Welcome to IMC 2018 International Mycological Congress
Correlations between biodiversity and secondary metabolism in endophytic and saprotrophic Xylariales
- M. Stadler
- S. Helaly
- B. Thongbai
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. Moreover, 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 correlations between biological and chemical diversity in this fungal order will be demonstrated, based on some striking examples. Furthermore, future challenges in their exploitation for applied mycology will be outlined. Those include the studies of their volatiles, as well as the exploitation of their secondary metabolome, using methods of bioinformatics, phylogenomics and transcriptomics. A major challenge for the future will be the development of concise, diagnostic PCR-based methods to identify the endophytic stages to species rank and elucidate their life cycle, using better-suited DNA loci than ITS sequence data, which have proven highly unreliable, leading to bad scientific practice and hence, chaos in the literature. The talk is based on the review “Diversity of biologically active secondary metabolites from endo-phytic and saprotrophic fungi of the ascomycete order Xylariales” by Soleiman E. Helaly, Benjarong Thongbai and Marc Stadler (submitted to Natural Products Reports).
Epichloë in South America: how many, where and why?
- L. Iannone
Some grasses in the subfamily Pooideae establish symbiotic associations with endophytes of the genus Epichloë (Clavicipitaceae). Whereas some species produce stromata with perithecia that abort the development of the florets causing total or partial sterility in the host, in other cases the endophyte colonizes the ovary to be spread in the seeds of the host. This association is considered to be mutualistic because the endophytes may provide the host with enhanced growth and resistance to abiotic stresses and produce alkaloids against herbivores. In this work, we studied the genus Epichloë in native grasses from South America, considering its diversity, host range, distribution and effect for the host plant. The presence of Epichloë was studied in field collected plants and herbarium material. When possible, the endophytes were isolated and characterized morphologically and genetically by mean of phylogenies of nuclear genes and detection of alkaloid-biosynthesis genes. The distribution of the different lineages was modelled using climatic variables. The effect of Epichloë on the growth of some hosts and its capacity to establish associations with mutualistic, pathogenic and soil fungi was studied by comparing Epichloë-infected plants (E+) with Epichloë-removed plants (E-). In Argentina and Uruguay, where surveys were performed more intensively, Epichloë was detected in 41 grass species, and at least eight more hosts species were identified along Cordillera de los Andes from Venezuela to Argentina. Although sexual species of Epichloë have not been reported from this region, molecular phylogenies have revealed that all the host species are associated with endophytes that evolved from the hybridization between different sexual species. Gene sequences and genetic profiles revealed at least 19 genotypes grouped in eight different lineages. In general, each host species can be associated with different endophytes and different endophytes may co-exist in the population as observed in Phleum alpinum or Bromus pictus, or to present different distribution areas as with endophytes of Bromus auleticus. Wide host-range endophyte lineages, endophyte diversity in individual host species, and relationships between endophytes from different host species in the same community suggest the occurrence of horizontal transmission between hosts, multiple independent hybridization events, or both. In Bromus setifolius and B. auleticus the endophyte increases plant growth and promotes seed germination. The association with Epichloë promotes mycorrhizal colonization and growth and confers resistance to the smut fungus, Ustilago bullata. Epichloë endophytes have also impact on the diversity of other endo-symbionts such as dark septated endophytes, mycorrhizal fungi, endophytic actinomycetes, and on free living soil fungi. Considering the high diversity of grasses and environments as well as the recent and fast radiation of the Pooideae in South America, it is likely that many other host and Epichloë species remain to be discovered in South America, a region that offers the opportunity to study evolutionary and ecological aspects of the symbiosis between grasses and Epichloë endophytes.
Amazing alkaloid diversity in Clavicipitaceae! How and why?
- C. Schardl
- D. Berry
- D. Cook
- S. Lee
- D. Panaccione
- B. Scott
- P. Tudzynski
- C. Young
Plant-associated members of the fungal family Clavicipitaceae — including species of Balansia, Claviceps, Epichloë, Metarhizium, Periglandula and others — are famous for the production of alkaloids belonging to five different classes: ergot alkaloids (named for the “ergot” sclerotia of Claviceps species), indole-diterpenes (e.g., lolitrems), aminopyrrolizidines (e.g., lolines), pyrrolopyrazines (e.g., peramine) and the indolizidines (e.g., swainsonine). As the genes and much of the pathways for biosynthesis of these alkaloids have been elucidated, it has become evident that profiles of alkaloids are extremely diverse among species and among strains, and also with respect to the alkaloid classes and specific end- and spur-products within each class. Furthermore, genomic studies have revealed details of several genetic mechanisms for evolutionary diversification. Surprisingly, loss of certain genes in alkaloid clusters is a very common mechanism to generate variation in alkaloid profiles. For example, some fungi have as many as 14 ergot alkaloid genes for biosynthesis of ergopeptines and other lysergic acid amides, whereas others have only 12, 11 or nine genes, or just the genes for the first four steps resulting in chanoclavine. Analogous situations are evident in the indole-diterpene and aminopyrrolizidine gene clusters. Another diversifying mechanism, gene duplication and neofunctionalization, has characterized certain enzyme classes including nonribosomal peptide synthetases (NRPS) for biosynthesis of ergopeptines and lysergyl amides (ergot alkaloids), as well as cytochrome P450 monooxygenases and prenyltransferases for biosynthesis of indole-diterpenes, thereby contributing to the huge chemical diversity of these alkaloids. Additionally, trans-species polymorphism is a feature of the loline alkaloid and pyrrolopyrazine biosynthesis genes, and in the latter case has worked in concert with interallelic recombination to give an array of pyrrolopyrazines in addition to peramine. Although horizontal gene transfer has been suggested for some of the alkaloid gene clusters, evidence is consistent with recombination, neofunctionalization, diversifying selection and perhaps frequency-dependent selection as causes of their unexpected phylogenies and distribution patterns in the fungi.
An ash dieback pathogen, Hymenoscyphus fraxineus, endophytically inhabits leaves of manshurian ash in Japan
- T. Inoue
- I. Okane
- T. Hosoya
- Y. Degawa
- Y. Ishiga
- Y. Yamaoka
A helotialean ascomycete, Hymenoscyphus fraxineus (T. Kowalski) Baral et al., causes a lethal disease known as ‘ash dieback’ in the common ash Fraxinus excelsior of Europe. The origin of this fungus is considered to be East Asia. The same fungus was found on leaf litter of manshurian ash, F. mandshurica, in Japan and reported to produce apothecia on pseudosclerotial plates formed on decomposing leaves. However, occurrence of dieback disease has not been reported in Japan. Therefore, the life cycle of H. fraxineus is largely unknown. The aim of this study was to clarify the behavior of H. fraxineus on F. mandshurica in Japan. Healthy leaves were collected from an adult tree and F. mandshurica planted at the Sugadaira Research Station of the University of Tsukuba in Nagano Prefecture, Japan for every one to two weeks from July to October. The sporulation period of the fungus is from middle July to early September at this site. Collected leaves were processed by surface sterilization method with sodium hypochlorite solution (1% available chlorine) to isolate endophytic fungi and by washing method with 0.005% aerosol OT to isolate both epiphytic and endophytic fungi. Isolates were identified based on morphology and sequence analysis of rDNA ITS regions. To detect the presence of H. fraxineus inhabiting leaves, DNA was extracted from leaf samples followed by DNA-based fungal species-specific real-time PCR assays by amplifying rDNA ITS regions with specific primers for H. fraxineus. In addition, the behavior of the fungus on fallen leaves was observed by using continuously retrieving fallen leaves from litter bags placed in the field. Results showed that one strain isolated by the surface-sterilization method was H. fraxineus. By the DNA detection test, the frequency of fungal DNA was low on fallen leaves collected from July to September, but the frequency sharply increased on adult tree leaves collected in October before defoliation. A high concentration of DNA was detected from all leaflets and rachises. Observations of fungal behavior on the fallen leaves revealed Chalara anamorph of H. fraxineus on blackish mycelial structure formed on the surface of rachises retrieved in December. Subsequently, a pseudosclerotial layer was found under the cortical layer in the rachises retrieved in January, and then, gradual degradation of the cortical layer was observed on rachises retrieved from February to June. Finally, a blackish pseudosclerotial plate appeared on the rachises. The near-UV light irradiation test successfully induced apothecia were on rachises retrieved from the litter bags in November and December. These results suggest that fertilization occurs swiftly after defoliation. In conclusion, H. fraxineus infects the living leaves of F. mandshurica by ascospores and endophytically inhabits the leaves until defoliation, and the fungus behaves saprophytically on fallen leaves after fertilization and produces apothecia on pseudosclerotial plates formed on the decomposing leaves.
Description of Darksidea, a novel genus within Pleosporales, and evaluation of their effects on grasses
- M. Romero-Jiménez
- A. Jumpponen
- J. Rudgers
- J. Herrera
- A. Porras-Alfaro
Dark septate endophytes are dominant colonizers in arid plants. Pleosporalean fungi that belong to the genus Darksidea were recently described, yet their distribution and function in semiarid grasses remains poorly known. The objective of this study was to describe novel Darksidea fungi and evaluate their effect on semiarid grasses. A total of 24 sites and 6 grass species were sampled across grassland ecosystems in central south states in the United States. We used a collection of 77 Darksidea isolates and characterized them using light microscopy and culturing in different media. Fungi were identified using the ITS rRNA region. The resulting sequences were compared with curated databases such as the Ribosomal Database Project and UNITE. Among the 77 congeneric Darksidea isolates, we identified a total of eight Operational Taxonomic Units (OTUs) defined at 97% similarity, representing likely yet unidentified diversity within the genus. Using representative isolates from each OTU, five potential novel clades of Darksidea were identified. Most isolates were obtained from the southwest field sites in Texas and were abundant in three of the sampled plant species (Bouteloua dactyloides, B. eriopoda and B. gracilis). The potential function of the fungal isolates was assessed using grass germination bioassays to evaluate direct contact and volatile organic compounds effects. Darksidea isolates vary in their role to improve plant growth and survival after 30 days. However, most fungal isolates produced VOCs that enhanced plant growth.
There’s treasure everywhere – putatively overlooked slow-growing fungi isolated from cereal cyst nematodes produce nematode-inhibiting compounds
- W. Maier
- S. Ashrafi
Cereal cyst nematodes (CCNs) can lead to significant yield reductions of grains. The use of nematicidal chemicals is banned in many countries due to their generally high non-target toxicity. Therefore, antagonistic microorganisms controlling nematodes are an important alternative. The search for such microorganisms, especially nematophagous fungi, has a long history extending back some 150 years. These studies provided a long list of nematode-associated fungi some of which showed great potential to be exploited as biological control agents. Microscopic observations of cyst samples of the CCN Heterodera filipjevi obtained from wheat fields in Turkey regularly revealed nematode cysts, which displayed fungal colonisation. The aim of our study was to (i) isolate the fungi from the nematode cysts and fulfil Koch’s postulates (ii) to classify the isolated fungi using light microscopic and molecular phylogenetic analyses (iii) to study the nematode-fungus interaction microscopically, (iv) to isolate and identify secondary metabolites produced by these fungi. Fungi were isolated from symptomatic cysts applying a specific single-egg isolation technique developed for this study. Fungal strains were identified using morphological studies and multi-locus molecular phylogenetic analyses. To fulfil Koch’s postulates, the pathogenicity of isolated fungal strains was examined against nematode eggs in vitro. Secondary metabolites of fungal isolates of interest were extracted and purified using EtOAc, and HPLC-based techniques. The bioactivity of obtained compounds was evaluated using nematode bioassays. This approach resulted in finding six new fungal species. All species are ascomycetes belonging to the Helotiales, Hypocreales and Pleosporales. The newly described Ijuhya vitellina and Monocillium gamsii belong to the families of Bionectriaceae and Niessliaceae, respectively. A new fungal genus was proposed to accommodate two new species. One of these, representing a dark septate endophyte (DSE), was isolated from nematode eggs. Two more species were preliminarily characterized as DSEs. These are the first DSEs found to parasitize nematode eggs and they might play a role in the plant defense against nematodes. All newly-found species could be successfully re-isolated from artificially infected nematodes and Koch’s postulates were thus fulfilled. Both I. vitellina and M. gamsii formed microslcerotia within the nematode eggs and in culture. Chaetoglobosin A, 19-O-acetylchaetoglobosin A and four novel compounds, among them cyclodepsipeptides, a class of compounds known for anthelmintic effects were isolated. Nematicidal and nematode-inhibiting activities were demonstrated for the isolated compounds. To conclude, using a specific isolation technique novel fungal species and novel compounds could be discovered from the CCN H. filipjevi that might be harnessed for biological control in the future.