First detection of Trichothecium roseum causing leaf spots on tomato in Germany.

Tomato (Solanum lycopersicum L.) is one of the most popular vegetables in Germany and the demand for organically grown tomatoes in particular is growing steadily. In seasonal low-cost organic tomato cultivation, the leaf mould disease caused by Fulvia fulva is an increasing problem (Meyer et al. 2021). In 2020-2021, during a survey for F. fulva infected tomato leaves in Germany, untypical, circular, and dark brown necrotic spots were observed. To identify the pathogen, symptomatic leaves were collected from three different geographic locations in the north and south of Germany. Specimens were observed under a stereomicroscope and immediately processed. After surface disinfection of symptomatic leaf material, the leaf tissues were placed on potato dextrose agar (PDA) and subsequently incubated at 25 °C in the dark allowing mycelium to grow. Single spore isolates from each location were prepared for morphological and molecular analyses, as well as for pathogenicity determination. Three single spore isolates (JKI-GFP-22-010 - 012) showed moderate to rapid growth rates that reached 4 cm in diameter after 7 days. Based solely on morphology (S1), they were identified as Trichothecium roseum (Pers.) Link (Link 1809) by having pink dense mycelium with circular and rough edge colonies. The conidiophores were simple or branched (105 - 254 × 2 - 4 µm). Conidia were 2-celled, smooth, thick-walled, hyaline, ellipsoid to pyriform with slanting and truncate basal mark, and were produced in clusters, (10 - 18 × 7 - 9.5 µm). To afford the genetic data of these fungi, fungal DNA was extracted using a DNAeasy® Kit Plant Mini Kit (QIAGEN®, Germany) and then subjected to PCR using the primer sets as follows, ITS1/ITS4 (White et al. 1990) and LROR/LR6 (Vilgalys et al. 1990). The consensus sequences generated in this study were deposited in GenBank under the accession numbers ON787818 - 20 and ON787821 - 23, for ITS and LSU, respectively, and compared via NCBI-BLAST. Initial BLAST searches of both ITS and LSU regions revealed that the isolates share 99 - 100% homology with T. roseum. The morphology and phylogeny (S2) supported assignment of all isolates to T. roseum. To fulfil Koch's postulate, the pathogenicity of selected isolates (JKI-GFP-22-010 and 012) were tested by spraying spore suspensions (105 conidia ml-1) on adaxial surface of 21-day-old tomato plants cv. 'Moneymaker' (n=10). Plants sprayed with sterile water served as controls. All tomato plants were grown under greenhouse conditions (average 25 °C, 98% relative humidity). After 21 days, symptoms were identical to those of the originally infected leaves associated with T. roseum. As the disease progressed, necrotic lesions reached approximately 35 mm in diameter. The fungus was re-isolated and identified as T. roseum. No symptoms developed in the control plants. Trichothecium roseum has been detected infecting fruit, particularly on tomatoes, e.g. in Argentina (Dal et al. 2008), Brazil (Inácio et al. 2011), Korea (Yun et al. 2013), and Pakistan (Hamid et al. 2014). To our knowledge, this is the first report of T. roseum infecting tomato in Germany. As the pathogen was found in several locations in the south and north, it is likely already well established in Germany. To date, it seems to play a minor role in pathogenicity of tomatoes but should be monitored, especially with regard to climate change as there are now several recent reports from the temperate region. The disease development and interaction between susceptible host and pathogen should be concerned.

Analogs of the carotane antibiotic fulvoferruginin from submerged cultures of a Thai Marasmius sp.

A recent find of a Marasmius species in Northern Thailand led to the isolation of five unprecedented derivatives of the carotane antibiotic fulvoferruginin (1), fulvoferruginins B-F (2-6). The structures of these sesquiterpenoids were elucidated using HRESIMS, 1D and 2D NMR, as well as CD spectroscopy. Assessing the bioactivity, fulvoferruginin emerged as a potent cytotoxic agent of potential pharmaceutical interest.

Diversity of biologically active secondary metabolites from endophytic and saprotrophic fungi of the ascomycete order Xylariales.

Covering: up to December 2017 The diversity of secondary metabolites in the fungal order Xylariales is reviewed with special emphasis on correlations between chemical diversity and biodiversity as inferred from recent taxonomic and phylogenetic studies. The Xylariales are arguably among the predominant fungal endophytes, which are the producer organisms of pharmaceutical lead compounds including the antimycotic sordarins and the antiparasitic nodulisporic acids, as well as the marketed drug, emodepside. Many Xylariales are "macromycetes", which form conspicuous fruiting bodies (stromata), and the metabolite profiles that are predominant in the stromata are often complementary to those encountered in corresponding mycelial cultures of a given species. Secondary metabolite profiles have recently been proven highly informative as additional parameters to support classical morphology and molecular phylogenetic approaches in order to reconstruct evolutionary relationships among these fungi. Even the recent taxonomic rearrangement of the Xylariales has been relying on such approaches, since certain groups of metabolites seem to have significance at the species, genus or family level, respectively, while others are only produced in certain taxa and their production is highly dependent on the culture conditions. The vast metabolic diversity that may be encountered in a single species or strain is illustrated based on examples like Daldinia eschscholtzii, Hypoxylon rickii, and Pestalotiopsis fici. In the future, it appears feasible to increase our knowledge of secondary metabolite diversity by embarking on certain genera that have so far been neglected, as well as by studying the volatile secondary metabolites more intensively. Methods of bioinformatics, phylogenomics and transcriptomics, which have been developed to study other fungi, are readily available for use in such scenarios.

Cysteine-Derived Pleurotin Congeners from the Nematode-Trapping Basidiomycete Hohenbuehelia grisea.

The discovery of a Hohenbuehelia grisea specimen during a field trip in Northern Thailand led to the isolation and identification of three novel sulfur-bearing derivatives of dihydropleurotinic acid (4). Thiopleurotinic acid A (1) was established by the interpretation of spectral data (HRESIMS, 2D-NMR) as a 2-hydroxy-3-mercaptopropanoic acid conjugate of dihydropleurotinic acid. Thiopleurotinic acid B (2) was shown to be the N-acetylcysteine conjugate of 4. A third compound (3) was established as a thiazole-containing derivative. Through feeding experiments with [U-13C3, 15N]-l-cysteine the formation of all three metabolites was shown to involve cysteine condensation with 4. The decreased cytotoxicity and antimicrobial activities of the new derivatives 1-3, compared to the parent compound 4, indicate a possible detoxification pathway of filamentous fungi.

Antiviral 4-Hydroxypleurogrisein and Antimicrobial Pleurotin Derivatives from Cultures of the Nematophagous Basidiomycete Hohenbuehelia grisea.

4-Hydroxypleurogrisein, a congener of the anticancer-lead compound pleurotin, as well as six further derivatives were isolated from the basidiomycete Hohenbuehelia grisea, strain MFLUCC 12-0451. The structures were elucidated utilizing high resolution electron spray ionization mass spectrometry (HRESIMS) and 1D and 2D nuclear magnetic resonance (NMR) spectral data and evaluated for their biological activities; for leucopleurotin, we provide Xray data. While most congeners showed moderate antimicrobial and cytotoxic activity, 4-hydroxypleurogrisein emerged as an inhibitor of hepatitis C virus infectivity in mammalian liver cells.

Pyristriatins A and B: Pyridino-Cyathane Antibiotics from the Basidiomycete Cyathus cf. striatus.

Two novel pyridino-cyathane diterpenoids, pyristriatins A and B (1 and 2), together with striatin C (3) were isolated from cultures of Cyathus cf. striatus, a basidiomycete that was found during a field trip in northern Thailand. The pyristriatins showed antimicrobial effects against Gram-positive bacteria and fungi. The isolation, structure elucidation, relative configuration, and biological and cytotoxic activity are described. Their structures were assigned by HRMS and NMR spectroscopy. We also describe the first 2D NMR assignment of striatin C. Pyristriatins A and B are the first cyathane natural products featuring a pyridine ring.

Deconins A-E: Cuparenic and Mevalonic or Propionic Acid Conjugates from the Basidiomycete Deconica sp. 471.

Bioassay-guided fractionation of antibacterial extracts from cultures of a basidiomycete from Northern Thailand, which represents a new species of the genus Deconica, yielded the terpenoid deconin A (1), whose structure was elucidated by spectral methods (NMR, HRMS) as a cuparenic/mevalonic acid conjugate. The absolute configuration of 1 was determined after saponification and comparison of specific rotations of the resulting cuparenic acid and mevalonolactone with authentic standards and literature data. Six minor congeners (2-7) were isolated and identified, and their antimicrobial and cytotoxic effects are reported. Compounds 1-4 are the first natural products featuring an unmodified mevalonic acid residue as a building block.

Gymnopalynes A and B, chloropropynyl-isocoumarin antibiotics from cultures of the basidiomycete Gymnopus sp.

A chlorinated isocoumarin with an acetylenyl side chain and its 3,4-dihydro derivative, named gymnopalynes A (1) and B (2), were isolated from cultures of a basidiomycete originating from the rain forest of Northern Thailand. The producing organism was identified as a species of Gymnopus (Marasmiaceae). Their structures were elucidated by spectroscopic methods including UV/vis and NMR spectroscopy as well as high-resolution mass spectrometry as 3-(3-chloroprop-1-yn-1-yl)-1H-isochromen-1-one (1) and 3-(3-chloroprop-1-yn-1-yl)-3,4-dihydro-1H-isochromen-1-one (2). The absolute stereochemistry of 2 was assigned as S by CD spectroscopy. Both compounds showed weak to moderate antimicrobial and pronounced cytotoxic activities.

Retiboletus (Boletaceae) in northern Thailand: one novel species and two first records.

Morphological characters and multi-gene phylogenetic analyses were used to identify Retiboletus specimens collected in northern Thailand. Retiboletus brevibasidiatus is described as new to science, whereas R. fuscus and R. nigrogriseus are reported for the first time from Thailand. Retiboletus brevibasidiatus produces medium-sized basidiomes, with a dark blonde to clay pileus and densely reticulate stipe mostly on the upper part with pale yellow to chrome yellow basal mycelium. It is difficult to separate R. brevibasidiatus from other closely related species on the basis of macroscopic characters. However, the new species can be distinguished by microscopic characters, mostly the shorter basidia. The macro- and micro-morphology of the R. fuscus and R. nigrogriseus collections from Thailand fit well with the previous descriptions of materials from China and Japan. Detailed descriptions, molecular phylogeny, and illustrations of the three species are provided.

Polyketide-Derived Secondary Metabolites from a Dothideomycetes Fungus, Pseudopalawania siamensisgen. et sp. nov., (Muyocopronales) with Antimicrobial and Cytotoxic Activities.

Pseudopalawania siamensisgen. et sp. nov., from northern Thailand, is introduced based on multi-gene analyses and morphological comparison. An isolate was fermented in yeast malt culture broth and explored for its secondary metabolite production. Chromatographic purification of the crude ethyl acetate (broth) extract yielded four tetrahydroxanthones comprised of a new heterodimeric bistetrahydroxanthone, pseudopalawanone (1), two known dimeric derivatives, 4,4'-secalonic acid D (2) and penicillixanthone A (3), the corresponding monomeric tetrahydroxanthone paecilin B (4), and the known benzophenone, cephalanone F (5). Compounds 1-3 showed potent inhibitory activity against Gram-positive bacteria. Compounds 2 and 3 were inhibitory against Bacillus subtilis with minimum inhibitory concentrations (MIC) of 1.0 and 4.2 µg/mL, respectively. Only compound 2 showed activity against Mycobacterium smegmatis. In addition, the dimeric compounds 1-3 also showed moderate cytotoxic effects on HeLa and mouse fibroblast cell lines, which makes them less attractive as candidates for development of selectively acting antibiotics.

Biological and chemical diversity go hand in hand: Basidiomycota as source of new pharmaceuticals and agrochemicals.

The Basidiomycota constitutes the second largest higher taxonomic group of the Fungi after the Ascomycota and comprises over 30.000 species. Mycelial cultures of Basidiomycota have already been studied since the 1950s for production of antibiotics and other beneficial secondary metabolites. Despite the fact that unique and selective compounds like pleuromutilin were obtained early on, it took several decades more until they were subjected to a systematic screening for antimicrobial and anticancer activities. These efforts led to the discovery of the strobilurins and several hundreds of further compounds that mainly constitute terpenoids. In parallel the traditional medicinal mushrooms of Asia were also studied intensively for metabolite production, aimed at finding new therapeutic agents for treatment of various diseases including metabolic disorders and the central nervous system. While the evaluation of this organism group has in general been more tedious as compared to the Ascomycota, the chances to discover new metabolites and to develop them further to candidates for drugs, agrochemicals and other products for the Life Science industry have substantially increased over the past decade. This is owing to the revolutionary developments in -OMICS techniques, bioinformatics, analytical chemistry and biotechnological process technology, which are steadily being developed further. On the other hand, the new developments in polythetic fungal taxonomy now also allow a more concise selection of previously untapped organisms. The current review is dedicated to summarize the state of the art and to give an outlook to further developments.

Study of three interesting Amanita species from Thailand: Morphology, multiple-gene phylogeny and toxin analysis.

Amanita ballerina and A. brunneitoxicaria spp. nov. are introduced from Thailand. Amanita fuligineoides is also reported for the first time from Thailand, increasing the known distribution of this taxon. Together, those findings support our view that many taxa are yet to be discovered in the region. While both morphological characters and a multiple-gene phylogeny clearly place A. brunneitoxicaria and A. fuligineoides in sect. Phalloideae (Fr.) Quél., the placement of A. ballerina is problematic. On the one hand, the morphology of A. ballerina shows clear affinities with stirps Limbatula of sect. Lepidella. On the other hand, in a multiple-gene phylogeny including taxa of all sections in subg. Lepidella, A. ballerina and two other species, including A. zangii, form a well-supported clade sister to the Phalloideae sensu Bas 1969, which include the lethal "death caps" and "destroying angels". Together, the A. ballerina-A. zangii clade and the Phalloideae sensu Bas 1969 also form a well-supported clade. We therefore screened for two of the most notorious toxins by HPLC-MS analysis of methanolic extracts from the basidiomata. Interestingly, neither α-amanitin nor phalloidin was found in A. ballerina, whereas Amanita fuligineoides was confirmed to contain both α-amanitin and phalloidin, and A. brunneitoxicaria contained only α-amanitin. Together with unique morphological characteristics, the position in the phylogeny indicates that A. ballerina is either an important link in the evolution of the deadly Amanita sect. Phalloideae species, or a member of a new section also including A. zangii.