Abstract

Dipterocarpaceae is an important tree family in Paleotropics that form ectomycorrhizal (EcM) symbiosis with fungi and it has been hypothesized that dipterocarps have been partnered in this mutualistic association prior to the separation of Gondwana. Two species of trees have been recently described from Neotropical ecosystems, Pakaraimae dipterocarp from Guyana and Venezuela, and Pseudomonotes tropenbosii from Colombia. We documented the EcM fungal diversity in a terra-firme forest with the endemic dipterocarp Pseudomonotes tropenbosii in the lowlands of Colombian Amazonia by collecting fruit bodies and by using rDNA sequence analysis of root material for identification of both fungal and plant symbionts. We addressed whether the fungal EcM community associated with P. tropenbosii exhibited spatial differences and if the fungal community revealed similar composition that other Neotropical hosts. A total of 83 species of EcM macrofungi were identified based on morphology-based techniques. These taxa represented 16 families and 27 genera. The most abundant families were Boletaceae (7 genera; 13 species), Clavulinaceae and Russulaceae (13 species). Fifteen species constituted new reports for Colombia and at least 18 species found in the study area new to science. Two of those have recently been described as Austroboletus amazonicus, Sarcodon colombiensis and others belong to genera such as Russula, Coltriciella, Coltricia, and Amanita. Species richness detected from 200 fragments of mycorrhizal roots tips revealed 34 species-level (ITS sequences). Seven species were detected three or more times such as Craterellus cinereofimbriatus, Uncultured Cortinarius 866root, Uncultured Tomentella 1452root and Uncultured Sebacina sp. 8. The hosts identified were P. tropenbosii, Coccoloba sp. and other plants not reported as EcM hosts as for example Brosimun, Ipomoea and Protium. Differences were observed in the composition of the EcM fungal community for 3 populations of P. tropenbosii in Colombia Amazonia. Most of the fungal species documented in this study have also been found in symbiotic associations with other legume or dipterocarp species from geographically distant forests located in Brazil, French Guyana, Guyana and Venezuela. The distribution of some fungal species that were previously considered restricted to the Guiana Shield was extended to P. tropenbosii forests in Colombia. The result highlights the low specificity of EcM fungi in relation to their host plants in Neotropical lowland forests. It is important to address further studies in understanding how factor such as forest structure, size of the host plants patches, host distributions and edaphic factors may drive the structure of the EcM fungal communities in Neotropical ecosystems.

Abstract

Ectomycorrhizal (ECM) fungi play a major role in tropical and subtropical African forest ecosystems, where many trees, often growing on N- and P-poor soils, completely depend on these associations. Based on above-ground biodiversity records, Russulaceae are the dominant group of ECM fungi in tropical African ecosystems, followed by Boletales and Cantharellaceae. The first soil diversity studies, however, show that Thelephoraceae follow Russulaceae in below-ground species richness. Sub-Saharan Africa is characterized by three vegetation types dominated by ECM associations: Central African Guineo-Congolian rainforests, West African Sudanian woodlands and East African Zambezian Miombo woodlands. Little is known concerning composition and distribution of ECM fungal communities in these vegetation types. We studied ECM fungal diversity of rainforest in Cameroon, Sudanian woodland in Togo and Miombo woodland in Zambia. Root tips were sampled in multiple plots per vegetation type and IonTorrent was used to sequence the ITS2 region from the root tips. Our results confirm Russulaceae as dominant ECM group below-ground in all three vegetation types, followed by Sebacinaceae and Thelephoraceae. ECM fungal community composition is strongly correlated with edaphic factors, with many fungi occurring in either woodland (high pH, together with low C, N and organic material) or rainforest (low pH, together with high C, N and organic material). ECM community composition thus differed amongst the three vegetation types, with the main regions of overlap occurring in the riparian forests in between the vegetation types. It is difficult to draw conclusions on species richness based on metagenomic sequences. Due to the absence of a taxonomical reference framework for most fungal groups, the majority of operational taxonomical units (OTU’s) could only be identified on genus level (>50%) or higher (>15%). This makes it difficult to see trends in species composition between vegetation types. Only for the milkcap genus Lactifluus (Russulaceae), for which we constructed a solid taxonomical framework over the years, most OTU’s could be identified on species level and more detailed conclusions on above- versus below-ground species richness could be drawn.

Abstract

Oreomunnea mexicana (Juglandaceae) is an ectomycorrhizal (ECM) tree distributed from Mexico to Panama. This species forms monodominant stands in tropical montane forests between 1000 and 1900 m a.s.l. Recent studies of O. mexicana in Panama indicate that this species is associated with a diverse community of ECM fungi, but this tree has been studied at only a few sites. We collected fruiting bodies and root tip samples from eight localities (four in Panama and four in Mexico) to elucidate the effect of geographic location, soil fertility, precipitation, temperature, and host abundance on the ECM fungal communities associated with O. mexicana. Fungal fruiting bodies were preserved and ITS sequences were generated for molecular identification. In addition, ECM roots were sampled from 60 O. mexicana trees (40 trees in Mexico and 20 in Panama) and the fungal communities were assessed based on Illumina ITS1 sequencing. We sequenced 874 ECM fruiting bodies and preliminary results suggest a high a diversity of ectomycorrhizal fungi with approximately 360 OTUs. Of these, 17 OTUs were shared between sites in Mexico and Panama. Fungal diversity in O. mexicana roots based on Illumina sequencing was high, with approximately 4000 OTUs (97% OTUs cutoff and all OTUs > 10 reads) with 1541 belonging to ECM genera. Alpha diversity of both the root-associated fungi and the ECM fungi were higher in Mexico than in Panama, consistent with a previously documented global pattern of decline in species richness of ECM fungi towards the equator. This lower species diversity of ECM fungi in Panama could be associated with a lower species richness and abundance of ECM plant species in the surrounding forests. Beta diversity was high at both regional and local scales with a significant turnover of fungal species. At a local scale, β-diversity was higher in Panamanian O. mexicana populations, with species composition changing rapidly over a short geographical distance. These results are consistent with previous findings based on Sanger sequencing where high β-diversity was found with O. mexicana in sites with contrasting soil fertility. Permutation tests determined that total soil nitrogen was the environmental variable that significantly explained the most variation of both the total root-associated fungal community and the ECM community. However, total soil nitrogen was also highly correlated with total soil carbon (98%). This is the first study to focus on the variation of ECM communities associated with a tropical tree at both regional and subcontinental scales. This work supports the hypothesis that soil nutrient availability is the main factor structuring local ECM fungal communities and that ECM fungal communities are less species rich towards the equator.

Abstract

Neotropical dry forest extends from Northern Mexico to Colombia, Brazil, and Peru, also including Caribbean islands. This ecosystem is imperiled by the land use change to agriculture or grazing. Plants and their symbionts from this forest have adaptations to resist long dry periods. Our main objective was to detect the ectomycorrhizal hosts and their mycobionts by sampling roots, fruitbodies and soil, and describe their interactions. We sampled roots from two places from the Mexican Pacific coast, Jalisco and Oaxaca. The roots were dissected and all the ectomycorrhizae (ECM) morphotypes were separated for anatomical observations and sequencing. We also sampled fruitbodies and soil, amplified ITS region, and sequenced by Sanger and Illumina respectively. To identify the photobiont we amplified rbcL, matK and trnL regions from the ECM. We found that ECM were restricted to particular plant genera, such as: Achatocarpus, Coccoloba, Guapira, Pisonia (Caryophyllales), and also some legumes, for example Lonchocarpus. These plants are frequent to scarce in the forest, none of them forms patches. From 19 ectomycorrhizal fungal species we amplified from roots, 18 were not in NCBI or UNITE data bases. ECM fungi belonged to Clavulina, Inocybe, Membranomyces, Russula, Sebacina, Thelephora, Tremelloscypha, Tomentella. We have described three new species including their fruitbodies and mycorrhizal associations: Thelephora versatilis, T. pseudoversatilis and Tomentella brunneoincrustata (Thelephoraceae). Illumina sequences found 121 ectomycorrhizal species as propagules in the soil. Tremelloscypha sp. was the most frequent on root-tips, forming fruit bodies and also in soil sequences. The ECM morphotypes were monopodial and rarely ramified, with a wide range of colors (whitish to dark hyphae), mantle with contact exploration type to mantle with abundant rhizomorphs. Coccoloba, Guapira and Pisonia morphotypes developed a paraepidermal Hartig net, however Achatocarpus does not form Hartig net. Under dry periods, ECM from Coccoloba have incomplete mantles, covering just some parts of the root. Our results showed that ECM fungi are mostly specific, its hosts are scarce in the forest, the ectomycorrhizal fungi are probably endemic, their morphotypes do not present the "typical" ectomycorrhizal characteristics and these vary depending on host and environmental conditions.

Abstract

Abstract

Abstract

Powdery mildew fungi (Erysiphales) are obligate biotrophic plant pathogens, infecting around 10,000 dicot species and also some members of the Poaceae. Important crops, including wheat, barley, grapevine, apple and a number of cultivated and ornamental plants, are amongst the major targets of powdery mildew fungi. Pycnidial fungi belonging to the genus Ampelomyces are commonly found in powdery mildew colonies in the field, and some selected strains have been developed as biocontrol agents of different powdery mildew species. To improve visualization of the interaction between Ampelomyces spp. and their mycohosts, we produced GFP expressing Ampelomyces transformants with Agrobacterium-mediated transformation using Agrobacterium tumefaciens strain AGL1 carrying a plasmid with the hygromycin resistance and GFP genes. Transformants were selected on hygromycin-containing medium and were checked for fluorescence after being grown in culture. Growth characteristics and mycoparasitic activity of transformants were measured and compared to those of the wild type. Selected transformants were used in mycoparasitic tests using five different powdery mildew species. In these experiments, sporulating powdery mildew colonies were inoculated with spore suspensions of transformants. We have also conducted persistence tests, in which experimental plants were inoculated first with GFP expressing transformants, and one week later with powdery mildew conidia. The transformation method was effective as several transformants emerged on the selective medium and exhibited strong green fluorescent signal. Transformants were genetically stable as they emitted strong green fluorescence after several subculturing in the absence of selective pressure. Most transformants did not differ in growth characteristics and mycoparasitic activity from the wild type. In mycoparasitic tests we observed extensive intracellular colonization of powdery mildew hyphae, conidiophores and conidia; intracellular Ampelomyces hyphae, as well as pycnidia and conidia produced in powdery mildew structures exhibited strong green signals when examined with fluorescence microscopy. In persistence tests Ampelomyces germinated on plant leaves in the absence of their mycohosts, and parasitized powdery mildew colonies as soon as these were available on the inoculated leaves. This work showed that Ampelomyces is amenable to Agrobacterium tumefaciens-mediated transformation and the commonly used heterologous marker and reporter genes like hygromycin resistance and GFP can be efficiently used. Transformation with GFP is useful for improving direct observation of this interfungal parasitic relationship. Our persistence tests demonstrated that Ampelomyces strains can act as biocontrol agents even if the target pathogen infects plants one week after Ampelomyces application.

This work was supported by a grant of the Hungarian Research, Development and Innovation Office (NKFIH NN100415), a grant of the Austrian-Hungarian Action Foundation (90öu16) and Janos Bolyai Research Fellowship to AP.

Abstract

Pucciniomycotina is a subphylum with a high diversity in terms of habitat and life history strategies that include plant parasites, animal associates (including opportunistic human pathogens), saprobes and antagonists of other fungi. Antagonistic interactions can occur through: 1) direct physical contact between two fungi, i.e., mycoparasitism; or, 2) the production of killer toxins and other agents (known as the killer-toxin phenomenon). Killer toxins are the less studied of these two types of interactions, yet may play a significant role in the development of community structure in natural environments. The killer-toxin phenomenon was first described in Saccharomyces cerevisiae and has been more extensively studied in ascomycetous yeasts, while in Basidiomycota only 50 yeast species have been reported as producers of killer toxins, including a few species in the Cystobasidiomycetes. In this class, direct physical antagonistic interaction which is associated with sexual states has been reported in species of Cystobasidium, Naohidea, Cyphobasidium and Occultifur. On the other hand, the killer-toxin phenomenon which mainly occurs between the yeast stage of the fungi and other organisms has only been reported in Cystobasidium minutum, C. pallidum and Hasegawazyma lactosa. We hypothesize that the common ancestor to Cystobasidiomycetes is a mycoparasite that also produced killer toxins. To test this hypothesis, we evaluated the presence of killer toxins for 54 strains belonging to 24 species of Cystobasidiomycetes (including 14 species new to science). A sensitive strain was allowed to grow for 24h on media containing 0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1.0% glucose, and 2.0% agar, supplemented with 0.003% methylene blue and pH 4.2. Each strain in the Cystobasidiomycetes tested for killer activity was also incubated for 24h on Yeast Malt agar and inoculated by making a single streak on the plate containing the sensitive strain. Cultures were evaluated every 24h for 5 days for the presence of an inhibition zone with no growth. In addition, we constructed a resolved phylogeny for the class based on six loci (ITS-including 5.8 rDNA, LSU rDNA, SSU rDNA, and the protein coding genes RPB1, RPB2, TEFα) to determine the evolutionary origins of mycoparasitism through ancestral character reconstruction.

Abstract

Soil fungi belonging to the ascomycete genus Trichoderma have the potential to provide environmental-friendly biocontrol of plant diseases. Biochemical and molecular genetic studies have clearly related the mycoparasitic behaviour of Trichoderma against phytopathogenic fungi and oomycetes with the secretion of chitinases, glucanases and proteases with the cell wall degrading activity. In addition, Trichoderma proteases can also hydrolyze nematode cuticles and eggs and inhibit enzymes produced by the pathogens to penetrate the plant. Comparative genome sequence analysis of biocontrol species of Trichoderma has revealed that the mycoparasitic activity in the rhizosphere facilitates the formation of endophytic associations and the evolution of positive interactions with plants, supporting the application of Trichoderma strains as plant biostimulants in agriculture and forestry. In this sense, it has been observed worldwide that, as a general rule, the Trichoderma positive impact is more apparent in plants subjected to some stress. Early transcriptomic responses of Trichoderma colonizing tomato roots have shown that genes related to the formation of infection structures in plant tissues resulted upregulated, and once the hyphal root attachment has already taken place, nutrient uptake and carbohydrate metabolism would be limited by plant defenses. This means that Trichoderma is capable of overcoming plant defense responses during the initial stages of the interaction, when the early systemic defense responses would not be reaching its full potential, allowing Trichoderma an intercellular apoplastic colonization. As a result, Trichoderma exerts beneficial effects on plants in terms of improvement or maintenance of soil productivity, increased percentages and rates of seed germination, nutrient uptake, growth promotion, alleviation of adverse effects caused by environmental damage and systemic defense stimulation against abiotic stress and pathogen attack, without the need of establishing any contact with the invader. Trichoderma-primed plants that have a priming memory are able to react more rapidly and more adequately when challenged by a stressor. We have recently observed that tomato progeny inherit resistance to pathogens linked to plant growth induced by Trichoderma, without compromising the level of defense.

Abstract

Biological control of plant diseases holds great promise for replacing chemical pesticides in future food production, as part of integrated pest management. The mycoparasitic fungus Clonostachys rosea is an efficient biological control agent under field conditions for a variety of plant diseases on agricultural crops. In order to improve our understanding of critical components of the mycoparasitic lifestyle of C. rosea, we sequenced the genome of C. rosea strain IK726 using Illumina/PacBio technology. Comparative genomics revealed a significant increase in the number of certain ABC-transporters, MFS-transporters, proteases, polyketide synthases, cytochrome P450 monooxygenases, pectin lyases and GMC oxidoreductases compared with other Hypocrealean fungi. Interestingly, the increase of membrane transporter gene number in C. rosea was primarily associated with efflux drug resistance transporters. Necrotrophic mycoparasites such as C. rosea are assumed to have broad host range with little specificity. However, transcriptomic analyses revealed that C. rosea responded with both common and specific gene expression during interactions with the plant pathogenic species Botrytis cinerea and Fusarium graminearum. In agreement with the data on increased gene copy numbers, the majority of the regulated genes were predicted to encode proteins involved in membrane transport, biosynthesis of secondary metabolites and carbohydrate-active enzymes. Whole-genome re-sequencing of 63 C. rosea strains followed by genome wide association studies of phenotypic variation related with biocontrol of Fusarium diseases on wheat further identified several membrane transporters, proteases and one polyketide synthase to be associated with biocontrol. Finally, gene deletion studies confirmed the involvement of several ABC-transporters, MFS-transporters and polyketide synthases in in vitro antagonism or biocontrol in C. rosea. In summary, our data emphasize the role of antibiosis in determining the outcome of biocontrol interactions. Efflux membrane transporters appear to play an important role in the biology of C. rosea, by providing tolerance towards secondary metabolites produced by the fungal prey or C. rosea itself.

Abstract

Trichoderma is well known for the ability of some species to act as i) important biocontrol agents against phytopathogenic fungi; ii) biofertilizers; iii) increasing tolerance of plants to biotic and abiotic stresses; and iv) inducer of plant defense responses via the production and secretion of elicitor molecules. Proteins of the Cerato-platanin (CP) family are released during the early developmental stages of filamentous fungi. They can act as elicitors and induce defense responses in plants. In this study, we analyzed the effects of the Trichoderma harzianum Epl-1 protein in the interaction process with the phytopathogen Botrytis cinerea and with tomato and common bean plants in short and long periods after Trichoderma strains inoculation. The results showed that T. harzianum Epl-1 protein affected the eliciting 1) B. cinerea virulence genes; 2) tomato defense-related genes; 3) the activation of the primer effect in tomato plants; 4) the interaction at the first stage of tomato roots colonization; and 5) the growth promotion of bean plants.

Abstract

Mycoparasitic species of the fungal genus Trichoderma are among the most successful biofungicides in today’s agriculture although our understanding of the exact molecular mechanisms of their activity still is fragmentary. The biological control of fungal plant diseases by Trichoderma includes direct antagonism of phytopathogenic fungi by mycoparasitism. This mycoparasitic attack comprises pre-contact sensing of the prey followed by activation of “molecular weapons” such as cell wall-lytic enzymes, secondary metabolites, and infection structures finally resulting in attack and killing of the prey fungus.

We used the strong mycoparasite Trichoderma atroviride as a model to study the mycoparasitic fungus-fungus interaction. Investigation of the early interaction stages employing T. atroviride labeled with fluorescent CRIB (Cdc42/Rac1-interactive binding) reporters revealed a switch between positive and negative chemotropism in the mycoparasites’ hyphae during the pre-contact sensing phase, a behavior indicative of a stress response probably triggered by prey-derived substances. Accordingly, secondary metabolites released by both interaction partners could be visualized in the interaction zone by mass spectrometry imaging pointing to a chemical cross-talk between Trichoderma and the prey fungus. Our data support the current model of pre-contact prey sensing; consequently, the receptors and signaling pathways that are involved in sensing and in governing the mycoparasitic attack are of special interest. As indicated by our studies, T. atroviride relies on signaling via the Gpr1 G protein-coupled receptor as well as MAP kinase and TOR kinase pathways for triggering the mycoparasitic response in the presence of a fungal prey.

Abstract

Coloration affects the fitness of organisms, e.g. via changes in thermal properties. Although color has been extensively studied in animals and plants, the role of color in mushrooms, the reproductive organs of many fungi, is unknown. We use citizen-science data consisting of 739 European grid cells with 3,054 fungal species, 3.2 million observations, 29,490 color samples and a mega-phylogeny to show that mushroom assemblage color lightness increases with temperature, meaning that assemblages are darker in cold than in warm climates. Our findings suggest that thermal adaptation via dark mushrooms facilitates reproduction of fungal species and the maintenance of populations that drive carbon and nutrient cycling in cold environments. We thus propose the ‘thermal pigmentation hypothesis’, stating a thermal-adaptation of mushroom-forming fungi.