information Criterion (BIC). The tree topology was optimized by subtree pruning and regrafting (SPR). Bootstrap support values (BS) were calculated from 1 000 replicates. The phylogenetic tree was rooted with Graphostroma platystomum and Hypoxylon fragiforme.

Cultivation procedures
Cultures were grown first on yeast-malt-glucose agar plates (YMG) at 23°C in darkness. Erlenmeyer flasks with 30 mL YM medium were inoculated with four mycelial plugs (0.5 × 0.5 cm 2 ) of well-grown plates to obtain seed cultures. For the preparation of submerged cultures flasks with 200 mL medium were inoculated with four to six mycelial plugs (0.5 × 0.5 cm 2 ), while flasks with 400 mL or 1 000 mL medium were inoculated with well-grown seed cultures (7.5 %). The flasks were kept at 23°C and 150 rpm in darkness and the consumption of glucose was monitored regularly using glucose test stripes (Macherey-Nagel, Germany). The following liquid media were used: YM [malt extract 10 g/L, yeast extract 4 g/L, D-glucose 4 g/L, pH 6.3], ZM/2 (Rupcic et al. 2018b) [molasses 5 g/L, oatmeal 5 g/L, sucrose 4 g/ L, mannitol 4 g/L, D-glucose 1.5 g/L, CaCO 3 1.5 mg/L, edamin (lactalbumin hydrolysate, LP0048 from Oxoid) 0.5 mg/L, Table 1. List of used taxa for chemical analysis and phylogenetic reconstruction. GenBank accession numbers, strain ID of public culture collections or herbaria (if available), origin and reference studies are given. Type specimens are labelled with HT (holotype) or ET (epitype). Strains included in the chemical study are marked in bold. *This strain was labelled Rosellinia britannica, but this is a later erected synonym of Rosellinia marcucciana Ces., Atti dell Accademia di Scienze Fisiche e Matematiche Napoli (1872) 5:13 fide Petrini (2013).

Species
Strain number Table S2). For the isolation of compounds the corresponding culture volumes were increased up to 4 L in total (200-1 000 mL per flask). The cultivation was stopped three to four days after total consumption of glucose.

Extraction and isolation of secondary metabolites
For the extraction of submerged cultures biomass and culture medium were separated by gauze filtration. The biomass was extracted with acetone in an ultrasonic bath (2 × 30 min) and after removing the organic solvent under reduced pressure at 40°C the residual aqueous phase was extracted twice with ethyl acetate. The combined ethyl acetate phases were dried over sodium sulphate and the solvent was removed under reduced pressure at 40°C to yield the crude extract. The culture medium was extracted with ethyl acetate twice and processed as described above. Culture plates were cut into pieces and extracted with 30 mL ethyl acetate twice on a magnetic stirrer for 30 min. After filtration, the organic solvent was removed under reduced pressure at 40°C to yield the crude extract. The crude extracts were subjected to analytical HPLC-UV/Vis-MS analyses. For the isolation of compounds, the crude extracts were dissolved in 0.5-4 mL methanol and applied to RP solid phase cartridges (Strata-X 33 mm, Polymeric Reversed Phase; Phenomenex, Aschaffenburg, Germany) and eluted with methanol and acetonitrile to remove highly nonpolar contaminants (e.g. fatty acids) before injection for preparative HPLC.

Structure elucidation
1D and 2D NMR spectra were recorded on a Bruker Avance III 700 spectrometer with a 5 mm TXI cryoprobe ( 1 H 700 MHz, 13 C 175 MHz) and a Bruker Avance III 500 ( 1 H 500 MHz, 13 C 125 MHz) spectrometer. UV-Vis spectra were recorded with a UV-2450 Shimadzu UV−Vis spectrophotometer and optical rotations were determined with a PerkinElmer 241 polarimeter.

Bioactivity assays
In vitro cytotoxic effects (IC 50 ) against mouse fibroblast cell line L929 and human carcinoma cell line KB-3-1 of the compounds (1 mg/mL stock solutions in methanol) were determined as described by Surup et al. (2018). Minimum inhibitory concentrations (MIC) were obtained in serial dilution assays with pure substances dissolved in methanol and various bacterial and fungal test organisms (Kuephadungphan et al. 2017). Nematicidal activity was detected using an assay with Caenorhabditis elegans as described by Rupcic et al. (2018a). The nematodes were inoculated monoxenically on nematode agar (soy peptone 2 g/L, NaCl 1 g/L, agar 20 g/L, cholesterol (1 mg/mL in EtOH, 0.5 mL), 1M CaCl 2 (1 mL), 1M MgSO 4 (1 mL) and 40 mM potassium phosphate buffer 12.5 mL; pH 6.8) with living Escherichia coli (DSM498) at 21°C for 4-5 d. For the assay the nematodes were taken up in M9 buffer (0.5 M KH 2 PO 4 /K 2 HPO 4 and 0.1 M NaCl, pH 7.2) and counted using a Malassez counting chamber. A final concentration was adjusted to 500 nematodes per mL and 1 mL of this suspension was added to each well of a 24-well microtiter plate. The pure compounds were dissolved in acetonitrile or ethanol and were tested at different concentrations (100 μg/mL, 50 μg/mL, 20 μg/mL, 10 μg/mL). Ivermectin (in the same concentrations in ethanol) was the positive control. The plate was incubated on a plate shaker for 18 h at 24°C.

RESULTS
To infer phylogenetic relationships (Fig. 1), 32 different strains were included, viz. one representative of the Graphostromataceae (G, Graphostroma plastystomum), one representing the Hypoxylaceae (H, Hypoxylon fragiforme), two representing the coprophilous Xylariaceae (P) and 28 strains for the remaining Xylariaceae, representing nine different genera (X, A, N, D, R). From these strains, 28 new sequences were generated for their relevant DNA loci, whilst the remaining loci were complemented with 80 sequences from GenBank. The resulting multigene alignment (MGA) consisted of 4 176 characters, with 469 and 1 273 positions originating from ITS and LSU ribosomal DNA, respectively, and 1 101 and 1 333 characters derived from protein coding regions (RPB2 and TUB2).
The initial alignments as well as the final inferred phylogenetic tree of the curated and concatenated single-gene alignments are available in the Supplementary Information. The inferred phylogeny shows six supported (>50 % BS) distinct clades with the Xylariaceae rendered as strongly supported (99 %). The coprophilous Podosordaria mexicana and Poronia punctata (P) are placed as sister clade to other saprotrophic and pathogenic representatives. Additional clades include Xylaria and Kretzschmaria spp. (X); Xylaria polymorpha clustered within a weakly supported (54 %) Amphirosellinia clade (A); and the clade consisting of Nemania spp. (N) forms a well-supported sister clade (94 %) to the Rosellinia and Dematophora clades (R, D). Clade N consists of two well supported subclades (100 %) of Euepixylon sphaeriostomum and Nemania beaumontii on the one hand and the type strains of Nemania abortiva and Nemania primolutea as well as a non-type strain of Nemania bipapillata on the other hand. The sister clade of clade N consisted of two subclades, the Dematophora (D) and Rosellinia (R) clades.

Studies on secondary metabolism of Rosellinia s. lat
In parallel to the molecular phylogenetic investigations several strains were cultivated in four different liquid media (see cultivation procedures and Table S2) and their corresponding extracts were analysed by HPLC-UV/Vis-MS. After comparing the secondary metabolite production of the submerged cultures, two Dematophora strains, D. bunodes CBS 123585 and D. pepo CBS 123592, were selected for the cultivation in CM medium in larger scale for the isolation of compounds due to a variety of interesting HPLC-UV/MS profiles in their extracts and in case of CBS 123585 relatively high quantities of substances (Fig. S1). Two new (1-2) and seven known isopimarane diterpenoids, libertellenone M (3) and the γ-lactone of libertellenone M (4) (Kildgaard et al. 2017), myrocin B (5) (Hsu et al. 1988), spiropolin
In order to investigate the production of cyclodepsipeptides of the PF1022 family (Scherkenbeck et al. 2002) by species of the genus Rosellinia, as indicated in a patent application by Harder et al. (2011), several Rosellinia strains and Astrocystis mirabilis (ATCC 66432) were cultivated on three different solid media (see cultivation procedures and Table S2). PF1022 A (12) (Fig. 4) was detected in extracts of R. corticium (STMA 15209), A. mirabilis (ATCC 66432) and in highest concentration in the extract of R. corticium (STMA 13324), (Fig. 6). The compound was isolated from submerged cultures of strain STMA 13324 in CS medium (3 L) and used as standard for the analysis of extracts obtained within this study (details are given in the Supplementary  Information). Additionally, compounds with the masses of PF1022 C and PF1022 D with similar retention times to PF1022 A were observed in the CS medium extracts of R. corticium (STMA13324). After comparison with a fermentation sample of the original PF1022 producer strain, we concluded that these compounds were most likely cyclodepsipeptides PF1022 C and PF1022 D (Fig. S6).

Structure elucidation
Dematophorane A (1) was obtained as a colourless oil (21.3 mg) and its molecular formula was deduced as C 20 H 30 O 5 by high resolution ESI-MS (Fig. S2), indicating six degrees of unsaturation. The 1 H and 1 H, 13 C-HSQC NMR spectra of (1) revealed one olefinic methine proton, one olefinic methylene group with a characteristic carbon at δ C 113.3 ppm, four methines (three of them oxygenated), five methylene groups (one of them oxygenated) and three methyl groups. Additionally, the 13 C NMR spectrum exhibited signals of one ketone, two olefinic quaternary carbons and three sp 3 hybridized quaternary carbons. The 1 H, 1 H-COSY correlations between the methylene groups CH 2 -11 and CH 2 -12 as well as between methine H-1, methylenes CH 2 -2 and CH 2 -3 and the analysis of the 1 H, 13 C-HMBC correlations of (1) indicated the presence of a tricyclic diterpene scaffold. Key HMBC correlations include H-7 to C q -6, C q -9, C q -8, H-5 to C q -6, C q -9, C-1, C-18, C q -10, C q -4, C-3, C-19 and H-14 to C q -9, C q -8, C-7, C q -13, C-12, C-17 (Fig. 3). Further COSY correlations between olefinic methine H-15 and methylene CH 2 -16 and HMBC correlations of H-15 to C q -13, C-17, C-12 and C-14 demonstrated a terminal vinyl group at C q -13 between methylene CH 2 -12 and oxygenated methine H-14, a structural feature of many pimarane-type diterpenoids . The oxygenated methylene group at δ 3.08 (d, 1H), 3.46 (d, 1H), C 70.7 ppm was identified as hydroxylated methylene CH 2 -19 at C q -4 via 1 H, 13 C long range correlations of H-19 to C-5, C q -4, C-3 and C-18. The relative stereochemistry of dematophorane A (1) was assigned by the analysis of ROESY data, which showed strong correlations between methyl protons H-17, methine H-14 and methylene proton H-11a as well as between methyl protons H-20, H-18 and H-11a, indicating that methyl groups CH 3 -17, CH 3 -20 and CH 3 -18 as well as methine H-14 are positioned at the same face of the molecule. On the opposite side, methine proton H-5 displayed strong correlations to H-7 and H-1 (Figs. S6-11). This stereochemistry is in accordance with the typical structure of an isopimarane-type diterpenoid .
Dematophorane B (2) was isolated as colourless film (2.1 mg) and displayed a major peak in the HR-ESIMS spectrum at m/z 373.1984 [M+Na] + , calcd. for C 20 H 30 NaO 5 , 373.1991), consistent with the molecular formula C 20 H 30 O 5 and indicating six degrees of unsaturation (Fig. S3). The 1 H and 1 H, 13 C-HSQC NMR spectra of (2) were similar to those of (1) and showed signals which were assigned to a terminal vinyl group with J = 17.6,10.6), δ C 149.4) and CH 2 -16 (δ H 4.92 (dd, J = 10.7, 1.4), 5.00 (dd, J = 17.5, 1.4), δ C 110.9), one olefinic methine, three further methines (two oxygenated), five methylene groups (one of them oxygenated) and three methyl groups. The 13 C NMR spectrum exhibited signals of one ketone, one olefinic quaternary carbon, three sp 3 hybridized quaternary carbons and one oxygenated quaternary carbon. Similar to dematophorane A (1) a pimarane-type diterpenoid scaffold was assigned due to 1 H, 1 H COSY correlations between CH 2 -11 and CH 2 -12 as well as between OH-1, H-1, CH 2 -2 and CH 2 -3 and corresponding 1 H, 13 C HMBC correlations. But in contrast to derivative (1) an additional olefinic methine (δ H 5.84 (s), δ C 130.6) and an oxygenated quaternary carbon (δ C 75.3) were detected instead of a second olefinic quaternary carbon. 1 H, 13 C HMBC correlations of this olefinic methine proton H-14 to C-15, C q -8, C-7, C q -9 and C-12 indicated a position of the olefin between the terminal vinyl group and hydroxyl group OH-7. HMBC correlations of H-20, H-12, H-11, H-5, H-1 and OH-9 to the quaternary carbon C q -9 confirmed the assignment of a hydroxyl group at position C-9. The analysis of the NOESY data showed strong correlations between protons H-17, H-11a, H-12a and H-20 as well as between H-20, OH-1 and H-18. Additional NOE correlations between protons H-7, OH-9, H-12b, H-1 and protons H-5, H-7, H-1 demonstrated the same relative stereochemistry as for compound (1), (Figs. S12-17). During the purification and analysis processes, the rearrangement of dematophorane B (2) in methanol solutions at room temperature was observed. After keeping (2) in methanol at 40°C for 19 d, approximately 70 % of the molecule converted into another major compound, which was identified as libertellenone C (7). The slightly basic conditions and/or thermal energy seem to induce a rearrangement of the alpha-hydroxyketone of (2) and a final stabilization of the molecule by dehydrogenation.

Biological activity of extracts and pure compounds
Extracts were screened for antimicrobial activity against Grampositive/Gram-negative bacteria (Bacilus subtilis/Escherichia coli), a yeast (Candida albicans) and the filamentous fungus Mucor plumbeus as well as for nematicidal activity against Caenorhabditis elegans. The pure compounds were tested against a larger panel of bacteria and fungi (Table 3) and for their cytotoxicity on mouse fibroblast cells L929, cervix carcinoma cells KB-3-1 or primary human umbilical vein endothelial cells HUVEC (Table 3). Due to its instability the γ-lactone of libertellenone M (4) was not tested.

Taxonomy
The phylogenetic study, in which various species of Rosellinia and allies were compared in a multi-locus phylogeny, indicated that the genus should be subdivided. While the majority of species in Rosellinia sensu Petrini (2013) remain to be incorporated into a comprehensive molecular phylogeny, we noted a strong correlation between the type of asexual morph and the results of our phylogenetic study. Table 4 gives a summary on the crucial characteristics of the genus Rosellinia sensu Petrini (2013), summarising the most important morphological features of the seven species groups defined in the latter monograph.
All Rosellinia spp. that are known to form a dematophora-like asexual morph are included in the "R. buxi Group" or the "R. necatrix Group", and at the same time, their DNA sequences clustered in clade D. On the other hand, sequences of those species studied that have a geniculosporium-like asexual morph, clustered in clade R along with the type species, R. aquila. In order to make the systematics of Rosellinia more compatible with the evidence obtained from the current molecular study and congruent with the morphological evidence, in particular considering the "One Fungus-One Name" concept (see also Stadler et al. 2013 for a respective treatment of the stromatic Xylariales), we therefore propose to resurrect the genus Dematophora for the two aforementioned "Groups". We propose the following taxonomic rearrangements: Typus: Sweden, "Småland", (today Skåne) Lund, E. Fries (UPS, sub Sphaeria aquila -holotype).

Notes:
The above generic description is identical to the one by Petrini (2013) except for the definition of asexual morph. The species that remain in Rosellinia after segregation of Dematophora cannot be segregated by morphological features of the asci, ascospores or stromata. The species that are retained in the genus Rosellinia comprise the R. aquila, R. mammaeformis, R. emergens, R. mammoidea and R. thelena "Groups" (the latter of which has been regarded as subgenus Corrugata) as defined by Petrini (2013). A lot of work remains to be done in order to clarify their affinities and the genus may possibly have to be further subdivided once more molecular data and more information about the morphology of the asexual morphs become available (see Figs 7,8). The segregation of Dematophora also has some practical implications, since almost all important pathogens among the rosellinoid Xylariaceae, except for Entoleuca mammata and the conifer pathogens of the R. thelena Group sensu Petrini (2013, subgenus Corrugata) are included in the genus. We hope that this taxonomic change may facilitate future work on the non-pathogenic species that are retained in Rosellinia. Even though the present study does not provide any direct evidence that the "beneficial" compound PF1022 A actually can play a role in the protection of the host plants by the saprotrophic/endophytic producers, there are only few reports on potential, weak pathogenicity of the species that were not transferred to Dematophora.  Petrini 2013).
Emended generic description: Differs from Rosellinia as defined further above in the presence of characteristic synnemata of the Dematophora type, while sharing a similar morphology of stromata, asci and ascospores. The asexual morph is geniculosporium-like. Nodulisporium-like conidiogenous structures are not observed.

Notes:
The genus comprises the following species (below), all of which were previously retained in Rosellinia by Petrini (2013) and are reported to possess a dematophora-like asexual morph.  the isolated isopimarane diterpenoids. Some of them (1, 3, 5) displayed moderate cytotoxic effects on different cell lines. They remain to be tested for phytotoxic activities in order to evaluate possible natural functions as they were predominant in the extracts of the pathogenic D. bunodes and D. pepo. However, many compounds detected in the extracts of the submerged cultivations are not yet clearly identified. Further isolation campaigns, along with the modification of culture media, and in particular the inclusion of additional species will shed further light on the true potential of these ascomycetes to produce interesting metabolites, which might be also useful for the taxonomic classification. Cyclodepsipeptide PF1022 A (12), which is known for its strong nematicidal activity (Conder et al. 1995) was detected in different extracts of R. corticium (and Astrocystis mirabilis), but in some of them titres were very low (Fig. 6). A patent application by Harder et al. (2011) had already indicated that the genera Rosellinia (in particular R. aquila and R. corticium) and Coniolariella hispanica were producer organisms of PF1022 A. However, the data were derived from strains that are unavailable in public collections, and the corresponding stromata were likewise not identified by experts or deposited in a public herbarium. Furthermore, the compound was only detected by HPLC-MS. We were unable to find a corresponding publication in a peerreviewed journal. The patent application mentioned that two strains of R. abscondita from the CBS collection were also examined for comparison, but no data were presented suggesting that those strains produced the cyclodepsipeptides. Another patent application by Harder et al. (2012) treated the isolation and identification of novel PF1022 derivatives and mentioned that various strains, including R. abscondita (strains CBS 447.89, CBS 448.89 and CBS 450.89), "R. britannica" (current valid name: R. marcucciana, strain CBS 446.89) and R. mammaeformis (strain CBS 445.89), R. nectrioides (strain CBS 449.89, all the former resulting from the study of Petrini 1992) were studied, as well as R. millegrana (strain CBS 111.75), and even strains of D. necatrix and Xylaria hypoxylon. The patent application does, however, not indicate which, if any, of the organisms studied actually yielded the known and new metabolites. In addition, even though semi-preparative fractionations of the crude extracts were described, the new compounds were only identified tentatively by their HPLC-MS characteristics and not isolated to purity. The authors also reported on "lines" they grew from the CBS cultures, which were able to overproduce the compounds (presumably by some means of classical strain optimisation), but did not state from which of the strains these "lines" were derived. Evidently, the patent application was filed in order to protect the new intellectual property on the newly detected cyclodepsipeptides, rather than to disclose information on their distribution. However confusing these data have been reported, the work gives corroborating evidence that PF1022 derivatives and other cyclodepsipeptides are widespread in Rosellinia. In any case, our present study is the first one in which PF1022 A has been isolated to purity from an ascospore-derived isolate that is derived from a taxonomically well-defined specimen and characterised by NMR spectroscopy. Nematicidal activity was observed for extracts of several Rosellinia species even though PF1022 A could not be detected in all of them or just in very small amounts, which indicated that PF1022 A is only one active principle in the produced metabolite mixtures. Other components, including derivatives described by Harder et al. (2012) may also contribute to the anthelmintic activity of extracts from these species.