Abstract

Genetic heterogeneity of Ophiocordyceps sinensis with 17 genotypes of O. sinensis in natural Cordyceps sinensis has been documented. But intraspecies genetic variations of Genotype #1 Hirsutella sinensis are unknown. Objective: To analyze the intraspecies genetic variations of H. sinensis. Methods: To compare 3 assembled H. sinensis genome sequences, ANOV00000000 of strain Co18 [Hu et al.2013], LKHE00000000 of strain 1229 [Li et al.2016], & LWBQ00000000 of strain ZJB12195 [Liu et al.2016], a set of unassembled shotgun genome sequences (JM973567-JM973820) of strain YN07-8 [Zhang et al.2012], 2 sets of transcriptome sequences, GAGW00000000 of natural C. sinensis [Xiang et al.2014] and GCQL00000000 of strain L0106 [Liu et al.2015]. Results: (1) There are 2 large DNA segment insertions/deletions in the genome sequences ANOV01002305, LWBQ01000138, & LKHE01001489. (2) Multiple, scattered point mutations were found between ANOV01021101 & LKHE01000642. (3) Multiple, scattered transition and transversion point mutations and DNA segment insertion/deletion mutations were noted between the genome sequences LKHE01000676, LWBQ01000084 and ANOV01001676, between the sequences LWBQ01000028, ANOV01000226/ANOV01006525, and LKHE01002847, and between the genome sequences LWBQ01000037, LKHE01000176/LKHE01002503/LKHE01003221, and ANOV0100526/ANOV01001961/ANOV01009408/ANOV01009409/ANOV01013329. (4) There are 42 of 254 unassembled shotgun sequences having low similarities with the 3 assembled genome sequences. (5) Unassembled shotgun sequences, JM973567, JM973713, JM973797 and JM973816, are absent in the 3 assembled genome sequences, while JM973567 is 100% homologous to Stenotrophomonas maltophilia CP011010. (6) JM973601 shares 96.5% and 94.2% similarities with the 2 transcriptome sequences of natural C. sinensis (GAGW01002159) and strain L0106 (GCQL01017221), and 91.7% similarities with the 3 genome sequences. (7) JM973579 is >99% homologous with the transcriptome sequence GAGW01000465 of natural C. sinensis and the genome sequence LKHE01000582, but only 87.2% and 86.2% with the transcriptome sequence GCQL01019215 of strain L0106 and the genome sequence ANOV01022831. (8) JM973799 is 99.7% homologous with the transcriptome sequence GCQL01011878 of strain L0106, but there is a 89-nt segment deletion in the transcriptome sequence GAGW01001648 of natural C. sinensis. (9) Aligning with translated genome and transcriptome sequences, the amino acid sequence EQL03991 of strain Co18 was found >97.5% homologous with the transcriptome sequence GCQL01017221 of strain L0106 and the 3 genome sequences, but 79.5% and 85.9% with the transcriptome sequence GAGW01002159 of natural C. sinensis and the unassembled shotgun sequence JM973601. Discussions: There are apparent intraspecies genetic variations among the H. sinensis strains, warning future molecular studies on the type strain HMAS 55469 of H. sinensis [Liu et al.1989], and suggesting avoidance of arbitrary selection of H. sinensis strains in research and probably also in industrial fermentation. (Supported by Grant #2017-SF-118 from the Science-Technology Department of Qinghai Province)

Abstract

The scientific literature reported the detections of 17 genotypes of Ophiocordyceps sinensis and the changes of their biomasses in a dynamic, asynchronous fashion in the stroma and caterpillar body of natural Cordyceps sinensis during maturation. Objective: To examine the ejection and fungal compositions of the C. sinensis ascospores. Methods: To collect and cultivate mature C. sinensis, to observe the ejections of the ascospores, and to examine the morphology of the ascocarps and ascospores under a microscope and the ascosporic fungal components, including the reported C. sinensis associated fungi: Geomyces pannorum, Hirsutella sinensis, Paecilomyces hepiali, Paecilomyces sinensis, Pseudogymnoascus roseus, and Tolypocladium sinense, and mutant genotypes of O. sinensis with using fungal- and genotype-specific primer PCR, amplicon cloning-sequencing, and SNP mass-spec genotyping. Results: Two types of the ascospores were observed from the same specimen of C. sinensis: the fully ejected ascospores and the semi-ejected ascospores that hang on and tightly connected to the outside surface of the openings of perithecia. Microscopic examination revealed multicellular heterokaryotic structure of the ascospores, consistent with the findings by Bushley et al.[2013] of the multicellular heterokaryotic ascospores with mono-, bi-, and tri-nucleates. PCR with using fungal specific primers and amplicon cloning-sequencing did not detect the ITS sequences of G. pannorum, P. chrysogenum, P. roseus, P. sinensis, or T. sinense in the genome DNA samples extracted from the 2 types of the C. sinensis ascospores, although these fungi were detected in C. sinensis and some of them dominate in the stroma or caterpillar body [Zhang et al.2010]. The ITS sequences of Genotypes #1 H. sinensis, #5-#6, #14, #16 of O. sinensis, AB067719-type O. sinensis and P. hepiali were identified in the fully ejected ascospores, but Genotype #13 instead of Genotypes #6, #14, #16 in the semi-ejected ascospores. The biomass of P. hepiali was significantly greater in semi-ejected ascospores than in fully ejected ascospores. Two newly discovered Genotypes #13 & #14 of O. sinensis feature hereditary variations with DNA segment reciprocal substitution between 2 parental fungi, Genotype #1 H. sinensis and AB067719-type O. sinensis. Discussions: The 2 types of multicellular heterokaryotic ascospores feature genetic heterogeneity, containing multiple genotypes of O. sinensis, AB067719-type O. sinensis, and P. hepiali. The findings of the Genotypes #13 & #14 of O. sinensis with the DNA segment substitution hereditary variations suggest fusions of chromosomes of 2 parental O. sinensis fungi, and even possible mating type(s) (heterothallic or hybrid mating) or hyperparasitism. The fugal components, both the various biomasses of P. hepiali and the presence of Genotype #13 or #14 in either type of the ascospores, appear to be of biological importance that determine the formation, maturation, and ejection processes of the C. sinensis ascospores. (Supported by Grant #2017-SF-118 from the Science-Technology Department of Qinghai Province)

Abstract

Numerous papers reported detections of 17 genotypes of Ophiocordyceps sinensis in natural Cordyceps sinensis that consists of a dead larva from the Family Hepialidae and multiple intrinsic fungi. Natural C. sinensis, a precious traditional Chinese substance, has been used in clinic as a folk medicine in China for 6 centuries. The 17 genotypes of O. sinensis include Genotype #1 Hirsutella sinensis, Genotypes #3-#6,#15-#17 (transition mutants), Genotypes #7-#11 (transversion mutants), Genotypes #2,#12 (insertion/deletion mutants), and Genotypes #13-#14 (DNA segment reciprocal substitution hereditary variations) [Zhu et al.2016]; Genotypes #1-#3,#7-#12 are GC-biased, while Genotypes #4-#6,#15-#17 are AT-biased. The multiple genotypes of O. sinensis coexist differentially in the stroma, caterpillar body, ascocarps, and ascospores of natural C. sinensis; their biomasses are changing in a dynamic, asynchronous fashion during the maturation of C. sinensis. The sequences of Genotypes #2-#17 belong not to the genome of Genotype #1 H. sinensis, but to the genomes of independent O. sinensis fungi. All 17 O. sinensis genotype fungi share the same Latin name, which has compounded the historical problems since the middle of 19^th century, associated with these fungi indiscriminately sharing the same Latin name with the natural insect-fungi complex. The history of and current perspectives on the indiscriminate use of the Latin names C. sinensis and O. sinensis are now for the multiple genotypes of O. sinensis fungi, for the anamorphs and teleomorphs of O. sinensis according to the fungal nomenclature rule established by the Amsterdam Declaration of “One Fungus=One Name”, and for the natural C. sinensis insect-fungi complex. For partially solving the problem of the indiscriminate practice, Zhang et al.[2012] proposed for using O. sinensis for the fungi and a non-Latin name “Chinese cordyceps” for C. sinensis insect-fungi complex. This proposal, however, did not reach a consensus because government regulations worldwide require every natural product used as a folk medicine or a dietary supplement to have an unique, exclusive Latin name and because of unnecessity of having an additional non-Latin name for natural C. sinensis when several such names have been used for centuries. Alternatively Ren et al.[2013] proposed for using “Ophiocordyceps & Hepialidae” to reflect the nature of C. sinensis fungal-insect complex. Discussion on the second proposal has to be postponed because all 17 genotype fungi currently under the name of O. sinensis may or may not be variants within the O. sinensis species or within the genus Ophiocordyceps Petch, also because Tolypocladium sinensis has been confirmed as one of the anamorphs of O. sinensis [Barseghyan et al.2011], and Quandt et al.[2014] have proposed “for Ophiocordycipitaceae (Hypocreales) with new combinations in Tolypocladium”. This taxonomy-nomenclature problem invites taxonomists across disciplines to characterize the multiple mutant genotype fungi, most of which are currently difficult to be cultured and identified through PCR with using common primers, and to end the centuries-old academic confusion on the indiscriminate use of the Latin name O. sinensis for multiple O. sinensis fungi and the natural insect-fungi complex. (Supported by Grant #2017-SF-118 from the Science-Technology Department of Qinghai Province)

Abstract

Light conditions during fungal growth are well known to cause several physiological adaptations in the produced conidia; thus, conidia of the insect-pathogenic fungi Metarhizium robertsii were produced on: 1) potato dextrose agar (PDA) medium in the dark; 2) PDA medium under white light; 3) PDA medium under blue light; and 4) PDA medium under red light. The conidial production, the speed of conidial germination, the virulence to the insect Tenebrio molitor, as well as gene expression, and tolerances to osmotic stress and to UV radiation were evaluated. Conidia produced under white light or blue light germinated faster and were the most tolerant to UV radiation and osmotic stress. White light improved conidial virulence as compared with conidia produced in the dark. Growth under blue light produced more conidia than the fungus grown in the dark. The small (Mrhsp30) and large (Mrhsp101) heat shock protein genes were highly up-regulated under white light condition, suggesting an active role of heat shock proteins in fungal exposition to the different visible spectrum components. The cytosolic catalase Mrcatc gene was not induced under all light conditions assayed. Conidia produced under red light germinated slower than conidia produced in the dark and were the least tolerant to osmotic stress and UV radiation. The virulence of conidia produced under red light was similar to conidia produced in the dark. In conclusion, white light produced conidia that germinated faster and killed the insects faster; in addition, blue light afforded the highest conidial production. Both white light and blue light afforded the highest tolerance to both stress conditions. This research was supported by grants from the National Council for Scientific and Technological Development (CNPq) of Brazil PQ2 302312/2011-0, and PQ1D 308436/2014-8 and the São Paulo Research Foundation (FAPESP) 2010/06374-1 and 2013/50518-6.

Abstract

Cryptococcus neoformans causes fatal cryptococcal meningoencephalitis in immunocompromised patients as well as immunocompetent people. Despite its clinical importance, the signaling networks governing its virulence remains elusive and therapeutic options for treatment of systemic cryptococcosis are limited. Here, to understand signaling networks regulating the virulence of C. neoformans, we aim to identify and functionally characterize the 139 putative phosphatases, which are major signaling components in the basidiomycete fungal pathogens. We selected putative phosphatases based on annotation in the C. neoformans var. grubii genome database provided by the Broad Institute and National center for Biotechnology Information (NCBI) and performed a BLAST search with their protein sequences to identify any corresponding orthologs in S. cerevisiae, A. nidulans, C. albicans and F. graminearum. We classified putative phosphatases into 16 groups based on InterPro phosphatase domain annotation. Thus far, we have successfully constructed 227 signature-tagged gene-deletion strains representing 114 putative phosphatases through homologous recombination methods. We are in the middle of examining their phenotypic traits under 30 different in vitro conditions, including growth, differentiation, stress response, antifungal resistance and virulence-factor production. Along with our previous functional genetic studies for C. neoformans transcription factors and kinases, this study will provide a comprehensive insight into the fungal signaling networks.

Abstract

Sterol lipid is essential for cell membrane structure in eukaryotic cells. In mammalian cells, sterol regulatory element binding proteins (SREBPs) act as principal regulators of cellular cholesterol which is essential for proper cell membrane fluidity and structure. SREBP and sterol regulation are related to levels of cellular oxygen because it is a major substrate for sterol synthesis. Upon cellular sterol and oxygen levels are depleted, SREBP is translocated to the Golgi where it undergoes proteolytic cleavage of N terminus, then it travels to the nucleus to play a role as transcription factor. In this study, we observed phenotypes in other strains of Cryptococcus species by constructing hob1Δ and sre1Δ mutants to confirm whether the functions of both genes are conserved in most serotypes. As a result, hob1Δ showed no noticeable phenotype under treatment of antifungal drugs and most environmental stresses in R265 (serotype C) and XL280 (serotype D), suggesting that Hob1 is related to sterol regulation only in H99 (serotype A). On the other hand, the function of Sre1 was found to be conserved in most serotypes. In conclusion, HOB1 and SRE1 play crucial role in regulating sterol-homeostasis in Cryptococcus neoformans, moreover, Hob1 is specific gene in Cryptococcus neoformans. It suggests that Hob1 is considered as potent factor-targeted new safety antifungal drug.

Abstract

Abstract

Methods of molecular biology are used in many different fields of scientific research. Archaeology is one of the disciplines that can benefit the most from it. Realization of main goals of archaeological research, which are i.e. drawing conclusions concerning people living in ancient ages, is a very difficult task since plethora of archaeological objects and traces was destroyed or significantly damaged. Our research focused on applying a molecular techniques for studying late Neolitic barrows located in south-eastern Poland. Our main goal was to verify whether high throughput sequencing of ITS2 fragment of rDNA can provide a full picture of fungal diversity and thus, provide new information concerning mankind history. Fungi are suitable organisms for studying the past because they interact with other organisms on every trophic level and moreover, there is an almost universal molecular barcode which can differentiate most fungi on at least genus level. We used MiSeq Illumina platform to sequence ITS2 amplicons and then, publicly accessible database Unite.ut.ee to assign taxonomic position of obtained sequences. We compared female and male graves, vessels buried alongside the non-preserved bodies, traces of wooden constructional element and ancient and modern soil from nearby. Although it is only preliminary study, some substantial differences between all the samples may be observed. The traces of Fomes fomentarius (common bracket fungus) were found only in the place where the wood was thousands years ago. There are also differences between vessels and other samples – water fungi (representatives of Rozellomycota and Chytridiomycota) and cereal pathogens (such as Giberella tricincta or G. zeae) were found only in the vessels. Concluding, analyzing patterns in fungal distribution on archaeological sites may lead us to creation of a complex tool which can be used to answer the questions about ancient diet, activities and funeral rituals.

Abstract

The International Nucleotide Sequence Database Collaboration (INSDC), is now more than 30 years old and comprise of three partners. These are the DNA Data Bank of Japan (DDBJ) at the National Institute for Genetics in Mishima, Japan; the European Nucleotide Archive (ENA) at the European Molecular Biology Laboratory's European Bioinformatics Institute (EMBL-EBI) in Hinxton, UK; and GenBank at the National Center for Biotechnology Information (NCBI), National Library of Medicine (NLM), National Institutes of Health (NIH) in Bethesda, Maryland, USA. A 1997 agreement to resolve taxonomic issues prior to the release of new data paved the way for the NCBI Taxonomy database to serve as a central organizing hub for the INSDC members. This database was thus intended for a specific, practical purpose - to provide nomenclature and classification information for the source organisms in the public sequence databases. This presentation will highlight and clarify recent improvements introduced to attain this goal, with a focus on Fungi.

Abstract

Decomposition of plant litter is essential for nutrient cycling and therefore a key process for ecosystem functioning. Our current understanding of this complex process suggests that leaf litter decomposition is driven by climate, litter quality and decomposer communities. Fungi are considered to be the main decomposers of leaf litter in forest ecosystems. They synthesize and secrete enzymes that change the chemical composition of the litter, and thus represent a major effect of the fungal community. However, fungal community composition and their metabolic activity have been rarely analyzed together, and so far never in combination with litter chemistry. To link fungal activity to decomposition chemistry, we characterized the chemical composition of freshly fallen autumn leaves of European beech (Fagus sylvatica) and the corresponding leaf litter after one year of decomposition by proton nuclear magnetic resonance (¹H-NMR) spectroscopy. The composition and transcriptional activity was assessed for fungal communities by high-throughput sequencing of amplicon barcodes and metatranscriptomes, respectively, in the same litter samples. These analyses were highly replicated across 14 different forest plots and cover three distant regions in Germany. We were able to successfully distinguish freshly fallen leaves from one-year-old litter with respect to their chemical composition. Leaves were chemically more distinct among regions than one-year-old litter. Fungal communities were locally structured, however, functionally redundant among regions, i.e. expressing genes coding for similar litter-degrading enzymes. We identified changes in the abundance of putative chemical compounds between freshly fallen autumn leaves and one-year-old that correlated to the transcription level of litter-degrading enzymes. Furthermore, transcription patterns were also correlated with the abundance of certain fungal species. Overall, we provide strong evidence of a dynamic interaction between substrate chemistry, expression of enzyme coding genes, and fungal community structure in nature.

Abstract

Although significant progress have been made in developing computational methods of binning and taxonomic profiling of metagenomic and microbiome samples, so far they were mostly restricted to the analysis of prokaryotic sequences. However many recent surveys of environmental metagenomic samples also include sequences from microbial eukaryotes, such as fungi, metazoans and protists in addition to usual bacterial and archael sequences. The accurate identification of eukaryotic sequences, in the large sets of short sequences is an important step for further characterization of their role in the microbialcommunity and evaluation of their metabolic potential. To aid with this task we conducted comparative analysis of ~900 eukaryotic genomes together with several thousand non-redundant reference prokaryotic genomes from IMG database in order to identify PFAM domains unique either to eukaryota or prokaryota domains. Additionally other identified unique eukaryotic and prokaryotic sequences without PFAM annotations were clustered into families, with subsequent building of their corresponding Hidden Markov Models (HMMs). These HMMs were calibrated and tested to predict with high accuracy eukaryote and prokaryote specific sequences. Based on found unique PFAMs and constructed new HMMs we developed a program that identifies and bins the potential eukaryotic sequences from metagenomic projects.

Abstract

The first genomes of Saccharomyces cerevisiae and Neurospora crassa were transformational for biology and development of molecular tools. The sequenced genomes of over 1000 fungal species offer new challenges and perspectives. In collaboration with research community from around the world the US Department of Energy Joint Genome Institute (JGI) has scaled-up fungal genomics in large-scale genomics initiatives like the 1000 Fungal Genomes project, which samples diversity across the entire Fungal Tree of Life; the 300 Aspergillus genomes, which drills down a single industrially important genus; the Fungal ENCODE, which produces multi-omics datasets to add new dimensions to the genomic data. These data equipped with analytical tools available from the JGI MycoCosm portal (jgi.doe.gov/fungi) are now offerred for over 1000 fungal genomes. What can we learn from the 1000 fungal genomes that we cannot from one or ten? Can we predict species lifestyles from their genome sequences after interpreting large collections of genomes? How can we reduce dimensionality and visualize Big Data to make new discoveries? Big Data in fungal genomics has a huge unexplored potential and we’ll explore groups of fungi to answer these questions using new computational approaches.

Abstract

Fungi are known to form associations with bacteria that grow either outside or within their hyphae. These associations can be functionally diverse, ranging from fungi that farm bacteria for nutrients to obligate endobacteria with dramatically reduced genomes that live within fungal hyphae. While there is an increasing number of examples of these bacterial/fungal interactions, not enough is known about the overall diversity of these fungal microbiomes or the physiological and ecological functions that result from the close interactions between fungal and bacterial species. Our objective here is to better understand the diversity of bacteria that are potentially associated with a wide range of fungal taxa. In order to do this, we have taken a bioinformatics approach by searching for bacteria-derived sequences within existing publicly available fungal genome sequencing data from a phylogenetically broad sampling of fungi, generated primarily through the 1000 Fungal Genomes project. Not all bacteria identified from fungal sequencing efforts will represent actual fungal associates, as some are almost certainly contaminants or mis-identified sequence reads. Therefore, we will use statistical and bioinformatics tools to attempt to identify and remove these reads from the analysis. To-date, we have analyzed genome sequencing data from a subset of fungal taxa that contain relatively few septa in their hyphae, which may foster the establishment and long-term persistence of endobacteria. We use standard sequence taxonomy classification methods to assemble a list of bacterial taxa represented in each fungal genome sequencing project. When these bacterial composition lists are analyzed in tandem with fungal phylogenetic relationships, we find that within bacterial groups such as the family Burkholderiaceae, some species can occur in a range of fungal genera, while others display statistically significant phylogenetic clustering in their associated fungal species. We are in the process of expanding this analysis to several hundred fungal genomes to gain a more complete understanding of the phylogenetic patterns and functional significance of these putative bacterial associates identified from fungal sequencing data. Our results begin to catalog patterns in bacterial/fungal associations that occur in diverse fungal species and that in the future may help to better understand and predict the functions of these bacterial/fungal associations.

Abstract

The FAIR guiding principles for data management and publication as described by Wilkinson et al. (2016) and Mons et al. (2017) are recommendations to make research data sustainable, which means long-term Findable, Accessible, Interoperable and Reusable. Currently, these principles are widely discussed and largely accepted by major stakeholders and initiatives of the various science domains, e.g., the European Science Cloud and national initiatives for research data publication, like the German Federation for Biological Data (GFBio; (https://www.gfbio.org/). These principles also directly affect primary research data producers and scientists managing mycological research data from their generation onwards. This is demonstrated by a work- and dataflow with the use case 'Mycological occurrence and community barcoding data'. For organizing such kind of data according FAIR principles, the relational database application DiversityDescriptions (DD) (https://diversityworkbench.net/Portal/DiversityDescriptions), a generic component of the Diversity Workbench environment (https://diversityworkbench.net), is applied. The newly published conceptual schema MOD-CO for 'Meta-Omics Data of Collection Objects' provides concepts and concept collections being appropriate as DD descriptor structure. MOD-CO (http://www.mod-co.net) has been established with the aim to describe operations and object properties along the work- and dataflow from gathering environmental samples, to the various transformation and measurement steps in the laboratory up to sample and data publication and archiving. By supporting parent-child-relationship, the MOD-CO schema (http://www.mod-co.net/wiki/Schema_Representations) allows for the entering and storage of individual records of each operational step (transformation, measurement and transaction) along a workflow. Based on a MOD-CO descriptor structure, DiversityDescriptions might be used as LIMS (Laboratory Information Management System), e.g., for organising 'Mycological occurrence and community barcoding data'. A DD data export interface enables data managers to provide content data in the formats CSV (https://en.wikipedia.org/wiki/Comma-separated_values) and XML (https://www.w3.org/XML/) according SDD metadata schema with EML and ABCD metadata schemata extensions (for biodiversity schemata and standards see https://gfbio.biowikifarm.net/wiki/Data_exchange_standards,_protocols_and_formats_relevant_for_the_collection_data_domain_within_the_GFBio_network). Domain-specific, service-oriented infrastructures for data publication like GFBio with its submission and brokering services and relation to EMBL-ENA and to several other recognized biodiversity and environmental data centers (https://www.gfbio.org/about/data-centers) rely on scientists with good data management skills, using such tools and workbenches as described above. In the addressed use case, data and metadata, curated in DD, will be checked by GFBio compliance and consistency tools for being published according to the FAIR data principles. The GFBio data submission and publication process includes terminology and parameter assignment and provides advanced search options for datasets ('findable'), data download services ('accessible'), services for machine-readability of data ('accessible', 'interoperable') and several visualization and analysis options ('accessible', 'interoperable', 'reusable'). The data package from the use case 'Mycological occurrence and community barcoding data' will be published and offered for reuse under a creative common license. A suggestion for standardized citation is provided, a DOI assignment is feasible and the long-term data archiving is ensured. In the GFBio context, mechanisms to provide metrics, count the impact of data publications and give credits to authors are going to be established.

Abstract

Agriculture and Agri-Food Canada (AAFC) has recently invested in a 5-year initiative to update and upgrade its vast collection of live and preserved fungal specimens. The Canadian Collection of Fungal Cultures houses over 19,000 live cultures and is a repository and distributor of fungal genetic resources. The Canadian National Mycological Herbarium contains over 350,000 preserved specimens, representing a historical archive of the existence and distribution of fungi and plant disease in Canada. Plans include full inventories, digitization of specimen metadata, and renovated infrastructure. Furthermore, selected specimens will subjected to characterization at the molecular level through DNA sequencing and analyses. Partial and whole genomes will be sequenced for ~2,500 and ~350 fungal species, respectively. These data will be leveraged by researchers for the development of tools for precise identification and detection of agriculturally important fungi and quarantine species, surveys of agricultural commodities, and the resolution of taxonomic and nomenclatural issues. DNA sequences for universal barcodes (eg ribosomal internal transcribed spacer) will be obtained from ~16,000 live fungal strains. These data will significantly expand the reference sequence databases that may be used in foundational work in the metagenomic and microbiome research fields. For example, these barcode sequences will support metabarcoding studies on topics that are currently of high interest to the agricultural and environmental sectors, such as the impacts of climate change and land use on soil microbial communities. All collection, specimen, and molecular data will be integrated into an information sharing system that will provide rapid and electronic, public access to AAFC’s biological collections.

Abstract

Selenium (Se) is an essential trace element in healthcare. It has many important bioactivities such as anti-oxidant, boosting immune function, anti-tumor and anti-metastatic. This study proposes a new cultivation method to produce Se-enriched mycelial biomass of Ophiocordyceps sinensis fungus. The result shown that the Se-adaptive cultivation method improved the ability of biomass production of O. sinensis AS (with a yield of 17.31 ± 1.12 g L^-1) at 25 mg L^-1 of Se⁺⁶ (Na₂SeO₄.12H₂O). The Se content of O. sinensis AS biomass reached about 1354.97 ± 54.54 µgSe g^-1 dry weight (dw). In addition, selenium-containing compounds of O. sinensis AS including selono-exopolysaccharides (Se-EPS), seleno-polysaccharides (Se-PS) and seleno-proteins (Se-Pr) were extracted. The fourier transform infrared spectroscopy (FT-IR) analysis demonstrated that these molecules had the presence of Se=O and C-O-Se stretching vibrations at the characteristic infrared absorption peaks, 1100 - 1050 cm^-1 and 550 – 620 cm^-1 bands respectively. Furthermore, there were a considerable rise in the anti-oxidant activities of Se-EPS such as ABTS^• and OH^• radical scavenging potential as well as reducing the intracellular ROS levels of HepG2 cells. In conclusion, not only does this study improve the pharmacological properties of the O. sinensis AS biomass, it also makes the supplementary Se source with high bioavailability.

Abstract

The aim of this research is to find a filamentous fungus with potential of bioremediation of metals, such as Pb, Cr, Zn, Hg, Ag, and Cd. For this, soil samples were taken from a mine, a strain of filamentous fungus was isolated and purified. The filamentous fungus was identified as Aspergillus fasciculatus based on molecular data. The tolerance index and minimum inhibitory concentration (MIC) for the metals Pb, Cr, Zn, Ag, Hg, and Cd were determined. A. fasciculatus was highly tolerant to 1, 5, 10, 15 and 20 mM of Pb, Cr, and Zn, respectively. While for metals Hg, Ag and Cd were highly tolerant to concentrations of 1 and 5 mM of these metals. Their MIC ranged from 5-10 mM for Hg, Ag, and Cd, moreover MIC from Pb, Cr, and Zn up 20 mM. With these results, it can be concluded that A. fasciculatus presents high potential of use to bioremediation the following metals: Pb, Cr, Zn, Ag, Hg, and Cd.

Abstract

Agriculture has become a multifaceted industry as the production of biofuels and the need for greater sustainability grow in prevalence. There is a finite amount of land available for either biofuel or food crop systems, and land availability is further limited due to the presence of major pollutants including heavy metal contaminants, leading to phytotoxicity and other concerns. Plants serve as hosts to a diverse community of microbes including fungi, which are capable of metabolizing and/or immobilizing soil compounds which the plant cannot. Some mycorrhizal fungi directly interact with heavy metal contaminants within the soil, and a fungal-plant symbiosis may increase a plant’s ability to survive in a soil containing high heavy metal concentrations. These relationships are often difficult to disentangle as a single plant species may be associated with hundreds of fungal species and each of these associations can display varying influences on host plant survival and growth that co-vary with the environmental and physical conditions of soil. One such symbiont, the Cenococcum geophilum, is ubiquitously distributed across multiple climates, soil types and plant species. The genome of C. geophilum is among the largest in the fungal kingdom, with a total estimated size of 178 megabases due to an abundance of transposable elements. This large size and highly repetitive genomic data have historically increased the difficulty of both sequencing and analyzing C. geophilum. New technologies, such as restriction enzyme associated DNA sequencing (RADseq) decrease both time and financial investment required for such analyses, and will be used to analyze over 200 wild isolates of C. geophilum obtained from soil samples across a range of 296 miles in the United States Pacific Northwest. These isolates will be used to determine if established host population structure will be reflected in the symbiont genomes, despite the generalist nature of C. geophilum. These wild isolates will also be screened to determine phylogenomic related correlates with the uptake and growth effects of heavy metal contaminants lead, strontium, copper, cadmium and zinc using laboratory growth assays. Heavy metal uptake will be analyzed using an X-Ray fluorescence device (XRF) on dried fungal biomass, and metabolomics analyses will be performed on representative isolates using gas chromatography–mass spectrometry (GC-MS). Finally, individual strains will be selected based on heavy metal resistance and inoculated onto living plant hosts of the potential biofuel crop Populus (Poplar). Plant physiological responses, growth patterns, and overall uptake of the contaminants will be monitored to determine if heavy metal resistant C. geophilum strains confer increased heavy metal contamination resistance to Poplar. If increases resistance is observed, this type of manipulation of host plant symbionts may increase plant tolerance to soil conditions that would otherwise be toxic to the plant, increasing overall land availability for use in agriculture. This would encourage the planting of biofuel crops within fields that may not be optimized for the growth of crops intended for consumption due to the contamination within the soil, thereby avoiding the “food-for-fuel” tradeoff that has driven agronomic policy concerns in the biofuel industry.

Abstract

Many mushroom species have been found to accumulate arsenic in fruit-bodies. Hyperaccumulation of this toxic metalloid was described in the ectomycorrhizal ascomycete Sarcosphaera coronaria. A common and often major chemical species of arsenic found in fruit-bodies is arsenobetaine (AB), a non-toxic methylated arsenic compound also known from marine biota. However, several other methylated arsenic species have been identified in fungal tissues, e.g., methylarsonic acid (MA), dimethylarsinic acid (DMA), and trimethylarsine oxide (TMAO). Based on a long-term analytical screening of arsenic in macrofungi by instrumental neutron activation analysis, we have revealed striking arsenic accumulation in Cyanoboletus pulverulentus and Elaphomyces spp. More samples of these two species were then collected and total arsenic concentrations were additionally determined with inductively coupled plasma mass spectrometry (ICPMS) after microwave assisted acid digestion. The chemical form of arsenic in fruit-bodies was investigated in aqueous extracts by high performance liquid chromatography coupled to ICPMS. In C. pulverulentus, arsenic concentrations in sporocarps may reach 1300 mg kg^-1 (dry mass, d.m.). No significant correlation between the soil arsenic content and arsenic concentrations in the associated sporocarps was found. Within the individual parts of the fruit-body, we found the majority of arsenic accumulated in the hymenium. Besides occasional traces of MA, the arsenic speciation in all Cyanoboletus samples consisted solely of DMA and no inorganic arsenic was detected. Because of the carcinogenic potential of DMA, C. pulverulentus should not be recommended as an edible mushroom and its consumption should be restricted. In Elaphomyces collected from unpolluted spruce plantations in the Czech Republic, the total arsenic concentrations ranged from 12 to 42 mg kg^-1 d.m. in samples of E. asperulus and from 120 to 660 mg kg^-1 d.m. in E. granulatus and E. muricatus. These concentrations are remarkably high for terrestrial organisms and demonstrate the arsenic accumulating ability of these ascomycetes. The dominating arsenic species in all samples was MA which accounted for more than 30 % of the extractable arsenic. AB, DMA, and inorganic arsenic were present as well, but only at trace concentrations. Surprisingly, we found high amounts of TMAO in all samples (0.32 – 28 % of the extractable arsenic). Even more remarkable was that the majority of samples contained significant amounts of the highly toxic trivalent arsenic compound methylarsonous acid (0.08 – 0.73 % of the extractable arsenic). This is the first report of the occurrence of trimethylarsine oxide and methylarsonous acid at significant concentrations in a terrestrial organism. From our results it becomes obvious that the arsenic speciation in fungal fruit-bodies is much more complicated than previously thought and a lot of work needs to be done before we understand the biological importance of arsenic accumulation and speciation in macrofungi.

Abstract

The ongoing increase of heavy metal concentration in soil environments requires the development and the improvement of soil remediation approaches. Microorganism-enhanced phytoremediation has been shown to be very efficient for soil rehabilitation. Here, we focus on ectomycorrhizosphere as a close interaction between plant roots, ectomycorrhizal fungi, microorganisms and soil to understand mechanisms of phyto- and bioremediation. To investigate microbial interactions, two-year pot experiment with 3 variants has been established using trees (birch, oak, pine), and soil substrate with natural occurring microflora from a former mining uranium site (Thuringia, Germany): (1) pots with soil substrate, (2) pots with soil substrate and planted tree seedlings, and (3) pots with soil substrate, planted and additionally inoculated with ectomycorrhizal fungi tree seedlings. The probable changes in metagenome and metatranscriptome of soil communities as well as heavy metal content in plant biomass and in soil adjacent to ectomycorrhizal roots after inoculation will be determined. Moreover, molecular mechanisms of interactions between mycorrhiza helper bacteria and ectomycorrhiza will be investigated using metal resistant Streptomyces strains isolated from test field on Tricholoma vaccinum – Picea abies symbiosis. Hydrophobins are unique surface-active fungal proteins. Up-regulation of several hydrophobin genes in the presence of heavy metals was demonstrated earlier; however, direct participation of hydrophobins in the alleviation of metal stress has not been addressed so far. Agrobacterium tumefaciens mediated transformation has been performed to produce hydrophobin 8 overexpressing mutants of T. vaccinum. The properties of hydrophobin 8 found in aerial mycelium of T. vaccinum and its participation in formation of ectomycorrhiza as well as in alleviation of metal stress will be studied. The preliminary results will allow to connect the microbial diversity of ectomycorrhizosphere of plants at contaminated site and performed functions. Furthermore, profound analysis of microbial communities will give insight into the molecular mechanisms of microbial biogeochemical functions which are related in many respects to microbial resistance mechanisms. In the future, the given results might be used for optimization of phyto- and bioremediation of soils contaminated with heavy metals.

Abstract

Ectomycorrhizal fungi plays an important role in protecting the host plants in heavy metal contaminated sites. They prevent the uptake of heavy metals into the cytosol by extracellular chelation through extruded ligands like tricarboxylic acid, oxalic acid or by biosorption of these metals to the fungal cell wall through chitin and glucosamine. Further, the metal accumulated in the cytosol is detoxified by synthesizing the range of thiol rich ligands like glutathione (GSH) and metallothioneins (MT). GSH is biosynthesized in two sequential ATP dependent reactions mediated by two enzymes, γ-glutamylcysteine synthetase (γ-GCS) and glutathione synthetase (GS). In the present investigation, genes involved in GSH synthesis were cloned from the ectomycorrhizal fungus Laccaria bicolor and studied their roles in metal detoxification. L. bicolor was subjected to different concentrations of cadmium and arsenic and their response was recorded using different parameters like dry weight, total metal uptake, the total glutathione produced, the enzyme activity of γ-GCS and GS and the relative expression of both γ-GCS and GS genes. The two genes involved in glutathione biosynthesis (γ-GCS and GS) were further expressed in E.coli cells and were characterized. Uptake of Cd increased up to 9 µM and then decreased due to the lethal effects, whereas in case of As the metal uptake increased with increase in arsenic concentration up to 15 mM. Total glutathione production increased as a function of external Cd. Cd induced the expression of γ-GCS 18 fold and GS 12 fold higher than control while As increased the expression of γ-GCS 10 fold and GS 6 fold higher compared to control mycelium. The functional characterization of the two genes, γ-GCS and GS in E.coli cells increased their metal tolerance up to 3 folds justifying the potential role of these genes in metal tolerance. The present study clearly depicts the potential role of glutathione in protecting L. bicolor under cadmium and arsenic stress, thus segregated the response of different thiols to different metals.

Abstract

Studies have revealed that mycorrhizal fungi, including ectomycorrhizal (EM) species, effectively mobilize heavy metals from soils and minerals. Mycorrhizal fungi further play an important dual role in plant metal homeostasis: scavenging of metal micronutrients and their supply to the host; detoxification of both the excess essential and physiologically irrelevant metals. The delineation of molecular basis of metal uptake and tolerance in metal-accumulating species may allow rating their metal cycling and host-protection/stimulation capacity, with certain significance for bioremediation purposes. This report focuses on the handling of metals in the EM species accumulating remarkably high concentrations of heavy metals. Searches of sequenced transcriptomes for metal related determinants, gene complementation studies in yeasts, metal speciation analyses in fungal tissues by using size exclusion chromatography and mass spectrometry, and gene expression analyses in the cultured mycelia allowed us to obtain the evidences supporting the following conclusions. In Ag-hyperaccumulating Amanita strobiliformis, at least two CTR transporters can efficiently recognize for import Ag in addition to Cu, P_1B-1-ATPase transporter can export Ag and Cu from the cytoplasm (presumably into the vacuoles) but virtually all the accumulated Ag occurs complexed by cytosolic metallothioneins (MTs). In Zn accumulating Russula atropurpurea, which has a high affinity ZIP transporter for the acquisition of Zn and CDF transporter for vacuolar sequestration of overaccumulated Zn, is 40% of accumulated Zn bound with unusual MT-like RaZBP peptides; RaZBP homologues are involved in binding of the excess of intracellular Zn also in other Zn accumulators of Russula spp., R. pumila, R. ochroleuca, and R. viscida. In contrast, Hebeloma mesophaeum, like several other Hebeloma species, preferentially funnels excess Zn (and Cd) into subcellular compartments, although it has the capacity to produce MTs. Altogether, our data suggest that there might be a link between cellular biology of Ag and Cu in EM fungi and show that EM fungi of different genera (Russula vs. Hebeloma) may employ different strategy to handle the excess of particular metal (Zn). Work currently supported by the Czech Science Foundation (16-15065S).

Abstract

The pollution of the environment with radionuclides is mainly caused by nuclear explosions. The most serious accident to date occurred on April 26, 1986 in Chernobyl in the Ukraine, when approximately 3 x 10⁶ trillion Bq were released into the environment. Although more than 30 years later, thousands of hectares of land are still contaminated, especially with cesium.

Since fungi are known to have high radiotolerance and are capable of accumulating various radionuclides, they are a good means of bioremediating the contaminated soil.

Within this study, tolerance or rather accumulation mechanisms for radionuclides should be identified in fungi.

For this purpose, an experimental set-up was established with the saprophytic white rot fungus Schizophyllum commune. It absorbs effectively radionuclides, grows well in soil, also under lab conditions, and mycelium free of soil particles can be harvested. Thus, RNA for mRNA-Seq can be isolated from the mycelium after growth on Chernobyl soil, control soil and on minimal medium with and without heavy metal composition found in Chernobyl soil. Thus, first conclusions can be drawn on the molecular mechanism behind the mycoremediation of S. commune.

Abstract

Intensification and inadequate agricultural management result in substantial losses of fertility and yield, and the accumulation of pathogens in soils. In order to maintain soil quality and health for the future in agricultural land, the development of more extensive and sustainable farming strategies is urgently needed. Hence, a better understanding of how agricultural management strategies affect soil and associated rhizosphere properties is the key to propose farming strategies for high plant productivity and plant health. We used three long-term field trials to analyze the impact of various management strategies on soil and its associated rhizosphere microbiome under consideration of plant productivity, plant health and the ability of the soil to suppress soil-borne pathogens. The soils of the long-term field trials were subjected to growth chamber pot experiments with lettuce (Lactuca sativa) as model plant. After a growth period of ten weeks, significant differences in lettuce shoot fresh mass and microbial biomass were observed among soils depending on long-term farming strategies. The rhizosphere exhibited different bacterial and fungal community compositions depending on soil sites as well as on the agricultural management history (tillage practice, crop rotation, fertilization strategy) of the soils. These factors influenced also relative abundances of distinct bacterial and fungal taxa. In addition the root exudation of the antifungal metabolite benzoic acid as well the expression of plant-defense related genes was affected by farming practice. This suggests a relationship between long-term agricultural management, soil microbiome and plant performance.

Abstract

Bromoalkanes, as 1-bromobutane, are persistent organic pollutants in water, sediments, and soils. They are commonly found in pesticides, herbicides, and industrial solvents. Recently, an aerobic pathway of bromoalkane degradation, based on initial dehalogenation, by an eukaryotic microbe was describe for the yeast Yarrowia lipolytica 3589. Our objective is to isolate fungi capable of degrading bromoalkanes. 1-bromobutane was added into mineral media as sole carbon on agar plates. The mineral media was used with and without nitrogen supplementation. Plates were randomly exposed to air at six locations in Puerto Rico and incubated at room temperature. After ten days, small fungal colonies were noted for three sites. Microscopic examination revealed six isolates exhibiting septated fine mycelium and conidiospore arrangements resembling different Penicillium strains. The growth suggest that 1-bromobutane can be used as carbon source with faint development. Biochemical characterization are in progress to demonstrate chemical transformations using and kinetics parameters and spectroscopy assays. Further studies will use haloalkanes differing in carbon chain length and position as sole carbon source to continue bioprospecting efforts. The ultimate goal is to propose strategies for decontamination of haloalkanes based on extended mycelial networks.

Abstract

Soil fungi play an essential role in plant growth and development, and the benefits they afford have recently seen them exploited commercially as bioinoculants. Improved phosphorous cycling is one such key benefit fungi can afford to crop production through a variety of modes of action, an understanding of which is important given the context of the unsustainability of rock P supply and the global food security situation. This study used a PSI scanner system to examine the efficacy of several commercial bioinoculants on Lolium perenne growth over a three month period, growth increments recorded daily by a monitored conveyor system and automated scales. Non-destructive cuts were taken at three week intervals. Plants treated with bioinoculants comprised of zeolite carriers demonstrated as much as 10x dry leaf and root mass of competitor products and controls. Soil, leaf and root P were recorded after a destructive cut to quantify P reclamation, which indicated soluble P concentrations up to six times higher in the soils where plants were treated with zeolite carrier bioinoculants, suggesting that the mode of action responsible for the growth improvements involved P mobilisation. Root:shoot ratio, root staining and elemental analysis were carried out to quantify other possible modes of action. Chlorophyll fluorescence from plants undergoing each treatment suggested lower amounts of non-photochemical quenching in zeolite-containing bioinoculant treated plants, suggesting greater resilience to climactic changes.

Abstract

ABSTRACT

In recent years there is growing trend towards organic farming to meet food security and food safety. Inorganic fertilizers are available to boost yield but there are environmental and public health concerns. Amongst various organic alternatives, using mycorrhizal species is one of the most promising option. Therefore, the present research was conducted to evaluate the influence of indigenous mycorrhizal species on the growth of wheat and soil properties under field conditions. In total 11 species of mycorrhizae were identified from the experimental areas, the most prominent genera being, Claroideoglomus, Rhizophagus and Funneliformis. For inoculation of mycorrhizae, their native density was maintained with a novel idea that these species work better in consortia when their native population density was maintained. There were eight different treatments having plot sizes as 6 meters x 2 meters, employing Randomized Complete Block Design (RCBD). The whole set of experiment was repeated at two different sites. Identified mycorrhizal species were mass cultured in pots using same variety of wheat that was used in field experiments. Bio-inoculation of consortia of different mycorrhizal species showed a significant increase in all growth parameters studied e.g., number of tiller per plant (up to 39 %), plant height (up to 13 %), dry biomass (up to 15 %), grain yield (up to 18 %) and hay weight (up to 15 %). Moreover, remarkable effects were recorded on soil fertility such as soil organic matter, available phosphorus and potassium were increased up to 143 %, 53 % and 25 %, respectively. The enhanced effects of different mycorrhizal species on different growth parameters in wheat were attributed to more number of infections these species caused in wheat roots in field experimentations. More over, it is also envisaged that these mycorrhizal species worked better while their native density was maintained in the field that also increased solubilization process of bound minerals. This increased solubilization resulted in pronounced increase of mineral content in the soil that was evident from soil tests of individual replicates from each treatment. The present study concludes that increasing the mycorrhizal inoculum in crop fields by keeping their density as native has tremendous effects on crop productivity and soil fertility status.

Abstract

The soybean cyst nematode (SCN) is a root pathogen of major concern for soybean growers worldwide and causes substantial yield losses in infested fields. A common practice to help manage SCN is rotation of soybeans with corn, a non-host of the SCN. Long-term soybean monoculture leads to a proliferation of fungal antagonists of the SCN in soil and cysts, but few studies have examined the root endophytic community for fungi with SCN-antagonistic activity. The objective of our study is to test whether metabolites produced by soybean and corn root endophytes can kill SCN juveniles, the life-stage most commonly encountered in soybean roots. Fungal endophytes were isolated from surface-sterilized corn and soybean roots from experimental plots in which plants had been grown under annual rotation and under 1, 3, 5, and 35 years of continuous soybean and corn monoculture. Fungal isolates were grouped into 413 morphotypes, and the full ITS region of one representative from each morphotype was sequenced using Sanger sequencing. Isolates with ITS regions sharing 99% sequence similarity were clustered using USearch, giving a total of 114 unique OTUs. Taxonomy was assigned to OTU representatives by using BLAST searches against the UNITE and NCBI databases. One or more isolates from each OTU were grown in malt extract broth and in a secondary metabolite-inducing medium for two weeks, and their culture filtrates were tested for nematicidal activity against SCN juveniles at 6, 24, 48, and 72 hours. Statistical analyses were performed to identify highly nematicidal isolates and to examine relationships between cropping sequences and overall nematicidal bioactivity of root endophytic communities. Results of this study will be used to identify potential biocontrol agents of SCN and to inform farming practices that foster the development of SCN-suppressive soils.

Abstract

Arbuscular mycorrhizal fungi (AMF) provide growth benefits to water-stressed plants. Carrots (Daucus carota) readily host AMF and are commonly used to model symbiosis as root organ cultures, but little is known of carrot-AMF symbiosis regarding whole plant outcomes. Water scarcity threatens production of highly nutritious vegetables due to global climate change; low- input systems are particularly vulnerable. Single inoculant greenhouse studies in sterile media often show that AMF benefit drought-stressed plants, but it is not known how field inoculation impacts carrot performance under low-water conditions. AMF contribution to plant water uptake likely differs among species, but these differences between intraspecific isolates remain unknown. The present study screened four popular carrot cultivars grown in low-nutrient soils inoculated with AMF isolates from geographically distinct locations. Carrots grew in an organic field site in plots amended with whole inoculants of eight AMF isolates. Mock inoculants served as negative controls. Four field studies occurred in 2016 and 2017 with early and late plantings in rain- excluding tunnels that enabled experimental control of water inputs. Carrots received ample water during establishment, and water-limitation occurred during taproot maturation in the six weeks preceding harvest. Biometric data were recorded for roots and shoots. Heirloom carrots differed from hybrid carrots in their response to water limitation and propensity to increase biomass with inoculation by AMF. Fungal isolates differed within species under both low- and high-water conditions. Carrot biomass allocation in response to mycorrhizal colonization differs from other plant models commonly used in mycorrhizal research. Inherent tolerance to water limitation influenced carrot benefits from AMF.

Abstract

The annual yield of fishery industries worldwide is approximately 200 million tons, of which, the catch using fishing boats amounts to approximately 90 million tons and has not increased since 1980. Pisciculture is required to support the increasing demand of fish. For securing a quantity of feedstock for the fish cultivation, harnessing new sources and methods converting them to feedstock is an urgent requirement. Currently, soybean meal (containing neutral detergent fiber [NDF] and crude protein [CP] of approximately 8% and 54%, respectively) and rapeseed meal (containing NDF and CP about at approximately 35% and 48%, respectively) are frequently used as alternative material for fish feed; these are ingredients of compound feeds for aquaculture. However, plant material is also used in animal husbandry; hence, there is limited prospect of securing a sufficient amount of plant material at a low price for aquaculture in future. Pueraria montana is a weed that grows in the temperate zones. The present study aimed to improve the performance of Pueraria montana on the raw ingredients of feedstock, using white rot fungus Pleurotus ostreatus. P. ostreatus was expected to degrade NDF and increase relative CP content. P. ostreatus was inoculated in 100 mL of ddH₂O or 100 mM urea containing 25 g (dry weight) of leaves of P. montana and incubated at 28ºC for 30 d. During incubation without urea, NDF content of P. montana leaves decreased from 38.9% to 23.3%, while CP content rate increased from 23.0% to 25.8%. Urea decreased NDF (21.9%) and increased CP (28.1%), more. Subsequently, P. ostreatus was cultured in glucose peptone (GP) medium with and without additional nitrogen at 28ºC for 7 d. Protease activity of the filtrates of the additional nitrogen-free culture was 0.128 unit/mL. 4 mM urea and 4 mM ammonia (2 mM ammonium tartrate) repressed protease activity to 0.022 unit/mL and 0.059 unit/ mL, respectively. To our knowledge, this is first study to report that basidomycete has a system of nitrogen-metabolism repression and the point at which urea was more effective than ammonia differs from that in ascomycetes such as Aspergillus. We are constructing a ∆AreA strain and a strain containing AreA-eGFP.

Abstract

The tripartite fruticose lichen Stereocaulon paschale is widespread and locally abundant in montane-alpine areas on the British Isles. This species forms cephalodia containing the cyanobacterium Stigonema and cephalodiate thalli have the capacity for moderate levels of nitrogenase activity. However, in upland regions exposed to high atmospheric N deposition cepalodia in S. vesuvianum are infrequent or absent and thalli are often covered by microbial biofilms. Nonetheless, S. vesuvianum remains plentiful in N-enriched areas in contrast to some cyanobacterium-containing lichens which are considered highly sensitive to N and acid-deposition. Accordingly, we have examined the relationship between nitrogen fixation and N-deposition in S. vesuvianum by comparing thallus chemistry (including ¹⁵N natural abundance), thallus morphology and nitrogenase activity among 10 montane sites with modelled N wet deposition (nitrate + ammonium) in the range 2- 36 kg per hectare per year. The common non-nitrogen fixing foliose lichen Parmelia saxatilis has been included in the study as a reference species for which thallus chemistry data have also been collected. Both S. vesuvianum and P. saxatilis occur on rock outcrops and boulders and intercept atmospheric deposits directly without modification by overhanging plant canopies. Data presented suggest that the N supply for S. vesuvianum switches from nitrogen fixation to combined nitrogen as atmospheric N load increases.

Abstract

Peltigera is a diverse and conspicuous genus of lichen-forming fungi, particularly in cold-temperate and boreal regions where macrolichens can constitute an important component of biomass and nutrient cycles. Previous studies on distribution and specificity of the main fungal and Nostoc partners in this genus have typically involved opportunistic sampling on a global scale or intensive sampling on a small spatial scale. In this study, we took a middle ground between these two approaches and evaluated the diversity and co-distribution of Peltigera species and their Nostoc symbionts across the Canadian province of Alberta, taking advantage of an existing collection of ca. 8500 Peltigera specimens collected from 1656 Alberta Biodiversity Monitoring Institute (ABMI) permanent monitoring sites spaced approximately 20 km apart across the province. In particular, we sought to understand: (1) which Peltigera species are present in the province, in light of recent work which has revealed numerous cryptic species within traditional morphological species; (2) what Nostoc phylogroups are associated with Alberta Peltigera species, as part of a global effort to understand patterns of Peltigera–Nostoc specificity; and (3) whether the distribution of Nostoc phylogroups has an influence, independent of environment, on the range of Peltigera species. We sequenced the ITS (Peltigera) and rbcLX (Nostoc) loci for c. 150 specimens and utilized ABMI's existing environmental data for the monitoring plots. ITS sequences showed that Peltigera identifications based on morphology were not always consistent with phylogenetic data. Sequence data indicate that P. britannica and P. wulingensis are more common in the province than previously thought, but distinguishing them from morphologically similar species remains a challenge. There is high genetic diversity among specimens morphologically identified as P. rufescens, P. leucophlebia, and P. canina. This diversity includes several potential new species (i.e., ITS genotypes distinct from anything else in our global database of Peltigera sequences) which will require further investigation using multiple loci, phylogenetic methods, and detailed morphological study. Globally common Nostoc phylogroups tend to be common within Alberta as well. There does not appear to be a relationship between Peltigera species richness at a particular site and the presence of specialist or generalist Nostoc phylogroups, contrary to our early working hypothesis. We present maps of the co-distribution of Peltigera species and Nostoc phylogroups across vegetation types and physiographic regions of Alberta. Our molecular analyses will be used to improve the morphological identifications of the unsequenced ABMI specimens, contributing to better monitoring and protection of lichen communities in the province.

Abstract

Lichens are dynamic and important components of ecosystems worldwide, yet large-scale understanding of functional trait patterns and how these relate to overall patterns of species richness across biomes is largely lacking. North America is a biologically diverse continent spanning numerous biomes from the arctic to the subtropics, and deserts to rainforests. We will present the results of analyses of a new trait dataset of 5,400 lichens and lichenicolous fungi known from the region. We will 1) address how patterns of species richness and endemism are distributed across the continent (north of Mexico) and whether these patterns mirror known hotspots of diversity for other groups, 2) examine correlations between traits, 3) examine patterns and correlations among traits across North American biomes, including as they relate to species range size.

Abstract

Mutualistic symbiotic relationships play important roles in the evolution of many ecologically successful groups of organisms despite their complexity and vulnerability. Lichens are well-known and reasonably well-studied examples of mutualistic symbiosis, consisting of a heterotrophic fungal partner, also called the mycobiont, and photosynthetic algae or cyanobacteria (photobiont). The relationships between the symbionts are shaped by, among others, environmental and evolutionary factors. However, it remains largely unanswered how and in what extent ecological preferences and phylogenetic constrains contribute in interactions between these intimate and long-term partners. We investigated genetic variation and symbiont network patterns of six common lichens along ecological and climatic gradients. Six epiphytic lichens, Hypogymnia physodes, Hypogymnia tubulosa, Lecanora aff. chlarotera, Parmelia sulcata, Physcia adscendens/tenella, and Pseudevernia furfuracea, were collected from the Swiss Alps and Estonia. The sampling sites were selected to include ecological (vegetation openness and host tree species) and climatic variation (mean yearly precipitation and temperature). We sequenced the full nuclear internal transcribed spacer region (ITS) for the mycobiont and the photobiont from the 852 collected specimens, resulting in total of 1622 sequences. We estimated genetic variation and calculated haplotypes for symbiotic partners in each species to evaluate network patterns in lichen microbiomes. Lecanora and Physcia showed the highest variation in mycobiont ITS sequences, with Lecanora dataset also including additional species besides L. aff. chlarotera. Lecanora pulicaris was the dominant species in colder and more humid sites, growing on acidic bark (mostly conifers), while Lecanora chlarotera dominated in dry valleybottoms in the Swiss Alps. The lichen photobionts divided into two groups – Trebouxia impressa and T. jamesii s.lat. Parmelia sulcata, Physcia adscendens/tenella, and Lecanora chlarotera included species-specific lineages of Trebouxia impressa. Pseudevernia furfuracea, Hypogymnia physodes, and H. tubulosa all included photobiont from a single Trebouxia jamesii haplogroup with very little genetic variation and no geographic or ecological distinction. Lecanora species, being the only sexually reproducing lichens among the studied, showed the lowest selectivity for photobiont, with four different photobiont lineages in Lecanora chlarotera and L. pulicaris. Symbiotic interactions in studied lichens seem to be determined by phylogenetic constrains, and are likely dependent on reproductive mode and availability of suitable partners in specific habitats.

Abstract

Factors shaping spatiotemporal patterns of associations in mutualistic systems are poorly understood. Here we used the lichen-forming fungi Peltigera and their cyanobacterial partners Nostoc to investigate the spatial structure of this symbiosis at an intra-biome scale and to identify potential factors shaping these associations. Ninety-three thalli were sampled in Québec, Canada along a South–North and an East–West transect of ca. 1300 km each. We identified the two main partners (Peltigera species and Nostoc phylogroups) and modeled the effect of environmental variables and partner occurrence on Peltigera/Nostoc distributions. Peltigera species showed a high degree of specialization towards cyanobionts, whereas two Nostoc phylogroups dominated both transects by associating with several Peltigera species. Peltigera species had narrower ranges than these two main cyanobionts. Distributions of three Peltigera species were highly associated with precipitation and temperature variables, which was not detected for Nostoc phylogroups at this spatial scale. For these cyanolichens, factors driving patterns of symbiotic associations are scale dependent. Contrary to global-scale findings, generalist Peltigera species were not more widely spread within the boreal biome than specialists, and Nostoc availability was not the only driver of Peltigera species geographic ranges, because environmental factors were also contributing to their intra-biome distributions.

Abstract

A range of selectivity for cyanobacterial symbionts has been demonstrated across investigated lichens, with some species apparently sharing Nostoc widely, and others highly selective, with little to no switching so far observed. We ask how ecological factors affect this symbiotic composition, testing climate, microsite, and environmental availability of symbionts, including substrate sampling and co-occurring ‘companion’ cyanolichens. In the Atlantic forest of Scotland, two Nephroma species often co-occur, the primarily sorediate N. parile and the primarily sexually-reproducing N. laevigatum. Data from rbcLX from Sanger sequences and partial sequences from using Illumina metabarcoding including bryophyte mats on trees, and bare bark were compared. We used ordination and analysis of variance to test sources of variation in the data from a nested sample comprising 6 sites across a steep climatic gradient, those with and without cyanolichen populations and including controls at several levels. In addition, several types of controls were included to check reliability of results and methods. No biases were detected between one-step and two-step PCR amplifications, nor among amplification of different length templates; and consistent results were found with repeat PCRs, and no template controls. Washed and unwashed single thallus extractions were compared. From more than 700K reads, we found ca 300 OTUs at 97% similiarity. There is strong Nostoc genotypic association with climate, and clear patterns of environmental availability of lichen symbionts from bryophyte mats in addition to co-occurring lichens with shared symbiont specificity.

Abstract

The specificity of Nostoc cyanobionts and lichenized fungi has been explored mostly in temperate and paleotropical regions. So far, little attention has been paid to neotropical environments, which are known for their ecological heterogeneity and high species diversity. Here we focused on exploring patterns of phylogenetic and ecological signals in symbiotic specificity of lichen specimens collected in four contrasting biomes of the Caldas department, in the center of Colombia: Andean Western Cordillera (2400-4000 m), Andean Central Cordillera (1670-3045 m), High Andean Central Cordillera (3470-4200 m) and Magdalena Inter Andean Valley (220-1100 m). To address the issue, we generated rbcLX sequences of Nostoc from 45 Leptogium thalli (representing 22 spp.) collected in those four biomes and aligned them with GenBank sequences of Collema and Leptogium cyanobionts. Phylogenetic analysis in a Bayesian framework were done using MrBayes 3. We found one haplotype of Nostoc per thallus, which suggests that each lichen included a single cyanobacterial species, at least at a detectable level. The phylogram obtained showed that the species follow a generalist pattern, as it has been shown before. However, we found that most of the cyanobacterial sequences of individuals from the Inter Andean Valley formed a monophyletic group, suggesting that the fungus associates with a locally adapted cyanobacterium. These results suggest that, whereas species from most habitats followed a similar generalist pattern that has been found in previous studies in temperate and paleotropical regions, the Inter Andean Valley differs. Potential explanations include adaptational value of locally adapted Nostoc strains or descreased cyaobiont diversity due to environmental stress, including high deforestation rate and the constant expansion of the agricultural border.

Abstract

Biodiversity hotspots such as the Atlantic Forest are among the areas most threatened by climate change. Loss of biodiversity is one possible effect, while species displacement and niche changes are others. Based on current niche preferences, future climate models allow to predict such changes. The Atlantic Forest in Brazil is the only region in the world where a continuous, tropical-temperate gradient can be observed in a narrow strip of closed forest from the equator to south of the Tropic of Capricorn, with the added advantage that this gradient can be studied within the confines of a single country. The Atlantic Forest is therefore an ideal target area to analyze the effects of climate change on shifts in species niches and displacements of metacommunities as a whole. Epiphytic lichens are an excellent study object in this context as they are highly sensitive to environmental changes. We thus used epiphytic lichens to estimate a potential spatial shift in the tropical-temperate transition of lichen metacommunities in the Atlantic Forest, selecting the families Graphidaceae, Lobariaceae, Parmeliaceae, and Trypetheliaceae, as these include the two largest families of lichen-forming fungi and two additional families that contain species typical of either tropical or temperate climates. Based on literature review, herbarium revisions, mining of occurrence data in online repositories [GBIF, speciesLink], and new field assessments in the north, southeast, and south of Brazil, we assembled a list of nearly 8,600 occurrence data for the four families in the Atlantic Forest, representing 1,055 taxa. Since existing records often lacked georeferences but included municipality data, we standardized geolocation by using centroid coordinates of municipalities representing the target area [https://www.ibge.gov.br], for a total of 3,241 municipalities. We performed niche modeling on present climate data using a grid raster of 30 degrees [QuantumGIS], resulting in 614 grids covering the target area, with one grid corresponding to approximately five to six municipalities on average. In addition to altitude and forest cover layers, bioclim variables [www.worldclim.org/current] were selected based on prior PCA [PC-Ord] to reduce redundancy effects, and niche modeling [MaxEnt] was performed on 251 taxa present in at least five municipalities each. Modeled, grid-based probability values for each taxon were used to assemble a two-dimensional matrix for grid cluster analysis [PC-Ord], with the main split indicating the line of maximum species turnover between tropical and temperate areas of the Atlantic Forest. We found that the main split based on present climate data corresponded precisely to the Tropic of Capricorn. Projecting the data onto the HadGEM2-ES future climate model [www.worldclim.org/cmip5_10m], we estimated a significant southward shift of the tropical-temperate transition, suggesting an expansion of tropical epiphytic microlichen communities southward and a reduction of available niche space for temperate macrolichen communities. Present niche models for the 251 lichen taxa were further used to characterize within-tropical and within-temperate meta-communities along the latitudinal gradient of the Atlantic Forest, with five meta-communities partially corresponding to the division of the Atlantic Forest into north, southeast, and south.

Abstract

Ectomycorrhizal (EM) fungi are thought to contribute to establishment of host plants after disturbance such as fire, especially in low-fertility xeric soils. Previous work has shown that while a diverse group of fungi are found on roots of pines in undisturbed settings, a different group of fungi are found on pine seedlings after a fire. The post-fire pine symbionts occur as a resistant spore bank that can be observed using soil bioassays. The objective of my study is to investigate if this same dynamic occurs in scrub oak (Quercus ilicifolia) ectomycorrhizal communities. I compare EM fungal assemblages on scrub oak roots collected from the Albany Pine Bush Preserve (APBP) where fire is an integral part of the plant community and is being reintroduced as a restoration tool. The APBP in east-central New York is a fire-managed, globally-rare inland pine barrens ecosystem that, like many fire-dependent ecosystems in the northeastern U.S., supports a disproportionally large number of rare or declining species. I collect scrub oak roots along with the surrounding soil from relatively undisturbed sites. The roots are carefully removed from oak root systems and sorted into morphological types (morphotypes). The soil associated with these roots is air dried to select resistant spore inoculum and used in a bioassay with lab grown scrub oak seedlings in a paired design with field and laboratory bioassay data linked. Fungi on root tips from field and bioassay samples are identified using the fungal barcode (nrITS region). Fungal DNA will be extracted to determine RFLP patterns. The DNA of each unique RFLP pattern is sequenced and submitted to GenBank. The mean proportion of root tips with EM fungi per seedling is analyzed using a paired t-test between field and bioassay seedlings to determine if there is a difference in EM fungal colonization on roots. Additionally mean Simpson’s species diversity is compared to see if there is a difference between treatments. Based on the results with pine I expect bioassay seedlings to be colonized by a different assemblage of EM fungi than seedlings harvested from the mature field sites, suggesting early successional fungi occur in soils as a resistant spore bank. I expect a different assemblage of EM fungi on oak roots collected from the field than roots harvested from the bioassay seedlings, with the field assemblage having a higher richness and a different assemblage of fungi on the roots. Preliminary analyses are promising, supporting this hypothesis. When looking only at morphology, field roots had greater morphological variety than bioassay seedlings alluding to this assemblage change. Field roots had an average of 5.000 (SE = 0.503) morphotypes per seedling while bioassay samples averaged 3.800 (SE = 0.115) morphotypes. Results of this experiment may elucidate how forests recover from disturbance and provide insight on restoration efforts of these threatened young forest habitats.

Abstract

Topography and fire can influence ecosystems' belowground attributes, including mycorrhizal fungi. In temperate and tropical regions, it has been well documented that soil physical and chemical characteristics change in complex ways with topographic position (ridge -valley positions) but that these changes can be modulated by fire. In contrast, we know little about the combined influence of topographic position and fire on mycorrhizal fungi in fine roots, particularly in montane tropical ecosystems. Understanding these complex interactions may be important in tropical montane forests that experience marked seasonal changes in precipitation given that global warming is converting fire in an important driver of change. The main goal of our work was to examine the combined effect of topographic position and fire on mycorrhizal fungi in fine roots of Pinus tecunumanii, including soil nutrients and root biomass in a tropical mesic forest located in the Sierra Las Minas, Guatemala. In paired ridges and slope positions affected and not affected by fire we identified two Pinus tecunumanii individuals and collected soil cores at two distances of the dripline. Roots were cleared and stained to estimated mycorrhizal abundance, whereas soils were analyzed for organic carbon and nitrogen, available phosphorus, extractable cations, and cation exchange capacity (CEC). A preliminary analyses of mycorrhizae showed that burned site have more abundance than unburned sites. On the other hand, most soil chemical attributes were greater in unburned than in burned sites. At the scale of this study fire, but not topographic position, influenced soil chemical properties. Based on these results, mycorrhizal fungi abundance increase with decrease of nutrient availability.

Abstract

Mycorrhizal fungi can affect the ability of their host plants to produce chemicals with which to resist insect and pathogen attack. How such resistance is affected by communities of soil fungi that include mycorrhizal, saprophytic, and phytopathogenic species is poorly understood. The composition of these communities can be altered by tree-killing forest disturbances, but how the resulting changes in soil fungi impact the functional roles these communities play in tree resistance is unknown. Clarifying such relationships is important to predicting the susceptibility of recovering post-disturbance forests to insect outbreaks and disease under predicted climate change-associated increases in the frequency of forest disturbances. We investigated how soil fungal communities altered by forest mortality caused by wildfire, clearcut logging, mountain pine beetle (Dendroctonus ponderosae) outbreak, and salvage logging following beetle outbreak in Alberta differentially affect the induced resistance of greenhouse-grown lodgepole pine (Pinus contorta var. latifolia) seedlings. Seedlings grown in pots inoculated with soil from disturbed forest stands were treated with defense-signaling hormones to elicit metabolomic induction responses associated with insect and pathogen attacks. The resulting seedling metabolomes were analyzed separately for above- and belowground tissues using untargeted metabolomic techniques. Results on how soil fungal community composition influences tree resistance-related metabolites will be discussed.

Abstract

The boreal forest is characterized and limited by the closed circulation of nutrients, related to abiotic factors such as low temperatures and low pH and biotic factors such as recalcitrant organic matter and suppression of saprotrophic activity. Nutrient cycling is regulated by ectomycorrhizal fungi, who associate with plants to trade nutrients for photosynthetically derived sugars. A large part of the boreal forest is managed for timber production, and common practice for increased production is a combination of thinning and fertilization. We used metabarcoding of the ITS region to investigate how the ectomycorrhizal fungal community and associated enzymatic activities are affected by different combinations of fertilization and thinning. We sampled forests along a latitudinal gradient across the entire boreal biome in Sweden. Fungal community assembly was characterized by metabarcoding of the ITS2 region using the PacBio Sequel platform. Ergosterol was measured to evaluate over-all effects on fungal biomass. Changes in fungal community composition were related to increased below-ground accumulation of organic matter in fertilized plots. Relationships are discussed in the context of functional guilds and ectomycorrhizal exploration types.

Abstract

Kauri (Agathis australis, Araucariaceae) is restricted in distribution to the northern tip of the North Island of New Zealand. Living to 1,500 years or more and having trunks up to 3 m diam., A. australis exerts enormous influence on surrounding forest composition and structure, providing varying habitat niches for complex fungal communities. However, information on the diversity of fungi associated with A. australis is sparse. Since the 1970s these trees have been under threat from the exotic invasive pathogen Phytophthora agathidicida that causes kauri dieback. Our study characterised the fungal leaf litter and root endophytic community of A. australis. We obtained root and leaf litter samples from diseased and asymptomatic trees around Auckland, New Zealand. We isolated over 400 cultures from kauri roots and obtained DNA sequences from all of the fungal cultures. We clustered the sequences as OTUs and assigned identities using BLAST. We also used metagenomics to investigate the kauri leaf litter fungi under asymptomatic trees and diseased trees. QIIME and UPARSE were used to assign sequence reads to OTUs. Root endophytic and litter-associated species were distributed throughout the fungal kingdom; most of the endophytic community were in Helotiales and most of the litter-associated community were in Mortierellales. The OTUs were analysed to understand the impact of the invasion of P. agathidicida on fungal diversity at a site, and to increase knowledge of kauri-influenced biodiversity. We compared the OTUs on the basis of habitat niche, geographic location, and disease status. These results give us an insight into the diversity of fungi associated with A. australis and suggest the possible impacts of P. agathidicida on the fungal community.

Abstract

Fungal communities play important roles in litter decomposition and nutrient cycling. The Canopy Trimming Experiment (CTE) began in 2003 at the Luquillo Experimental Forest in Puerto Rico, with the intention of collecting samples that would provide relevant data. The CTE focused on the immediate effects of hurricanes on forest floor processes and their recovery, in a tropical wet forest ecosystem. Changes to the forest’s fungal community structure of litter layers may influence ecosystem recovery. Canopy trimming was performed again in October-November 2014 with the purpose of understanding long-term effects of increased hurricane frequency on forest productivity and carbon sequestration. Our objective was to evaluate if, and how, a hurricane affected fungal communities in the litter. Leaf litter samples were collected in three blocks, at various times up to two years. Based in the results of the first trimming, two treatments were considered for the second: unmanipulated control and trim plus debris. DNA was extracted using MoBio Power Soil DNA Isolation kit. The TRFLP technique was used to obtain profiles of the fungal communities in each sample using the fungal ITS region. Changes in fungal community structure between samples were analyzed using NMDS and Two-Way PERMANOVA. The fungal diversity in the leaf litter increased with the addition of canopy deposits. Fungal diversity decreases as the decomposition of litter progresses. The results indicate significant differences in fungal communities between treatments and though time. Fungal communities were heterogeneous among the treatments and through time indicative of a high turnover of species during the decomposition process. The results support previous observations obtain with the first trimming. In the future, we will analyze the effect of a recent hurricane on the structure of leaf litter fungal communities and the characterization of specific taxa. Climate change will cause an increase in intense hurricanes and understanding their effect in leaf and soil microbial communities will help us understand how resilient or vulnerable tropical forest are to natural disturbances.

Abstract

Climate variability models predict increase in incidence and intensity of hurricanes. In Puerto Rico, hurricanes have impacted the Luquillo Rain Forest in many occasions resulting on canopy debris deposited in the forest floor. As consequence, the microclimate of the forest floor changed by the increase of direct sunlight, addition of complex plant biomass, alteration of microbial activity and, ultimately, the operation of biogeochemical cycles. A Canopy Trimming Experiment, that simulated the pass of a hurricane, has been done in the Tabonuco forest. It was designed to understand the effect, resistance and resilience of a tropical forest ecosystem after the impact of a hurricane. Our objective is to determine temporal heterogeneity of three microbial groups (fungi, bacteria, and sulfidogens) in response to detritus deposition of simulated hurricane effect. Two treatments are considered: with and without detritus deposition trimmed from the local canopy. Soil samples are being collected from plots, at various times for a period of two years. Bacteria, fungi, and sulfidogens are being characterized independently by the molecular analyses of three distinctive genes (16S rDNA, ITS, and dissimilatory sulfite reductase) using Terminal Restriction Fragment Length Polymorphisms. Fungal diversity was greater than the bacteria over time. Bacteria was homogeneous over time for the same plot suggesting microbial succession in which rare microbiota became more prevalent over time. Bacterial and fungal communities exhibited spatial variation regardless the availability of plant debris. Diversity of sulfidogenic bacteria decreased over time where detritus was added. Richness for sulfidogens was lower in the absence of detritus. Diversity trends describe the dominance of fungi after deposition of debris and the gradual involvement of the anaerobic sulfidogenic bacteria. Fungal decomposition of complex substrates in the canopy debris seems to foster anoxic conditions where anaerobes thrive using more labile carbon sources. Further studies include the effect of recent hurricanes on the structure of microbial communities, characterization of specific taxa and quantification of microbial guilds examined.

Abstract

Seedling regeneration after forest dieback events is crucial for forest recovery. In a subalpine coniferous forest in Kii Peninsula in Japan, however, spruce seedling regeneration has been seldom observed after a wide-range forest disturbance caused by a typhoon in 1959. Deer-proof fence constructed to reduce grazing by deer has not improved regeneration. Given that spruce seedlings need coarse woody debris (CWD) to colonize, their establishment is greatly affected by CWD condition which largely depending on decay activity of decomposer fungal community. Recent studies in Europe found that CWD decayed by brown rot fungi negatively affects spruce seedling density, and that frequency of occurrence of brown rot fungi tends to be increase after forest dieback. These results lead a hypothesis that forest dieback in Kii Peninsula also lead dominance of brown rot fungi and the logs decayed by them reduce spruce seedling regeneration. To test this hypothesis, we compared fungal communities within CWD and spruce seedling density among three sites of different dieback levels (control, intermediately dieback, and intensively dieback). Fungal communities within CWDs were documented using Illumina sequencing. Seedling and epiphytic bryophyte communities were recorded and were analyzed with CWD properties such as wood decay type (white rot, brown rot, and soft rot), pH, moisture, and bryophyte coverage. Sequencing showed that the frequency of brown rot fungi was higher in intensively dieback site than intermediately dieback site and control site. Among the CWD properties, frequency of brown rot wood showed the same trend found in brown rot fungi. Spruce seedlings and bryophyte coverage were higher on CWDs in the control and intermediately dieback sites than that in the intensively dieback site. Spruce seedling density was negatively associated with brown rot in sapwood and positively with bryophyte coverage. Brown rot in heartwood negatively associated with bryophyte coverage. These results suggest that dominance of brown rot fungi after forest dieback event in Kii Peninsula negatively affected spruce seedling regeneration.

Abstract

The Mortierellaceae are an ecologically and industrially relevant lineage of early diverging fungi comprised of six genera classified within what is now recognized as phylum Mucoromycota. This polyphyletic family is estimated to contain at least 100 species, the phylogeny of which cannot be fully resolved with ribosomal markers. The ITS region is too divergent to align across the entire lineage and while the 28S region can be aligned across the family, there is poor backbone support for the resulting phylogenetic tree. In this research, we tested the use of high-throughput targeted amplicon sequencing for generating multi-locus sequence data to improve phylogenetic resolution of diverse lineages within the Mortierellaceae. First, we analyzed three de novo sequenced Mortierella genomes with a non-biased bioinformatic pipeline to identify potential non-ribosomal markers and designed PCR primers for multiplexed PCR amplification. We identified 13 loci that performed well across a diverse test panel of Mortierella isolates. We amplified these loci across 330 Mortierellaceae isolates, prepared libraries with an Illumina Nextera kit, and sequenced them on an Illumina MiSeq platform. Our non-biased locus selection successfully identified established phylogenetic markers (i.e., TEF1 and RPB1); however, 7 loci were members of gene families or under selective pressure and therefore inappropriate to use as phylogenetic markers. This multilocus sequence typing (MLST) approach is very robust in extracting phylogenetic information from isolates contaminated by non-target organisms (e.g., Fusarium or Mucorales), an advantage over Sanger sequencing of universal barcodes or genome sequencing. However, we were less successful in resolving cross-contamination by other Mortierellaceae. Low-coverage genomes that were available for 60 of our isolates were analyzed to identify 400 informative markers. These markers were used to build a strongly supported genome-based phylogeny. We are identifying MLST loci within the low coverage genomes to serve as a backbone constraint for MLST phylogenetic analyses. This combination of approaches leverages both the sequencing depth of genomics and the sampling depth of amplicon-based sequencing. A resolved phylogeny and identification of non-ribosomal markers will improve identification and placement of novel species and genotypes as they are isolated, and inform higher taxonomy of this family. We will discuss the resulting phylogeny, informative non-ribosomal loci, our evaluation of the MLST approach, and implications for other lineages and organisms.

Abstract

Arbuscular mycorrhizal fungi (AMF) are ecologically and agronomically important, but global data on their species abundance and diversity are scarce. From ~290 morphologically described AMF species only a part is sequenced or available in culture collections, due to their obligate symbiotic lifestyle. The amount of ‘taxa’ based on environmental DNA sequences largely exceeds the number of described species. Different research teams often use different DNA marker regions for AMF diversity and taxonomy studies, therefore it is impossible to compare their results and make conclusions on global AMF species distribution, ecology and biogeography. Thus, research is needed to further define the AMF species on the molecular level and to understand their genetic variability. We optimized an AMF-specific nested PCR approach to amplify part of the rDNA spanning majority of the SSU, complete ITS region and part of the LSU (~2.5 kb). This target fragment was amplified from morphologically described taxa in cultures representing main AMF lineages and selected AMF species-rich root and soil field samples from across the globe. PCR products were multiplexed into libraries and sequenced on the PacBio platform using the SMRT (Single Molecule Real Time) sequencing. High accuracy of such long reads was achieved due to construction of circular consensus reads (>5 passes) with 99% predicted accuracy. Sequences spanning the target fragment will enable us to evaluate genetic variability and determine bioinformatic thresholds for species, genus, family and order- level resolution for the different rDNA marker regions used (SSU, ITS and LSU) for all main AMF lineages, to conduct a robust backbone phylogeny, to compare results of AMF community studies targeting different marker regions and search for general patterns in AMF biogeographic and ecological distribution at global scale. The results will improve the environmental sequence assignment to the species-level and thus serve for a better interpretation of AMF community data from next generation sequencing studies.

Abstract

Neocallimastigomycota is a distinct fungal phylum consist of micro-fungal symbionts in mammalian rumen and gastrointestinal tracts (e.g. Elephant, Horse, Sheep etc.). Neocallimastigomycota differ from other zoosporic fungi (Chytridiomycota and Blastocladiomycota) in many aspects including the strict requirement for the anaerobic environment. There is still limited knowledge about the biology and ecology of these fungi, except for their pivotal role in the plant biomass degradation for their animal hosts. To better understand the evolutionary history and genomic context of the Neocallimastigomycota we sequenced 22 transcriptomes from 7 distinct taxonomic groups (Anaeromyces, Caecomyces, Feramyces, Neocallimastix, Orpinomyces, Pecoramyces, and Piromyces). Combining publically available genomes of Neocallimastigomycota and Chytridiomycota, we conducted phylogenomics and comparative genomic analyses of 32 taxa to better understand gene content and phylogenetic relationships of these lineages. Neocallimastigomycota is estimated to emerge as a monophyletic group (~74 Ma) with the herbivory in mammals (72-100 Ma) based on 426 conserved orthologous using BEAST analyses. Comparative genomics identified that Neocallimastigomycota fungi have lost functional Protein Family (Pfam) domains retained in Chytridiomycota related to oxygenase, dioxygenase, photolyase, and uric acid metabolism. We also identified Pfam domains that are specific to Neocallimastigomycota but missing in other chytrids. These include many carbohydrate binding modules, glycoside hydrolase, metal transporters, and plant cell wall binding domains. Three domains (“Cthe_2159”, “Gal_lectin”, “YoeB_toxin”) are uniquely found in these lineages and have not been identified in any other fungi. One anaerobic functional domain HemN (oxygen-independent coproporphyrinogen III oxidase) is found to be missing from the higher classes of Animal and Fungi (Dikarya). Using phylogenetic methods we aim to better understand the biological functions of these domains which appear most dramatically different in these fungi. These include galactose-binding lectin, oxygen-independent coproporphyrinogen III oxidase, and the polysaccharide lyase Cthe_2159 domains. Using these approach we hope to better understand what roles they play in the animal rumen ecosystem, whether these originate through horizontal gene transfer, and how these dynamic gene content changes inform evolution and emergence of the enigmatic Neocallimastigomycota fungi.

Abstract

There are an estimated 10 million extant species of fungi, each of which contains a genome with unique functional genetic diversity and capacity. Genomic resources provide research opportunities for medicine, toxins/anti-toxins, and food, and a deeper understanding of the world in which we live. While genomes are being sequenced at an unprecedented rate, genomic sequencing is still contingent upon cellular extractions of high molecular weight DNA that are neither sheared nor degraded, and are free from RNA and other contaminants. Factors known to impact DNA quality are the pre-extraction culture conditions, including media contents and concentration, mycelial growth rate, and age of the culture. The NSF-funded ZyGoLife consortium has prioritized the genome sequencing of Mucoromycota fungi, since these fungi are under-sampled and less studied than Dikarya (Ascomycota, Basidiomycota). For these reasons, we optimized a CTAB-PVP genomic DNA extraction method for generating high-quality DNA extractions from fungi in the Mucoromycota (Rhizopus and six clades of Mortierella) for genomic sequencing through the Department of Energy Joint Genome Institute. DNA quality was measured using a Nanodrop to compare the ratio of absorbance of nucleic acids versus protein (optimal 260/280nm ratio: 1.6-2.0), and agarose gel electrophoresis for ensuring concentrated high-molecular weight DNA free from RNA contamination. DNA quantity was assessed with a Qubit fluorimeter (target amount: 12 μg). Overall, we found that the quality of DNA extraction varied with species and taxonomic group, with the best results from Mortierella lignicola and M. ambigua. Some of the factors that improved DNA quality involved: abundant fungal tissue, multiple replicates of a sample – with less tissue per tube, ‘Snap-freezing’ tissues in liquid nitrogen to prevent DNA degradation, and treating the DNA with RNase before precipitating. This approach has yielded pure, high molecular weight genomic DNA and improved upon previous CTAB-PVP protocols. We anticipate that this approach will enable future work with previously unsequenced fungi in Mucoromycota, and will be applicable to genome sequencing in other Phyla.

Abstract

The adoption of metagenomic environmental sampling technology by mycologists for community analyses have uncovered entirely new lineages within the fungal tree of life such as the Archaeorhizomycetes and the Cryptomycota. Metagenomic ecological analyses of fungi have also enabled broad insight into the global and large-scale distribution of fungi. These studies have led to testable hypotheses about the factors that influence biogeographic patterns in fungi, such as fungal distributions shaped by dispersal limitation and climate. However, awareness of inherent methodological biases such as primer mismatch, taxonomic representation in reference databases used for operational taxonomic unit (OTU) assignment, and sequencing and barcoding errors have resulted in modified protocols for certain groups. Many surveys of soil-inhabiting fungi have focused primarily on ectomycorrhizae or other fungi in Dikarya. Despite being diverse and ecologically important members of the soil environment, Mucoromycota and Zoopagomycota are often briefly mentioned or included only as a taxonomic rank in OTU richness figures in metagenomics studies. Though members of the Zoopagomycotina are often encountered in culture-based surveys, their detection in environmental sampling surveys is limited. Here we present an empirical test of Illumina sequencing which demonstrates the strong bias against long internal transcribed spacer regions (ITS) of the mycoparasitic Zoopagomycotina taxa Piptocephalis and Syncephalis. Previously-analyzed soil DNA samples were spiked with DNAs of isolates of known ITS length ranging from short (~600 bp) to medium (~750-900 bp) to long (>900 bp). A treatment comprised of a mixture of Piptocephalis and Syncephalis DNAs with varying ITS lengths and a negative control were added to the experimental design, resulting in five treatments that were each replicated three times. All treatments were amplified with the fungal ITS primers ITS1F and ITS2 and sequenced with Illumina MiSeq. Our results show minimal to no detection of the longest sequences, variable detection of medium sequences, and preferential detection of short sequences. We also provide preliminary analyses of the Zoopagomycotina community in environmental samples from Florida based on group-specific, modified metagenomic protocols.

Abstract

Incursions from exotic Phytophthora species are considered one of the principal threats to forest ecosystems around the world. New Zealand kauri (Agathis australis) is currently threatened by a recently described, exotic Phytophthora species, P. agathidicida. Pre-emptive detection of novel Phytophthora species could provide an early warning of potential disease threats before they become established. While we have knowledge of the post-border soilborne Phytophthora species present in the kauri forest, we lack a complete understanding of the diversity of Phytophthora species naturally occurring in this system. Research published in Western Australia, Europe and the USA has demonstrated that stream-based catchment-wide surveys of Phytophthora can provided useful baseline data on the diversity of Phytophthora species, and have identified novel taxa not previously known to science.

In this study, we used leaves of either native species (e.g. Agathis australis and Pittosporum tenuifolium) or exotic species (e.g. Cedrus atlantica, Cedrus deodara, Pinus radiata, Rhododendron arboretum and/or blue lupin cotyledons Lupinus angustifolius L.) to capture the diversity of Phytophthora species occurring in the Cascades Kauri Park (west Auckland) and Whangaparoa (Coromandel) catchments – both catchments contain forests with the kauri dieback disease, and include kauri in the riparian zone. We found that stream baiting can detect Phytophthora species year-around, and in total 12 different species of Phytophthora were retrieved across the west-Auckland and Coromandel catchments. The diversity of species recovered included species considered to be part of the “aquatic” Clade 6, however, the further isolation of P. multivora and P. cinnamomi from the baits means that soil-borne Phytophthora species can also be detected via stream surveillance. Species not previously recovered in New Zealand from soil baiting in the kauri forest were also detected in the streams. A previously undescribed species in clade 9 was also characterised, and a member of the recently described Nothophytophthora.

The “bait” species with the most utility to attract the greatest diversity of Phytophthora species were shown to be the Himalayan cedar Cedrus deodara and kauri Agathis australis. This study demonstrates the potential for stream baiting to be used for passive-surveillance, and to monitor changes in Phytophthora diversity in forested water catchments. As part of the research project, we developed a re-usable, plastic “bait-cassette” which helped standardise the experimental approach, and made the technique easily operated by school students and “citizen scientists”. This will help build awareness around kauri dieback and the pathways through which it is spread utilising a participatory, science platform.

Abstract

Health risks connected to the indoor microbiome are mostly associated with poor indoor air quality. Wherever excess moisture is available, fungi and other microorganisms start to grow. These microorganisms can act as a source of indoor pollutants and cause poor indoor air quality that is associated with adverse health effects, such as allergies, asthma and other respiratory symptoms. In other parts of the world, several studies have been performed trying to identify the main determinants of the indoor mycobiome and important factors influencing the mycobiome. These factors commonly include building type, geography, ventilation and outdoor air influence amongst others. Therefore, the overarching aim of our project is to improve the knowledge about the indoor mycobiome in the Northern Europe using DNA metabarcoding analyses. By analyses of dust samples, the indoor mycobiomes are characterized at different spatiotemporal scales; within buildings, across buildings at larger geographical scales, as well as throughout different seasons. Sampling has been performed within Norwegian Kindergartens throughout a year by biweekly sampling at four different floors. Dust has been collected from specific areas on identical glass plates by using floq swabs and tape. Real-time PCR has been performed covering the 20 most common fungal species in the indoor air, including Aspergillus, Penicillium, Alternaria and Chaetomium amongst others. The Real-time PCR results can then be compared to the metabarcoding data. We are also evaluating the usage of internal standards for improved quantitative information from DNA metabarcoding data. Compositional differences in the indoor mycobiomes will be coupled to local and regional environmental variation and building characteristics through multivariate analyses.

Abstract

In the riparian forests, a number of extremely important functions of the ecosystems are carried out maintaining the quality of water and stability of soils. In the riparian portions of rain forest fragments, large quantities of organic matter are produced that guarantee nutrients to the maintenance of these ecosystems, besides offering essential environmental conditions for fungal development. Among the fungi capable of decomposing organic matter are the hyphomycetes, the largest (morpho-)group of conidial fungi which main importance to the ecosystems is to promote nutrient cycling through decomposition of dead organic matter. Therefore, the aim of this work is to quantify fungal biomass in the leaf litter and in the soil of riparian regions in areas of the rain forest that are under environmental protection in the Northeast of Brazil. Three sets of samples (soil leaf litter, submerged leaf litter and soil) were taken between August 2014 and July 2017, in four conservation unities (REBIO-Saltinho, RVS-Gurjaú, REBIOmu-Mata da Chuva and APA-Lagoa da Mata) in Pernambuco, Northeast of Brazil. Each area was represented by six sampling points that were sampled at least six times. These samples were kept in the freezer until extraction of ergosterol and analysis of this biomarker by HPLC were carried out. Statistical analysis showed that there are differences between the areas for each type of substrate. The soil presented largest amounts of fungal biomass than the decomposing leaf litter independent of litter origin. Submerged leaf litter presented the lowest amounts of fungal biomass. The amplitude of variation between samples of each type of substrate was largest for the soil samples, indicating the importance of microhabitats. There was alternating higher and lower values of ergosterol in all substrates along the period of study, therefore no pattern of ergosterol amounts could be established when comparing with the occurrence of fungal species that have been detected by morphological analysis. The use of a multiplicative conversion factor proposed in the literature resulted in a variation of fungal biomass in the studied natural substrates ranging from 21.84 to 409.5 micrograms of mycelial mass per gram of soil or leaf litter, however, these values can be considered overestimated by a factor of approximately 20%, according to the literature. Nonetheless, ergosterol is considered a good biomarker to estimate fungal biomass in complex substrates. Financial support: CAPES and CNPq.

Abstract

The host-associated microbiome, the community of microbes living on or within a host organism, has become a popular area of study in animal health research. Recent breakthroughs in DNA sequencing technology have greatly expanded our understanding of the diversity, distribution, and abundance of host-associated microbes. However, basic biological questions about microbiome structure remain to be answered. For example, it remains to be understood what role host genetics plays in determining microbiome assembly and diversity. In addition, microbiome studies rarely examine how both eukaryotes and bacteria contribute to overall microbiome structure. In this study, we (1) determined the impacts of host genetics on the microbiome, and (2) examined the positive and negative associations between bacterial and eukaryotic taxa found in the microbiome. We used a model system of Brazilian land-bridge island frog (Thoropa taophora) populations. Isolated island T. taophora frog populations were previously shown to contain very low genetic diversity relative to mainland populations. We collected skin swabs and genetic samples from frogs to respectively analyze the skin microbiome and frog host genetics using DNA sequencing. To determine associations between microbiome structure and host genetics structure, we examined the effects on microbiome assembly and diversity of two aspects of host genetics: (1) population-level neutral genetic diversity, and (2) genotype at the MHC immune locus, which we hypothesized would play a significant role in the ecological selection of microbes. Microbiome structure was significantly related to both host neutral genetic diversity and individual MHC immune genotype. Frogs from genetically diverse populations hosted a higher diversity of microbes. In addition, within each population, frogs that were heterozygous at the MHC locus hosted a higher proportion of likely commensal (non- parasitic) microbes. We then employed a network-based analysis to determine the positive and negative associations between bacterial and eukaryotic taxa across hosts. Specifically we were interested in testing whether bacteria previously shown to inhibit fungal growth were negatively associated with fungal diversity. This analysis is still in progress, but preliminary results suggest no negative associations between the presence of “antifungal” bacteria and the abundance of microbiome fungi. The results of this study contribute to our understanding of how host genetics and ecological interactions between microbes impact the structure of the host-associated microbiome.

Abstract

Batrachochytrium dendrobatidis (Bd) is a dermatophytic pathogen that has caused enigmatic declines in amphibian populations worldwide. Salamander populations in the eastern US persist in the presence of Bd with surprisingly low rates of infection, despite relatively high rates in co-occurring anuran populations, which may be due to cutaneous probiotic defenses. We conducted a foundational study to better understand effects of microbial community dynamics on pathogenicity of Bd, and to identify keystone probiotic members of the salamander skin microbiome. Objectives were to (1) use high-throughput DNA sequencing to characterize the structure of the skin microbiome, (2) isolate and identify bacterial isolates that inhibit Bd, and (3) evaluate dominance of antifungal isolates within the microbial community as a whole. During May and July in both 2016 and 2017, salamanders from the genera Plethodon (n=67), Eurycea (n=65), and Desmognathus (n=83) were captured from nine sites in Tennessee, and skin swab samples were obtained. We used high-throughput DNA sequencing techniques and bioinformatics analyses to define the composition and structure of the resident skin microbial community across salamander taxa, seasons, and ecoregions. Results indicate that both geography and host taxon significantly affect the structure of the microbiome, but seasonality does not. Additionally, we isolated a total of 476 bacterial colonies in pure culture. Of the 63 isolates that have been challenged against Bd, 16 formed measurable zones of inhibition and were identified through Sanger sequencing. We compared genotypes to a published database of antifungal isolates, and we identified three new anti-Bd isolates. After conducting indicator species analyses within salamander skin communities, we pinpointed a core member of the microbiome that is also a strong antagonist of Bd. During future research, we will identify additional core probiotic members of the skin microbiome, infer their ecological roles through construction of interaction networks, and continue screening to evaluate candidacy for in vivo treatment trials. Results may directly impact future conservation efforts for North American wildlife.

Abstract

Understanding the generation, maintenance, and distribution of genetic diversity is fundamental to predicting the ecological trajectory of emerging diseases. Chytridiomycosis, caused by the chytrid Batrachochytrium dendrobatidis (Bd), is the emerging infectious disease implicated in recent population declines and extinctions of amphibian species worldwide. In regions where Bd has been recently introduced to naïve host populations resulting in disease outbreaks, only a single hypervirulent clonal genotype has been observed (BdGPL). In the Atlantic Forest of southeastern Brazil, however, a deeply divergent – and potentially endemic – lineage has been recently described (BdBrazil). This newly discovered population of Bd provides a critical opportunity to characterize standing genetic variation, and to probe the dynamics of virulence evolution in this emerging pathogen. Here we characterize the population dynamics of Bd lineages in the Brazilian Atlantic Forest using whole genome resequencing of field-collected isolates. Our sequencing reveals a high degree of genomic plasticity, with variable chromosomal copy number and mitotic recombination as major drivers of variation among isolates. Our population genomic analyses suggest that the two lineages have been brought into secondary contact by human activity, with the BdGPL having recently arrived in the historical range of BdBrazil. While the long-term consequences of this secondary contact remain largely unknown, our field studies recovered a number of hybrid strains resulting from outcrossing events between BdGPL and BdBrazil. Using our genomic dataset, we assess the patterns of inheritance in hybrid strains to address the degree of incipient reproductive incompatibility between these Bd lineages. Our study provides insight into the genetic basis of divergence, adaptation, and establishment of fungal pathogens; and we highlight the utility of comparative genomic tools in understanding the evolutionary ecology of novel emerging diseases.

Abstract

The fungus Pseudogymnoascus destructans (Ascomycota: Leotiomycetes) is invades the skin of European and Asian hibernating bats. This pathogen was recently introduced to USA, where it causes so called “white nose syndrome”, resulting in the bat mortality. Our team has long time interest in the multidisciplinary research on P. desctructans genetics and physiology and ecology. We studied the genetic relatedness of European and Asian (Ural) strains using six variable genetic loci including the mating type factors. Both mating types were found in the Czech Republic in the equal proportions, what showed to its cryptic sexuality. Among the five recognised haplotypes, one was found in the Czech R., USA as well as in Ural, thereby expanding the source region of this fungus. Further, we compared the extracellular enzymatic activities and secondary metabolite profiles of six virulent strains (Europe, Asia, USA) and six non-virulent Pseudogymnoascus species. The characters specific to P. destructans were the lipase activity, production of siderophores and hyper production of the riboflavin (vitamin B₂). This shows, that siderophores, which are common virulence factors in microorganisms, play also the important role in P. destructans pathogenesis. Riboflavin is hyper accumulated in the skin lesions, and is responsible for their distinctive yellow-orange fluorescence. Its local concentrations reached the values toxic for the bats cells and its massive presence can significantly weaken the infected bats. The other aim of our study was to clarify properties responsible for unique ecology of P. destructans by comparison with ecological related or unrelated pathogenic or nonpathogenic fungi. This part includes study of tolerance to physiological stresses and recognition of spectrum of utilizating nutrients (compounds of carbon, nitrogen, phosphorus, sulphur and nutrient supplements). Influence of several types of physiological stress (e.g. UVA, UVA with UVB, 25 °C, 30 °C, 37 °C and dryness) was investigated with fluorescent stain propidium iodide by flow cytometry. The spores of Pseudogymnoascus destructans and three fungi from underground spaces were not viable after 3 weeks at 37 °C. Other stresses did not cause a decreasing of viability or some stresses caused a decreasing of viability only in some strains of fungi. System Biolog showed, that isolates of P. destructans differ in utilization of sources of carbon, nitrogen, phosphorus, sulphur and nutrient supplements from other testing fungi. The selective isolations medium was developed in the course of the study and tested in real conditions.

Abstract

There are many symbiotic and parasitic relationships known between fungi and humans. For instance, some fungi are components of the normal flora in mammalian intestinal tracks, and some are parasitic inside the body and/or on skin tissues. Additionally, micro- and macro-fungi are prominent components of soil. Some of these soil fungi prefer ammonia and lipid rich soils, such as are produced by decomposing bodies. Knowing these previously mentioned attributes of fungi, we asked the question: how can mycology help solve problems in forensic science? Forensics is used in every day crime investigation. Many sub-disciplines are well known and researched, such as forensic entomology; however, mycology is rarely used and is not extensively researched in a forensics context. Fungi can be used to determine time of death by looking at fungal succession on deceased individuals. Also, they can serve as trace evidence to support location of a crime by linking perpetrator to a crime scene by comparing fungal traces, such as spores and mycelium, and yeasts in soil and on materials such as clothes or tires. Our goal is to determine a relationship between fungal flora changes, in terms of community composition, and post-mortem interval (PMI) based on human swab samples. Samples were obtained from a medical examiner from 65 different bodies that were undergoing natural decomposition (were not embalmed). Individuals selected were of varied PMI (between 12 and 456) hours. To broaden our sampling, individuals were included of differing sexes, races and ages, and from different seasons, locations, and causes of death. These samples were collected from different regions of the body, including ears, nose, umbilicus, mouth, eyes, rectum, arms, and skull. The DNA extracted from the body samples was amplified at the fungal barcode (ITS region), and sequenced using Illumina MiSeq. The fungal flora associated with human decomposition will be presented and correlations with PMI will be shown.

Abstract

Two entomopathogenic fungi, Verticillium lecanii and Beauveria bassiana, were tested against Rhipicephalus annulatus. Mycotal® was the source of Verticillium lecanii while Biosect® was the source of Beauveria bassiana. Five concentrations (1×10⁷, 5×10⁸, 2.5×10⁹, 1×10¹⁰ and 4×10¹⁰ spore/ml) of Verticillium lecanii as well as five different concentrations (5×10⁷, 2×10⁸, 8×10^9, 3.2×10¹⁰ and 12.8x10¹⁰/ml) of Beauveria bassiana were prepared and tested against adult female tick, eggs and larvae. The mortality in adult ticks was 60.60 to 72.00% after 2 weeks of application for V. lecanii at concentration ≥ 5x10⁸spore/ml, while B. bassiana showed no mortality at any concentrations. The treated tick revealed nutritional index significantly lower than control untreated one for both fungi. Furthermore, V. lecanii showed no effect on eggs, while, B. bassiana delayed and reduced the egg hatching. In addition, both fungi caused 100% mortality of larvae. The effective concentration was ≥ 10⁸spore/ml for both fungi with no significant difference among the highest concentrations. Moreover, the fungal extract had no effect on adult tick. In conclusion, V. lecanii is lethal to adult tick and B. bassiana caused larvae mortality and reduced egg hatching. A prospective application of fungi in the pasture or animal farm is possible for tick control.

Abstract

Tea shot hole borer (TSHB, Euwallacea fornicatus) and polyphagous shot hole borer (PSHB, Euwallacea sp. nr. fornicatus) are morphologically identical wood boring ambrosia beetles that vector Fusarium spp. from the ambrosia Fusaria clade (AFC) in female mycangia, and have been reported as invasive pests in multiple regions around the world including: Madagascar, Costa Rica, Guatemala, Panama, Hawaii, Florida, California and Israel. These xylomycetophagous pests are native to southeast Asia and are a cause for concern as they attack multiple hosts in natural, urban and agricultural environments using a suite of fungal mutualists including Fusarium spp., Graphium spp., and Paracremonium spp. In agriculture, TSHB is best known for causing damage on tea (Camellia sinensis) in India and Sri Lanka, while PSHB has been reported causing damage on avocado in California and Israel. In these locations, TSHB and PSHB have been found to be associated with heterothallic Fusarium ambrosium and Fusarium euwallaceae respectively, suggesting an exclusive mutualism with these AFC members. The aim of this study was to investigate the symbiotic association of AFC fungi and Euwallacea spp. from a field location in Taiwan where the two species are sympatric. Ten female beetles from Danei District Tainan City, Taiwan were sampled from six infested avocado groves. The females were surface sterilized with 70% ethanol and the heads, containing mycangia, were removed from the bodies to isolate fungal mutualists. Single colonies of Fusarium spp. were recovered from each respective beetle and identified using multi-gene phylogenetic analyses of the internal transcribed spacer region (ITS), translation elongation factor-1 alpha (TEF1-α), and RNA polymerase II subunit (RPB1, RPB2) regions. The body of each beetle was subsequently used to establish its specific identity via a high-resolution melting assay. Of the recovered specimens from Taiwan, four TSHB and six PSHB were determined to be vectoring two Fusarium spp., from two divergent clades within the AFC. Within the two AFC clades, PSHB and TSHB were found to vector the same Fusarium spp., indicating the mutualism within Euwallacea spp. is not exclusive, but promiscuous in this region. The Fusarium spp. sampled also indicate that both Fusarium phylogenetic species are heterothallic and both mating types are present within the Fusarium spp. recovered from the beetles. These findings show evidence for a non-exclusive mutualism in heterothallic Fusarium spp. vectored by Euwallacea spp. in native areas within Taiwan.

Abstract

Fungus-farming ambrosia beetles are a globally distributed, abundant, and diverse group of wood-boring beetles, boasting more than 3,000 species in the weevil subfamilies Platypodinae and Scolytinae. Ambrosia beetles cultivate gardens of nutritional fungi to feed their young. They have complex organs, termed mycangia, to carry living fungal colonies while dispersing from their natal galleries to new trees. The fungal symbionts of less than 5% of these beetles have been described and aside from a few known plant pathogens, the ecological impacts of these fungi are largely unknown. The majority of the described fungal symbionts of ambrosia beetles belong to Ascomycota and are not known to decay the complex structural components of wood. Recently, however, an aggressive wood-decaying member of Basidiomycota, Flavodon ambrosius, has been described as the nutritional symbiont of a diverse, globally-distributed clade of ambrosia beetles (Ambrosiodmus and Ambrosiophilus). These beetles are common and infest a diverse range of host trees. Thus, this symbiosis may have broad implications for wood-decay processes world-wide. Previous culture-based work on beetle-associated Flavodon ambrosius has been largely restricted to Asian beetles in Asia and introduced to North America and suggested that these beetles farm a single Flavodon species worldwide. We examined Ambrosiodmus and Ambrosiophilus beetles native to multiple continents, as well as the closely related genus Beaverium. In addition to culture-based assays on freshly-collected beetles, we use next-generation sequencing (Illumina and PacBio) approaches on both fresh and preserved specimens from existing collections. This combined approach allows us to look for multiple symbionts within each beetle and also facilitates broader geographic and taxonomic sampling. Moreover, we compare the results obtained from each method and we suggest an efficient combined approach for future surveys. We incorporated single-copy fungal mock communities to help parameterize our bioinformatics pipelines for both sequencing platforms. We further support our results with mock communities composed of fungal taxa that are closely related to Flavodon ambrosius. We discovered new diversity in Flavodon from beetles native to both Asia and North America, including a new association between an undescribed Flavodon and the beetle Beaverium. We also found the presence of Asian Flavodon within the mycangia of North American beetle species, suggesting an introgression of invasive fungi into native beetles. By shedding light on the dark diversity of decay fungi present in beetle mycangia, our results demonstrate that the symbiosis between globally distributed ambrosia beetles and aggressive wood decay fungi is more diverse and widespread than previously thought. These interactions may be important to decomposition and nutrient cycles in forest ecosystems. Our results call attention to the potential ecological impacts of widespread introductions of these beetles and their symbionts.

Abstract

Recent studies have shown that ant-fungi interactions may be much more common, than previously thought. Studies on ‘carton ants’ and ‘black yeasts’; Formica aquilonia and its yeast community; or F. pratensis and the fungus Mortierella formicae lead us to believe that a number of fungal species engaged in interactions with ants still could be undiscovered. Moreover, previous research on ant-fungus interactions, with the notable exception of leaf-cutting ants and their fungal gardens, focused mostly on visible, lethal, ant-parasitic fungal species (e.g. Pandora, Cordyceps spp.), neglecting the mycobiota that inhabits the cuticular surface of ants. Furthermore, in ants occur an age-dependent division of labour which, more or less, goes hand in hand with spatial distribution of individuals, with young ones being confined mostly to the inner part of the colony and old ones foraging outside, coming in contact with different fungal species. Thus, an age-dependent compositional change of mycobiota would be expected. The purpose of this study was to investigate the yet unknown, mycobiota of the ant Myrmica scabrinodis and to compare the mycobiota of young and old individuals. We analyzed the mycobiota of 324 M. scabrinodis ants from nine ant colonies. Five colonies were also infected with another fungus, Rickia wasmanii, a mild parasite belonging to the family Laboulbeniales. Ants were classified as either young (yellowish) or old (brownish) and then successfully placed on culture media (SDA). Fungal colonies, which grew from the ants’ cadavers were verified and assigned to 66 morphotypes. The strains from the genera Cunninghamela, Penicillium, Mortierella, Absidia and Cladosporium were the most frequently isolated and occured in all colonies. Additionally, rare Mucoromycota fungal species (e.g. Gongronella) were also identified in the mycobiota of ants. No differences were found between the mycobiota growing from infected with R. wasmanii and uninfected ants. On average 1,75 fungal colony was growing from every individual, and old individuals contained significantly more colonies. Interestingly, Mortierella strains were growing more often from young ants, while Absidia colonies were isolated mostly from old individuals. This is the first study to show that the mycobiota of ants changes with age.

Abstract

The obligate lifestyle and large genome size of many members of the Zoopagomycota have long precluded their study. Modern –omics-based tools including metagenomics, transciptomics and metabolomics have facilitated a better understanding of these fungi despite barriers that still prevent traditional lab-based studies. Massospora is one of several members of the Entomophthorales that have not yet been studied with a systems biology approach. Global and targeted metabolomics were used to compare two contemporary Massospora – cicada symbioses, M. cicadina-infected periodical cicadas (Magicicada spp.) and M. platypediae-infected banger-wing cicadas (Platypedia putnami) to identify candidate secondary metabolites influencing host colonization and recently described behavioral modification. Results of the global metabolomics uncovered a diverse assortment of monoamine alkaloids including psilocybin, a psychedelic compound, from freshly collected M. platypediae and archived samples of M. levispora and cathinone, an amphetamine, from M. cicadina. Fragmentation of these analytes in pooled samples further validated their identification and targeted quantification confirmed absolute concentration of cathinone, psilocybin and, and psilocin. Additional MS/MS analysis will be performed to confirm intermediates within the psilocybin biosynthesis pathway across all samples. Metagenomics and targeted LC-MS proteomics are currently underway to further validate these findings. These psychotropic compounds may enhance stamina of infected cicadas to ensure continued conidial dispersal over several days despite debilitating infections that likely result in fatigue, decreased muscle strength and general malaise. The presence of these compounds, particularly psilocybin, may also support the hypothesis that they serve to deter predation. Phylogenetic studies showed M. platypediae and M. levispora were not genealogically exclusive but instead a single clonal lineage with both species yielding psilocybin. Such discoveries as shown here have only begun to scratch the surface of the vast metabolic capacity of these enigmatic fungi. Follow-up studies on Strongwellsea, another member of the Zoopagomycota, are currently underway.

Abstract

Small black disc fungi, discomycetes, are not commonly collected in Australia, possibly because of the problems they present. They are cryptic, usually cannot be identified without detailed microscopy, relevant literature is often scant and molecular sequences with which to compare collected specimens may not be available. During twenty years of surveying the macrofungi in South Australia, the authors have collected a number of small, black, saucer-shaped fungi from a number of genera. These include under-collected Sphaerosoma trispora McLennan & Cookson, Ruhlandiella berolinensis Henn., several species of phoenicoid Plicaria, a new species Smardaea australis P.S.Catches. & D.E.A.Catches. and a new genus and species Antrelloides atroceraceum P.S.Catches. & D.E.A.Catches.. Descriptions of macro- and microscopic characters, known distribution in Australia and molecular data are presented.

Abstract

We carried out this pilot study in a tropical cloud forest at Estación Biológica Yanayacu, Napo province, western Amazonia. During three field campaigns in 2006 we established two 20 m² permanent plots where all envisaged sporocarps (fruting bodies) growing on the ground and over rotting tree-trunks were collected. We classified the fungi in four “developmental states”: immature, mature, decomposed and highly decomposed. In situ, we manually extracted from each sporocarp as much adult hexapods (insects and allies) as possible which were preserved in ethanol for posterior taxonomic treatment. Posterior extractions were carried out under controlled conditions (artificial light, temperature and humidity) in the laboratory, allowing thus remaining larvae to emerge as adults. Our goals were to i. describe the invertebrate fauna hosted in the sporocarps, and ii. evaluate the preference for the guests to their hosts. We recorded ca. 400 specimens in eight exapod groups and identified 65 morphospecies in 30 families. The beetles were dominant representing ca. 50% of the specimens collected, and among these the rove beetles (Staphylinidae) were highly abundant. Regarding the fungi, we identified 12 families, 21 genera and 25 species, plus ca. 10 morphospecies whose identification is pending. The insects showed an apparent preference for mostly three fungi families: Marasmiaceae (white-hut mushrooms), Polyporaceae (poroid mushrooms) and the paraphyletic Tricholomataceae (white, yellow, pink-spored agaricales). We found more flies (mainly Drosophilidae) in the first group possibly because the Diptera have high protein requirements during fight foraging, whereas the beetles were abundant in the other two fungi families. We have no current ecological clues for the latter apparent preference. Principal component analyses showed a tendency for the beetles (mostly Staphylinidae) to “select” mature fruiting bodies, while the flies were more frequently collected at highly decomposed and mature fruiting bodies. Based on literature search we classified the insect families in the following trophic guilds: primary fungivores (2), secondary fungivores (15), detritivores (9) and sporocaps predators (4). This study was supported by Escuela Politécnica Nacional and Instituto Nacional de Biodiversidad (INABIO_QCNE).

Abstract

Harpellales is a fungal group which are inhabiting in the digestive tracts of aquatic insects (Ephemeroptera, Plecoptera, Diptera). More than 250 species have been recorded in the world. On the other hand, only 17 species have been recorded in Japan, which have been derived mainly Diptera. In this study, we focused on the family Capniidae (Plecoptera) which has not been examined as host for Harpellales in Japan. Nymphs of Capniidae appear in late autumn and emerge in winter, which are living between the submerged leaves in the very slow flow of the streams. We made collection in Fukushima, Ibaraki and Nagano Prefectures. Submerged leaves were collected and brought back with ice to the laboratory. The nymphs were sorted from the leaves, and were dissected by a forceps to derive the hindgut. Derived gut was rinsed in a drop of distilled water on a glass slide and was observed with water mount by a light microscope with differential interference apparatus. The distilled water was replaced by lactophenol for preservation. Here we report the two noteworthy undescribed species. The first one was a species of Ejectosporus (Legeriomycetaceae). Thallus consisted of a central axis, thin branches and thick branches. The basal part of the main axis attaches to the host gut lining with holdfast mass of secretion. The lateral part of the main axis has several protuberances in a line and attaches to gut lining secreted materials. Two types (long and short) of trichospores were produced (ca 30 x 5µm and 10 x 3µm). The generative cells in the distal end of the branch produced only one type of the trichospores. A branch with long trichospores and a branch with the short trichospores were occasionally produced from one common branch. Long trichospore has two appendages. Thick branches produce long cylindrical spores (vegetative spores) (ca 80 x 10µm). Zygospores have not been observed. As all of the known species of Ejectosporus do not have two types of trichospores, this species appears to be undescribed. The second undescribed species also belongs to Legeriomycetaceae. This species also has a main axis and with thin branches. The basal part of the main axis attaches to the host lining with mass of secreted holdfast. The lateral part of the main axis also has protuberances with secretion. In this species, many trichospores are produced. More than 20 generative cells are in a line at the distal end of a branch and produces long elliptical trichospores, ca 10 x 2.5µm, with one appendage. Zygosporophores arises from the conjugation tube, triangle in shape, between two parent hyphae. Zygospore is biconical, 30 x 8.5µm, and connected to the zygosporophore at right angle. The most remarkable character is detached zygospores. Detached spores were surrounded by round clear zone. This zone can be regarded as an expanded appendage. This clear zone reminds us a skirt-like appendage of zygospore of Zygopolaris but can be clearly distinguished from it, which is a very unique character and has never been known in Harpellales.

Abstract

The trichomycetes is an ecological group of fungi (and protists) associated with arthropods. One fungal order Harpellales has been the target of collecting Idaho, where some dry regions make collecting these fungi challenging. Representatives of two genera, Smittium and Zancudomyces, were not uncommon in Idaho mosquito (Culicidae) larvae when their puddle (lentic) habitat was associated with stream (lotic) systems. The same was not true in lentic systems free of lotic input, which revealed no fungal infestation. Surveys of gut fungi in Idaho have led to further unraveling of one species that attaches precociously in the midgut, but does not produce spores. Pure isolates were obtained by suspending the larval mosquito midgut linings with immature unbranched thalli on BHI agar plates to promote the continued growth, branching and maturation of thalli and spores for identification as Zancudomyces culisetae. Curiously, each particular gut fungus seems to present consistently with its own unique penetrating holdfast during precocious extrusion in the midgut, a type of holdfast which is not found during growth in the hindgut. Among trichomycetes, Z. culisetae is a common gut fungus associated with multiple mosquito genera in freshwater lentic and slow moving lotic systems and serves as a model organism for study. Other hosts of this microfungus include: Simuliidae, Chironomidae, Psychodidae, Ceratopogonidae (Diptera), Ephemeroptera and a new report of Z. culisetae for the first time in solitary midges (Thaumaleidae). Although its life history and capacity to infest has not been fully recognized, it is loosely held that Z. culisetae should be found in almost any type of lentic system (including discarded bottles, tires, roadside puddles, etc.) due to a putative ovarian cyst or chlamydospore stages in the flying adult female. However, our findings indicate that adult inoculation of puddles is either not as common or perhaps even rare in mosquitoes and could suggest some other factors are in play. When mosquito larvae were collected from puddles associated with lotic systems, Z. culisetae was recovered with nearly a 100% infestation rate. This fosters the idea that fungal spores in some way depend on lotic systems for their successful dispersal to adjacent lentic systems, and leaves us pondering the ecological relevance this might have in dry regions such as in southern Idaho.

Abstract

Arbuscular mycorrhizal fungi (AMF) are the most ubiquitous plant-symbiotic fungiin the global ecosystem. Owing to their enhanced nutrient absorption capacity, AMF significantly contribute to the survival of individual plants and the ecosystem functioning. Community structures of AMF are affected by many environmental factors Inland wetlands have a different environment from common forest soils, therfore, plants inhabiting wetlands may have characteristic AMF communities. The purpose of this study was to compare the AMF communities in wetlands, among the species of host plants. We sampled the roots of 3 host plant species, Phragmites communis, Miscanthus sacchariflorus, and Trisetum bifidum, inhabiting the Upo wetland, Korea. We extracted DNA from the roots and amplified 18S rDNA of AMF using AMF specific primers and identified 5 AMF species from 3 genera. Diversispora aurantia was the most dominant species, and the AMF community of P. communis was different from other host plants. The results showed that the AMF community had specific to host plants in the inland wetland.

Abstract

Arbuscular mycorrhizal fungi (AMF) are known to be beneficial symbionts of most terrestrial plants, exchanging soil-derived nutrients for photosynthates within the root. Dark septate endophytes (DSE) colonize plant roots in a variety of environments, though their ecological role is less understood. Due to the anoxic conditions and variable inundation regimes characteristic of wetland ecosystems, AMF and DSE functional traits in these environments are not well known. Studies of Southeastern USA wetlands show that baldcypress (Taxodium distichum), an important foundational species in swamps, has extensive AMF and DSE root colonization. However, no study has previously assessed root fungal infection of baldcypress under environmental stress, and the implication of colonization to plant performance. Our main goal was to evaluate the role of mycorrhizal fungi and root fungal endophytes in alleviating drought and inundation stress of T. distichum seedlings. We predicted to observe higher AMF and DSE colonization in drought versus flood-stressed seedlings, due to preference for an aerobic environment. We also expected a decline in plant performance under stressful hydrological conditions in the absence of AMF and DSE. A manipulative flooding experiment was conducted under controlled temperature and humidity conditions. Two-month-old seedlings were inoculated with live and sterile swamp soil from Bayou Chevreuil, a Louisiana swamp with seasonally flooded soil banks. After a month of inoculation, seedlings were exposed to three simulated hydrological regimes: flood, drought, and regular water levels. Water levels were calibrated from two decades of hydrological data from Bayou Chevreuil. Plant growth rate and physiological performance were tracked during the course of the experiment, and plant relative fitness in terms of total dry biomass was estimated post-harvest. Roots were stained for microscopy and AMF/DSE were quantified using a modified magnified intersects method. Contrary to our predictions, plant performance of non-inoculated plants was significantly higher than plants grown with live soil. Interestingly, plants grown in the presence of AMF and DSE showed equal fitness under the different hydrological regimes, while plant growth and fitness was reduced under inundation stress in the absence of AMF and DSE. This result suggests that fungi may help regulate plant response to extreme conditions. No significant differences were found in total AMF and DSE colonization among hydrological regimes of inoculated plants, demonstrating that the presence of AMF and DSE is not affected by an anaerobic environment. Our findings show that seedling root fungi in variably anoxic environments play a more important role than previously estimated, and may help regulate seedling establishment and success under extreme conditions. This study will inform current restoration efforts of Louisiana coastal swamps by providing a mechanism (i.e. AMF inoculation) that can confer baldcypress seedling resilience under threatening climate events.

Abstract

Saltmarshes are highly productive ecosystems in decline globally. Historically, many saltmarshes worldwide became areas for waste disposal or were converted to agricultural, commercial or recreational lands due to their relative flatness and fertile soil. These activities have had negative effects on saltmarsh ecosystems which are essential nursery and refuge habitat for many juvenile fishes, invertebrates, and birds. Saltmarshes also stabilize coastlines, provide a means of storm buffering and nutrient recycling, and are crucial contributors to primary production in marine ecosystems. Restoration efforts of saltmarsh habitats are of interest worldwide due to new threats imposed by current climate change patterns. Unfortunately, these restoration efforts have had mixed success, with many being unsuccessful in long term coastal stability. This research will improve restoration efforts by assessing the role of fungal communities within saltmarsh sediments and the roots of the dominant high saltmarsh species Sporobolus pumilus (Poaceae) in Nova Scotia. Arbuscular mycorrhizal fungi (AMF) can improve the salt tolerance of many plant species, but are often overlooked in saltmarsh restoration projects. Our research focuses on the identification and abundance of AMF species in the roots of S. pumilus, from three saltmarsh sites around the Minas Basin, Nova Scotia. We are also interested in the role of AMF in the establishment and growth of S. pumilus. We used ITS rDNA barcoding, ink/vinegar staining and microscopy, and qPCR to assess the mycorrhizal status of S. pumilus in the field. An AMF species (Funneliformis geosporum, Glomeraceae), we previously identified from S. pumilus in Nova Scotia, was propagated in trap pots and used in inoculation experiments to determine whether this fungus can increase the establishment and growth of S. pumilus under simulated field conditions (tidal mesocosms). As the only identified AMF species currently known to colonize S. pumilus in Nova Scotia, this species may play a crucial role in the grass’s ability to tolerate the dynamic, saline environment of Nova Scotia’s megatidal saltmarshes.

Abstract

A system of agriculture called jhumming which involves clear cutting and buring of plant debris thereof is a customary practice in Mizoram, Northeast india. Once the land becomes unproductive, it is left to be reclaimed by regeneration of natural vegetation, or sometimes converted to different long-term cyclical farming practices. Seasonal dynamics of arbuscular mycorrhizal (AM) fungal community composition in three different jhum fallows in Mizoram were investigated. The jhum fallows are of three different years i.e., 1-3 years, 4-6 years and 7-10 years. In all three seasons variation in AM fungal spore density was observed. Maximum spore density and AM species richness were recorded during the monsoon season. A total of 15 AM fungal species representing 4 genera were recorded where Glomus species were the dominant species.

Abstract

This macroproject studies the interaction of symbiotic or endophytic high mountain fungi species associated with Abies religiosa in temperate forest in Veracruz, Mexico. We study mycorrhizal colonization in adult trees and seedlings. The diversity of arbuscular mycorrhizal fungi (AMF) spores in the adult rhizosphere was monitored and the effect of autochthonous mycorrhizal fungus inoculation on the development of seedlings was studied. Ectomycorrhizal fungi were also studied. We present the first catalog with more than 30 species of AMF and 35 species of ectomycorrhizal fungi associated with A. religiosa in high mountain. The molecular and morphological identification of the associated mycorrhizal fungi is presented. When estimating mycorrhizal colonization, the characteristic structures of AMF were observed in roots of seedlings and adult trees. Colonization by endophytic fungi was very low. Inoculation of AMF in seedlings increases its growth rate. Arbuscular mycorrhiza is present in many woody perennial species, including the Pinaceae family. The AM can be important during the establishment of the seedlings in places where the nutrients are limited, because their contribution to increase the absorption of mineral nutrients of the soil, which finally is reflected in a greater growth and development of the plants. The ectomycorrhizal colonization was very high in seedlings and adults of A. religiosa. To date little is known about the effects that HSOs have on the seedlings of A. religiosa, although it has been reported that these organisms can be similar to mycorrhizal fungi. These results allow to select species to produce inoculated seedlings for restoration or plantations.

Abstract

Slopes strongly modify biogeographical patterns of species and ecosystem functions through causing significant climate change and spatial heterogeneity. The effect of slope aspect on the composition of belowground microoranisms of arbuscular mycorrhizal (AM) fungi is still unclear. Here, we investigated the distribution patterns of AM fungi, a type of functionally important soil microorganism, along an alpine ecosystem of a steep environmental gradient from northwest-facing slope (SS) to southeast-facing slope (NS), and inferred the ecological processes assembling the fungal communities according to the phylogenetic patterns. We detected 32 distinct AM fungal virtual taxa (VT) totally, which mainly belong to the genus Glomus. Both the species and phylogenetic composition of AM fungi differed evidently between SS and NS, the majorly being driven by niche (environment and host) filter, including soil pH, microclimate and plant species composition. Instead, the role of geographical distance was a very slightly contributed factor. With slope aspect conversion from SS to NS. Significantly reduced AM fungal richness was observed and such distinct niche shift caused a marked loss of Glomus taxa, and increase of Rhizophagus spp. In addition, AM fungal communities were phylogenetically clustered on SS and random on NS, respectively, suggesting that the central ecological process governing AM fungal assemblage shifted from niche-dominated filter to one both of niche filter and competitive exclusion. These findings, of the strong local environmental effects of structuring AM fungal community, shed light on the significant susceptibility of these fungi to environmental change.

Abstract

Ericoid mycorrhiza is arguably the least investigated mycorrhizal type specific for Ericaceae. It has traditionally been considered as a domain of ascomycetous fungi, e.g., Meliniomyces spp., Oidiodendron maius and Rhizoscyphus (=Pezoloma) ericae. Recently, however, a member of Serendipitaceae (Sebacinales, Basidiomycota) has been experimentally confirmed as ericoid mycorrhizal and anatomically and morphologically unique sheathed ericoid mycorrhiza formed by a basidiomycetous mycobiont has been discovered and described in Ericaceae from mid-Norway. Subsequently, molecular tools revealed that this mycobiont belongs to the Kurtia argillacea (=Hyphoderma argillaceum) complex within the Hymenochaetales. Such an intriguing placement among mostly saprobic and parasitic fungi begs further investigation on the ecophysiology of K. argillacea and related fungi. A series of resynthesis experiments with ericaceous hosts has been therefore set up to confirm/reveal ericoid mycorrhizal potential of K. argillacea, its close relative Hyphoderma orphanellum, Hyphodontia subalutacea (all Hymenochaetales) and Hyphoderma cf. medioburiense (Polyporales). Kurtia argillacea formed typical sheathed ericoid mycorrhiza with vigorous intracellular hyphal colonization and hyphal sheaths on the root surface but also H. orphanellum formed superficial hyphal sheaths and intracellular colonization typical for ericoid mycorrhiza. Additionally, also the two remaining fungi colonized host rhizodermal cells forming loose hyphal loops with no harm to the host plants. These results indicate that certain lineages in Hymenochaetales, and possibly also in Polyporales, have an apparent but hitherto overlooked potential to colonize Ericaceae roots and form ericoid mycorrhizae. This should be taken into account when investigating these mostly saprobic/parasitic groups of fungi.

Abstract

We present one of the first few reports providing evidence of strong interaction between the soil environment, mycorrhizal communities within roots, and population demography of a rare terrestrial orchid. Orchids utilize complex ecological niches including obligate mycorrhizal interaction. The assembly of orchid mycorrhizal fungal (OMF) communities within roots likely reflects a combination of influences from host-fungus compatibility, the structure of OMF in soil, edaphic characters, and the microenvironment. Untangling the direct and/or indirect effects of one or more of these explanatory variables on plant populations is challenging yet necessary to understand the fundamental niche selection strategies in orchids. We sought to reveal the interaction among an array of biotic and abiotic traits of soil, OMF communities in roots, and population demography of a terrestrial orchid endemic to the California Floristic Province. Platanthera (Piperia) cooperi displays wide spatial and temporal variation in population size and demography and presents an opportunity to address our specific questions including if: 1) soil OMF communities, microclimate and edaphic regimes interact to drive the assembly of root OMF communities? and 2) if spatio-temporal differences in root OMF assemblages lead to distinct populations demographic patterns? To answer these questions, P. cooperi roots were sampled in February (seedlings and vegetative individuals) and April (reproductive individuals) in each of the three consecutive years (2015-2017) from six disjunct populations. Simultaneously, soil samples were collected to profile OMF communities in soil from the study populations and two sites where plants of P. cooperi do not occur. Soil samples were also collected to generate physicochemical profiles and physical environment data was recorded at four populations and one control site. Fungal metabarcoding libraries were generated with ITS3/ITS4OF primer pair which primarily amplifies the internal transcribed spacer 2 (ITS2) region of the nuclear ribosomal DNA and were sequenced on Illumina MiSeq platform. Subsequently, data were analyzed using QIIME and VSEARCH bioinformatic tools along with biostatical analyses in R. We identified 955 OMF operational taxonomic units (OTUs) after clustering ITS2 sequences from P. cooperi roots at 97% similarity threshold. The 30 most abundant OTUs contributed to 75-95% of abundances across sampling events. The most abundant OTU in roots belonged to Tulasnellaceae and the second most to Ceratobasidiaceae. Further, permutational analyses of variance (PERMAVOVA) revealed the differences in distribution and abundance of root OTUs across populations and years (P=0.001 for both), whereas OMF communities were similar across three phenological stages. Further, PERMANOVA also revealed that 30 most abundant root-associated OTUs had differential distribution and abundances in soil across populations (P=0.001), and years (P=0.004). Additionally, hierarchical clustering grouped populations with similar dynamics (size and demography) together for both, root and soil OMF communities. Similarly, redundant analyses (RDA) also clustered populations with similar dynamics together based on edaphic factors and environmental variables. In conclusion, our data suggest that physical, biological and chemical soil environment influence the assembly of root OMF communities, and the spatial and temporal fluctuations in root OMF communities in response to these drivers likely influence the spatial and temporal populations dynamics of P. cooperi.

Abstract

Subalpine forests in subtropical Taiwan are bounded by Taiwan fir (Abies kawakamii) tree-line at their upper margin. Taiwan fir is native to Taiwan and one of the southernmost true firs. Sharing of mycorrhizal fungi could facilitate the coexistence of plant species and the stability of the multispecies assemblage. We evaluated how varied arbuscular mycorrhizal fungi (AMF) and ectomycorrhizal fungi (EMF) were shared among 9 plant taxa in a forest using denaturing gradient gel electrophoresis (DGGE) and high-throughput sequencing techniques. In this study, the long-term research plot (3,000 m a.s.l.) on Mt. He-Huan is an Abies kawakamii - Tsuga chinensis var. formosana (Taiwan hemlock) forest, Yushania niitakayamensis (Yushan arrow bamboo) fills in the middle level, and Rubus pungens, Viola sp., ferns, moss, and mycoheterotrophic plants Monotropastrum humile var. humile and M. humile var. glaberrimum are on the forest floor. Results of DGGE indicated that about one-quarter mycorrhizal OTUs associated with single host, 16 of total 20 AMF and 32 of total 43 EMF OTUs were cross-host and some of them interacted with 8 plant taxa. The architecture of AMF and EMF networks seemed to be nested structure. We provided the evidences that i) ectomycorrhizal plant T. chinensis and A. kawakamii host AMF simultaneously. ii) Yushania niitakayamensis harbored high diversity of mycorrhizal fungi, 12 AMF and 27 EMF OTUs, including half AMF/EMF specialists in the forest. iii) Two mycoheterotrophic plants, M. humile var. humile and M. humile var. glaberrimum were reported as EM plants. We found they were AM plants as well, they associated with similar AMF community. They shared the same habitat but their EMF communities are quite different, it could be the mechanism of sympatric speciation of these two mycoheterotrophic species. We are going to describe the mycorrhizal fungal diversity and the AMF, EMF interaction network topologies among 9 plant taxa at a higher resolution level with NGS data.

Abstract

Members of the orchid family form mycorrhizal associations defined by anatomy, nutrient exchanges and fungal taxa that are distinct from other types of mycorrhizae. In particular, mycoheterotrophy at the protocorm stage of photosynthetic orchids, and lifelong in non-photosynthetic orchids, demonstrates unusual fungus to plant net carbon flow. Significant advances have been made in identifying the fungi associated with non-photosynthetic orchids and understanding the ecology and evolution of these associations. However, the cellular and molecular mechanisms by which orchids achieve narrow specificity and carbon acquisition are unknown. In a search for clues, we undertook an RNA-Seq global transcriptome analysis of interactions between the fully mycoheterotrophic orchid Corallorhiza striata and its specific mycobiont, Tomentella fuscocinerea. Here we report differential gene expression in the fungus. Using de novo transcriptome assembly combined with mapping to a draft genome, we identified 281 differentially regulated genes when comparing pure culture, early mycorrhizal colonization and late mycorrhizal colonization. Transporters and other genes related to nutrient exchange that are often upregulated in mycorrhizal symbioses were not identified. Instead, genes belonging to families associated with apoptotic programmed cell death (PCD) signal cascades, such as Het-C, WD-40, STYKc, tetratricopeptide repeat domain, and NACHT were strongly upregulated when the fungus colonized the orchid. These results raise the possibility that orchids enhance their mycotrophic nutrition by triggering fungal PCD pathways.

Abstract

The transition from saprotrophic to mycorrhizal lifestyles has occurred several times independently during evolution in the fungal kingdom. Recent studies also indicate that the border between a saprotrophic and mycorrhizal lifestyle is far more blurry than earlier appreciated and that numerous saprotrophic fungi can associate with plant roots as facultative biotrophs in vitro. Mycena, one of the most speciose genera in Agaricales, has uniformly been described as saprotrophic, and have profound ecological importance in forest ecosystems as litter decomposers. However, several recent studies have suggested that some Mycena spp. may have a biotropic lifestyle. Firstly, recent high-throughput sequencing surveys have revealed high abundances of Mycena spp. in living ectomycorrhizal plant roots, secondly, some Mycena species are difficult to culture, what is typical for biotrophic fungi, and lastly, one Mycena species was suggested to form beneficial associations with ericoid plant roots in vitro. These findings encourage us to further investigate Mycena’s association with plant roots. This study is part of a larger project, where we aim to disentangle the ecology of the genus Mycena using genomic, isotopic, physiological and imaging analyses. In this study, we investigate the putative interaction between Mycena spp. and plant roots using in vitro growth experiments. Sterile seedlings of the ectomycorrhizal plant Betula pendula and axenic cultures of 17 Mycena species were grown together in microcosms for eight weeks. Images were obtained biweekly, to record changes in seedling growth. We used cryomicrotome sectioning, differential staining and fluorescent microscope imaging to visualize the extent of fungal growth in the fine roots. Mycena spp. associated closely with plant roots in all microcosms; we observed evidence of hyphal penetration into the plant fine roots in all microcosms, and in some cases intracellularly. A few Mycena spp. formed mantel-like structures and caused shortening of plant fine roots. Our preliminarily data suggest that with most species of Mycena, seedling growth was impaired, and there was no evidence of growth benefits for the seedlings in associating with Mycena spp. Several Mycena spp. seem to be facultative biotrophic in vitro. Results from a second, ongoing experiment investigating potential nutrient transfer between B. pendula and Mycena spp. using ¹⁴C and ³²P radioactive isotopes will be presented. Our preliminary results show the need to reconsider the previous view on the genus Mycena as being uniformly saprotrophic. The ecology and nutritional modes of Mycena is likely far more complex, plastic and flexible than earlier believed.

Abstract

Mycena sensu stricto is one of the largest genera in Agaricales, comprising over 500 species, many of which are widespread and commonly found in all types of habitats across all climate zones. They are quantitatively important litter and wood debris decomposers. Traditionally, they have been uniformly described as saprotrophs, and the genus contains several very broad generalist species such as Mycena epipterygia and M. pura growing nearly everywhere, needle litter specialists (M. rosella) and hardwood specialists (M.haematopus). In addition there are extreme specialists like M. belliae that only grows on dead Phragmites stalks. However, recent research has suggested that Mycena members may have a biotrophic or symbiotic relationship with ericoid plants.

Here, we combine several lines of evidence, including datamining of high-throughput amplicon ITS sequences from plant roots, stable isotope signatures of fungal communities and whole genome sequencing - to construct a new and detailed picture of the evolution and nutrition of Mycena.

A large datamining study of ITS1 and ITS2 amplicons from roots from Betula, Salix, Pinus, Cassiope, Bistorta, Dryas and Arctostaphylos from temperate and Arctic latitudes showed that Mycenas can be found in often significant quantities inside living plant roots, from 2-4% of the total sequence count in temperate Pinus silvestris up to 20-50% in Arctic Cassiope tetragona, with apparently higher frequencies towards higher latitudes.

We then surveyed the stable isotope ratios (¹³C/¹²C and ¹⁵N/¹⁴N) of fruit bodies of Mycena taxa compared to available host plants and other present known saprotrophic and mycorrhizal taxa at five selected locations (three from temperate Scandinavia and two from subarctic/arctic Norway). We find no Mycena species that convincingly displayed the combination of relative ¹³C depletion and ¹⁵N enrichment which could suggest with a mycorrhizal lifestyle, but some that do have a relative ¹³C depletion, consistent with obtaining carbon from a living source. This suggests that some Mycena do have an opportunistically biotrophic, but not mutualistic, lifestyle, which is also in line with recent (unpublished) co-cultivation studies of Mycena and Betula pendula.

Finally, we are currently assembling full genomes from 23 Mycena species, 16 of which are complete. Their genome sizes are surprisingly variable, ranging from a moderate size of ~40 Mbp in highly specialised M. belliae to over 120 Mbp in generalist M. epipterygia and up to a very large 4-500 Mbp for several Arctic Mycena species. Several genomes show evidence of transposable element overload, which is mainly known from biotrophic mushrooms and associated genetic bottlenecks.

Taken together, our results show Mycena s.s. to be ecologically diverse with a much higher adaptive versatility than traditionally believed.

Abstract

The Ascomycete genus Morchella is fascinating for a number of reasons. Morchella species are globally popular mushrooms collected for the table so they represent a curious recreational activity and a potential income source for mushroom growers. Despite this popularity and a concerted effort to understand their propagation, they have not been consistently cultivated on a large scale. Perhaps this is because the life history of Morchella species remains elusive. It has been hypothesized that they form mycorrhizal associations with plant roots, but these associations have not been unequivocally proven. By using a comparative genomics approach, we are sequencing numerous taxa, including Morchella esculenta, Morchella conica, Morchella rufobrunnea, Morchella americana, and three strains on unknown lineage. In addition to this sequencing, we have used numerous members of the Morchellaceae to characterize the evolution of family specific metabolic pathways. Phylogenetic analyses reveal the relative position of each strain within Morchella and the ascomycetes. Gene annotation methods were used to identify candidate genes for interaction with plants (effectors and other small secreted protein production), novel biochemical pathways and secondary metabolite production. In addition to these genomic approaches, we have data mined 18S & ITS amplicon data, as well as binned metagenomic reads, from plant endophyte studies. This data clarifies the role of Morchella species as plant endophytes in the Poaceae and other plant lineages.

Abstract

Fungi play essential roles in ecosystems. They facilitate plant access to nutrients and water, serve as decay agents that cycle carbon and nutrients through soil and atmosphere, and are major regulators of macro-organismal populations. Although technological advances are improving the detection and identification of fungi, there still exist key gaps in our ecological knowledge of this kingdom, especially related to function. Trait-based approaches have been instrumental in strengthening our understanding of plant and animal functional ecology and, as such, provide excellent models for deepening understanding of fungal functional ecology in ways that complement insights being gained from traditional and -omics-based techniques. Here we present a synthesis of the current knowledge of the functional ecology, taxonomy, and systematics of plant-associated fungi by addressing three fundamental questions: what is currently 1) known and 2) unknown about fungal guilds, and 3) how we can use a trait-based approach to fill in gaps in our knowledge. We then introduce a novel database of fungal functional traits (Fun^Fun). Fun^Fun is designed to be a living dataset for which taxonomy and guild definitions update as they change and new information can be easily incorporated, as trait data currently lags behind other databases. We include approximately 870 traits encompassing genetic, enzymatic, morphological, stoichiometric, life history, and physiological aspects of fungi to highlight the state of empirical fungal functional ecology. The current version of this database uses Index Fungorum taxonomy and contains 25,864 measurements for 3,104 species distributed across 1,611 genera, 267 families, and 107 orders. Fun^Fun also directly links to the curated and open annotation fungal ecological guild database (FUNGuild), which provides tools for researchers to explore and predict how fungal functional diversity varies across fungal guilds. Finally, we will present several examples of insights that can be gained from our database and a trait-based approach. Our new database will allow researchers to explore critical dimensions of fungal functional diversity and map taxonomic, genomic, and functional data within and across fungal guilds, and will lay the framework for rapid progress on fungal functional ecology in the years to come.

Abstract

Yeasts and other microfungi are highly diverse and almost ubiquitous members of the microbiota in nearly all habitats on the planet. The study of fungal microbiota (mycobiome) of multicellular organisms and their complex relationships with their hosts is a relatively new and rapidly developing field. Data on host-microbe interactions are still very sporadic considering the sheer number of plants and animals that potentially sustain diverse and dynamic mycobiomes. The immense diversity of tropical arthropods represents an interesting target to study the species richness and host-relationships of microfungi, but detailed observations have only focused on a few arthropod groups, such as passalid beetles (Coleoptera: Passalidae) and flower-visiting insects. Isolation from the host enables the characterization of culturable members of the mycobiome, but their interactions with the host are often unclear. During fieldwork in Panama, we collected arthropods by hand and using a mouth-operated aspirator. Habitats surveyed include caves (La Cueva de Chilibre) and tropical rainforests (Isla Barro Colorado, Gamboa, Parque Nacional Soberanía). We collected hosts belonging to various orders (Arthropoda: Araneae, Blattodea, Coleoptera, Julida). Host were surface-sterilized, and midguts were removed and homogenized before streaking onto plates with acidified yeast-extract malt (AYM) agar. Colonies were isolated and species were identified based on ribosomal DNA. Here, we report several species of yeasts, dimorphic fungi and filamentous species (Fungi: Ascomycota and Mucoromycotina) isolated from some of these hosts. Among the cultures of yeasts, we discovered two new species, one related to Diutina and one to Kodamaea. Using the Galleria melonella larva pathogenicity model, we found that most yeast and dimorphic species isolated during this study cause high mortality, despite the versatile immune system and antifungal defense mechanisms described for these larvae. Our results suggest that easily culturable microfungi in arthropod guts may represent pathogens capable of circumventing insect antifungal response. With these findings we show that the Galleria model is suitable to make implications about the ecology of arthropod-associated yeasts. Some of the hosts from our survey carried fruiting bodies of non-filamentous, ectosymbiotic fungi (Ascomycota: Laboulbeniomycetes). We collected Coreomycetopsis oedipus on a Nasutitermes sp. termite (Blattodea: Termitidae); Herpomyces paranensis on Blaberus giganteus cockroaches (Blattodea: Blaberidae), which represents a new country record; and Laboulbenia sp. nov. on semiaquatic bugs (Heteroptera: Veliidae). Of 328 screened Nasutitermes sp. termites, 20 (= 6.1%) carried fruiting bodies of Antennopsis gallica, a „minute mycological mystery” with uncertain affinities. Finally, we found a single fruiting body of Amphoromorpha sp. on a millipede (Julida).

Abstract

Brachycybe (Wood) is a genus of fungivorous millipedes. To date, the fungal associates of these millipedes have never been characterized. In an attempt to resolve these relationships, culture-based approaches combined with DNA barcode sequencing were used. Sampling of 313 individuals collected from three of four B. lecontii clades and 20 sites across seven states uncovered at least 183 genera in 40 orders from four fungal phyla. At least seven putative new species were recovered in this study, despite the use of more classical culture-based approaches. Three of these fungi were phylogenetically resolved using ITS + LSU and include two new species, aff. Fonsecaea sp. and Mortierella aff. ambigua, and a new genus related to Apophysomyces. Overall, the results of this study highlight the vast amount of undescribed fungal biodiversity associated with millipedes. Twelve fungal genera from nine orders showed high connectivity across the entire B. lecontii-associated fungal network, indicating a central role for these fungi in their association with these millipedes. These twelve include the two putative new species described above. The ecology of these and other fungal associates were also explored, using fungal cohort pairings and entomopathogenicity trials. Over 40% of all fungal pairings resulted in competitive interactions, a majority of which involved inhibition or overgrowth by fungi in the Hypocreales and Polyporales, respectively. The abundance of these competitive interactions in these two orders indicated differing ecological strategies. Interactions with Hypocreales frequently resulted in a zone of growth inhibition around the Hypocrealean colony, indicating accumulation of chemicals to competitively exclude other fungi, while Polyporales physically overgrew their competitors, indicating greater competitiveness for resources and resistance to potential antifungal chemicals. Mucoromycotan fungi used a similar strategy to the Polyporales. Results of a series of entomopathogenicity trials indicated that B. lecontii was less susceptible to entomopathogenic Hypocreales than an insect model (Galleria mellonella), even though these fungi are known to attack several classes of arthropods. Furthermore, the absence of a negative interaction between B. lecontii and entomopathogenic Hypocreales may allow B. lecontii to associate with these fungi in way that provides them with a benefit, such as protection from predators. When challenged with some Polyporales, B. lecontii exhibited high mortality, while G. mellonella was unaffected. This stands in sharp contrast to previous casual observations of the feeding behavior of B. lecontii. Recent discoveries of previously overlooked fungal diversity have been groundbreaking and hint at substantial cryptic fungal biodiversity across the globe. The 200-300 million-year-old association between fungi and the Colobognatha, which includes Brachycybe lecontii, provides an ideal system to uncover biodiversity and examine function of these fungi in a highly understudied and ancient association.

Abstract

Leaf galls can be formed by a variety of agents, including bacteria, insects and fungi. There is very little known regarding formation of leaf galls on mangrove trees. In studies to determine the incidence of diseases of mangrove trees in South Africa, several Avicennia marina trees with leaf galls were observed. Unidentified adults and larvae of midges (Cecidomyiidae) were consistently associated with the galls. In addition, fungal fruiting structures were observed colonizing the galls and isolations were made from these. Phylogenetic analyses of multigene sequence data from the isolates, including the internal transcribed spacer (ITS), a portion of the actin gene region (ACT), mitochondrial large subunit (LSU) and translation elongation factor -1α (TEF-1α), revealed that the fungal fruiting structures represent a new taxon in the Mycosphaerellaceae. Morphological observations supported the separation of the new species, which is in the process of being described as Periconiella galla sp. nov. This is the first report of a species in the Mycosphaerellaceae isolated from cecidomyiid galls on leaves of A. marina. Furthermore, the results of this study contribute to limited knowledge of fungi associated with mangrove trees in Africa and worldwide.

Abstract

Soils play important roles in forests, such as organic matter decomposition and mineral nutrient cycling. The interactions among soil organisms are critical for understanding underlying soil mechanics. Myxomycetes and Collembola are abundant and play an essential role in organic matter decomposition in forest soils. As such, their interactions are important for soils. Some Collembola species of the family Neanuridae feed on myxomycete plasmodia, while some plasmodia ride on Collembola and suppress their movement in response to Collembola grazing. However, the consequences of their species-specific interactions are poorly understood. We investigated whether other Collembola species graze on plasmodia and whether the behaviors of plasmodia differ depending on the Collembola and Myxomycetes species. We cultured different combinations of the plasmodia of two Myxomycetes species (Physarum melleum and Didymiaceae sp.) and three Collembola species (Vitronura pygmaea, Ceratophysella denticulata, and Sinella umesaoi) for 3 weeks and measured the changes in body size, egg number, and survival rate for the Collembola, and size, body color brightness, frequency of sclerotium formation, and fragmentation for the Myxomycetes. The response to the plasmodia varied with the Collembola species. Vitronura pygmaea showed significantly higher growth and egg production with the plasmodia treatments. Although C. denticulata grew and produced eggs in the plasmodia treatments, these were significantly increased in the dry yeast treatment. Ceratophysella denticulata appeared to eat the waste products of plasmodia. Sinella umesaoi did not show any differences between the treatments. The responses of the plasmodia varied with the Collembola species. Although the frequency of sclerotium formation was higher in P. melleum than in Didymiaceae sp., it was not affected by the presence or species of Collembola. In the presence of V. pygmaea, both P. melleum and Didymiaceae sp. frequently dropped the attacked parts of their bodies. These results suggest that V. pygmaea feeds on living plasmodia, whereas C. denticulata feeds mainly on fungi, but can utilize the waste products of plasmodia. Dropping the attacked part of the plasmodia body may be a strategy for avoiding consumption by V. pygmaea.

Abstract

Recent studies of mushroom diversity in the forests of northern Thailand have documented a great number of new species as well as new distribution records of known species. The present study is part of an inventory of Amanita in Thailand over a five-year period (2012–2016). As a result, the number of Amanita species from various sections, both new to science or new to Thailand has dramatically increased. This study dealt with section Vaginatae and section Phalloideae. Remarkably, fifteen specimens studied belong to nine new species in sect. Vaginatae, namely, A. brunneoprocera, A. brunneosquamata, A. brunneoumbonata, A. cinnamomea, A. flavidocerea, A. flavidogrisea, A. luteoparva, A. subovalispora, and A. suborientifulva, confirming that the diversity of Amanita in Thailand is high, with likely many more remaining undescribed. Specimens were identified based on morphology and DNA sequence analyses of β-tubulin, LSU, nrITS, and rpb2 loci. Those nine species are fully described and illustrated with line drawings and color photographs. Morphological characteristics of related taxa are compared and discussed, and a dichotomous key of Amanita sect. Vaginatae in Thailand is provided.

Several new species and records were also found that belong to sect. Phalloideae. Two species, namely Amanita ballerina and A. brunneitoxicaria spp. nov., were described as new. Their taxonomic placement is supported by both morphological and phylogenetic evidence. Moreover, A. fuligineoides is also reported for the first time from Thailand, increasing the known distribution of this taxon, which supports our view that many taxa are likely yet to be discovered in the region. The three taxa are placed in sect. Phalloideae based on a multigene phylogeny (β-tubulin, rpb2, nrITS, and nr5.8S), morphological descriptions, photographs, and line drawings. This section contains many of the deadliest poisonous mushrooms in the world. We therefore screened for two of the most notorious toxins by HPLC-MS analysis of methanolic extracts from the basidiomata. Amanita fuligineoides was found to contain both deadly toxins α-amanitin and phalloidin, while A. brunneitoxicaria contained only α-amanitin. Interestingly, neither α-amanitin or phalloidin was found in A. ballerina, the first taxon with white basidioma lacking these toxins in sect. Phalloideae. Together with unique morphological characteristics, the basal position in the phylogeny indicates that A. ballerina is either an important link in the evolution of the deadly poisonous Amanita species, or perhaps a member of a new section also including A. zangii. In conclusion, further exploration of Amanita diversity and historical biogeography in South-East Asia and Australia, which seems to be a hotspot of early diverging Amanita lineages, is critical and could reveal more members of this clade, and help elucidate morphological and molecular synapomorphies to support or refute the hypothesis of a new section.

Abstract

Two new species in the mushroom genus Amanita are described and illustrated from Northwestern Pakistan. Phylogenetic data derived from nuclear ribosomal ITS and LSU regions along with morphological characterizations indicate these species are novel. Amanita cinis is a member of Amanita section Lepidella, while Amanita olivovaginata is a representative of section Vaginatae. Amanita emodotrygon was recently described from the state of Uttarakhand, India; a new record of this species is reported herein for the first time from Paskistan.

Abstract

Mizoram lies in Northeast India. It is regarded as one of the biodiversity hotspots of the world. However, study of mushrooms from this region is very limited. A taxonomic study of the genus Amanita from Mizoram was undertaken by morphological characteristics. Specimens were collected from the different forest of Mizoram. From the study, five species of the genus Amanita were collected and identified, which were Amanita jacksonii, A. pachycolea, A. vaginata, A. cokeri and A. spissacea. The study is a first detailed report on the genus from this region.

Abstract

Amanita is a species rich genus of fungi in Amanitaceae (Basidiomycota, Agaricales) with more than 500 known species distributed worldwide. The genus is important because of its mycorrhizal symbiosis with trees belonging to different genera and families. Many taxa of the genus have been reported as edibles, though some others are deadly poisonous. During the investigation for community studies of ectomycorrhizal macrofungi, several species of Amanita have been collected and described on the basis of morphological characters and molecular data based on Internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene and 28S nuclear ribosomal large subunit RNA gene (LSU). For the characterization of Amanita species, combining ITS with LSU data have been proved best to discriminates species. During this study, five Amanita species were identified. Among these three species have not been described previously while the two species have been reported from Pakistan on the basis of morphological characters. This investigation provides a detailed account of their morphology as well as molecular phylogeny.

Abstract

The purpose of this research project is to use genetic sequence data to understand more about the Amanita pantherina var. multisquamosa, that is endemic to the Southern Rockies of North America. Compared to other A. pantherina species, var. multisquamosa is set apart by its pale beige phenotype. Previous studies have demonstrates that the N. American A. pantherina is a separate clade from the A. pantherina sensu stricto, which originates from Eurasia. The purpose of this study is to explore whether North American A. pantherina var. multisquamosa is a species that is phylogenetically distinct from other North American A. pantherina sensu lato. More broadly this study will explore the relatedness of North American A. pantherina relative to their Eurasean counterparts. To evaluate these questions, molecular sequence data from ITS, 28s and RPB2 regions will be studied under maximum likelihood and Bayesian methods of phylogenetic inference. This data will help to better understand the "A. pantherina Complex" and fungal biodiversity in North America.

Abstract

The primarily ectomycorrhizal (ECM) mushroom genus Amanita (Amanitaceae, Agaricales, Basidiomycota) may number ~1000 species worldwide, though only ~600 species have been formally described. Many Amanita species have broad distributions in higher latitude forests, while Amanita species in the Neo- and Afro-tropics have restricted ranges in lowland rainforests primarily dominated by ECM trees of Fabaceae subfam. Detarioideae (“detarioids”). Collecting expeditions in rainforests dominated by the ECM detarioids Dicymbe corymbosa in Guyana from 2000–2017 and Gilbertiodendron dewevrei in Cameroon from 2014–2017 indicated that at local spatial scales both regions are diverse for Amanita: 25 morphospecies were discovered in an area of 10 km² in Guyana, and 35 morphospecies from an area of ~3 km² in Cameroon. Collections were compared with published descriptions and fungarium specimens of previously described Amanita taxa. This preliminary work indicates that numerous Amanita morphospecies from Guyana and Cameroon are currently undescribed and common in their local habitats. Macro- and micromorphological features and multi-locus DNA sequence data have been compiled from collections of each species, and multiple species from each region will be formally described or epitypified. This work will enhance a broader understanding of fungal biodiversity in tropical ecosystems, and contribute to phylogeographic analyses of post-Gondwanan ECM fungal communities. An overview of the proposed new taxa will be presented.

Abstract

Species of Gymnohydnotrya are of ectomycorrhizal, exothecial fungi in the order Pezizales. Exothecial fungi are basically ‘inside-out truffles’, i.e. their fruit bodies lack a peridium and are covered with a hymenium layer instead. Gymnohydnotrya species have ornamented ascospores and asci that lack opercula. Morphologically, Gymnohydnotrya is similar to species of Hydnotrya except that the Hydnotrya species typically have a peridium. Gymnohydnotrya species are also characterized by hyaline, ornamented ascospores that lack an outer spore wall. The genus was described by Zhang and Minter in 1989 based on herbarium specimens from Australia. Currently, there are three recognized species in this genus: G. australiana, G. echinulata, and G. ellipsospora. Zhang and Minter placed Gymnohydnotrya in the Helvellaceae. However, a recent study showed that these species were part of a distinct monophyletic lineage that also included Southern Hemisphere species of the epigeous genus Underwoodia. This group was identified as the /gymnohydnotrya lineage, but no further taxonomic or phylogenetic work has addressed the systematics of this group. We recently discovered specimens of an unknown exothecial fungus in Patagonian Nothofagus forests in South America. These fungi are morphologically similar to taxa in the genus Gymnohydnotrya but there are no previous reports of this genus from South America. Morphological and molecular analyses indicate that there is at least one novel species of Gymnohydnotrya among our collections from South America. Here we describe the morphology of this South American species and place it within a multi-locus phylogenetic framework that also includes new samples of Underwoodia and Helvellaceae species. We also discuss the implications of this work on the evolution of truffle-like fungi, the biogeography of the /gymnohynotrya lineage, and the taxonomy of Helvellaceae.

Abstract

The genus Amylascus Trappe was described from Australia in 1971. The only two described species, A. herbertianus and A. tasmanicus have globose spiny spores, an ascocarp with a basal tuft, and a gleba with meandering veins or chambers lined with an epithecium, similar to what is seen in the Northern Hemisphere genus Genea. Depending on the species, the spores may or may not have a perispore. The warted excipulum is composed of large-cells and the amyloid asci (for which this genus was named) are distributed throughout the gleba. Molecular phylogenetic studies of Amylascus indicate that this genus is closely related to the Northern Hemisphere ectomycorrhizal genera Pachyphlodes, Plicariella (=Scabropezia) and Luteoamylascus. Amylascus species have been collected in forests dominated by Eucalyptus and other ectomycorrhizal Myrtaceae and are assumed to form ectomycorrhizas. ITS sequences from ectomycorrhizal root tips of South American Nothofagaceae are also phylogenetically close to sequences from Australian Amylascus specimens, suggesting that the genus has a wider geographic and host range than has been previously documented. Over several decades we have obtained specimens from Australia and southern South America that generally fit the morphology of Amylascus. We have also discovered diverse collections of mitospore mats from South American Nothofagaceae forests that are phylogenetically affiliated with Amylascus, and morphologically similar to mitospore mats of Pachyphlodes and Plicariella. Using molecular and morphological techniques, we document our findings of new Amylascus species from Australia and South America and document the range of morphologies that we observed in ascomata and mitospore mats. The morphological characteristics of our new species expand the definition of the genus to include taxa with a wide range of morphological features, including inamyloid asci. We also present a phylogenetic reconstruction of Amylascus and related lineages based on ITS, LSU, RPB2 and TEF markers, to trace the most recent common ancestors of these lineages and infer the evolutionary history of Amylascus in the southern hemisphere. We compare the morphology, biogeography and host associations of this genus with those of related genera.

Abstract

The most obvious and perhaps most critical instrument of fungal dispersal is the spore, whose size and shape may critically affect movement over large distances. There is likely a compromise between small spore size, which can enable dispersal over longer distances, and large spore size, which can facilitate settling onto a favorable substrate. In principle smaller spores should remain aloft for greater time intervals, but their reduced mass makes landing more difficult and increases susceptibility to adverse environmental stresses. But while data on spore size and shape are available for many species, greater and improved data on a range of other spore parameters––emphasizing physiological resilience to atmospheric stresses including: UV radiation, relative humidity (RH), and temperature (T)––may be required to more carefully model dispersal. This study focuses on two Alternaria species with dramatically different conidial dimensions: A. solani and A. alternata. Conidia of A. solani are nearly 10x larger than those of A. alternata (~50-100 vs. ~10µm, respectively), yet little is known about their conidial aerodynamics or survival under atmospheric conditions; the larger A. solani is hypothesized to be a short distance disperser and the smaller A. alternata a long distance disperser. These Alternaria species have emerged as major threats to Wisconsin potato crops in recent years, and despite known differences in spore size, conidia of both species often co-infect the same plant, challenging current hypotheses and suggesting other dispersal dynamics and strategies are at play, including perhaps strategies associated with evolved tolerance to atmospheric stresses. We quantified spore mortality under varied UV intensities and wavelengths (365 nm and 302 nm; 5024±23 to 742±17 µm/cm2), relative humidities (10-90%), and temperatures (10-25ºC) typical of the lower-mid troposphere in order to identify the “optimal” dispersal conditions of the longest surviving, “luckiest spores.” A machine learning approach was used to count live and dead spores from digital images of experimental spores according to the presence or absence, respectively, of a hyphal germ tube 24 hours after treatment. In-house scripts were used to measure germ tube length and to map spore viability as a function of conspecific spore proximity. Biophysical simulations were then performed using NOAA’s HYSPLIT trajectory models, and in-field dispersal measurements collected from lesions on live potato plants arranged radially about an infected host plant. Conidial mortality data was then used to inform both simulated and field-based dispersal measurements to provide a physiologically constrained dispersal kernel of both A. solani and A. alternata.

Abstract

Adaptation of fungal pathogens to colonize plants often involves escape from host recognition. This can be mediated by sequence polymorphism in avirulence factors that evolve to prevent recognition by host resistance genes. Despite the ubiquity and significance of avirulence factors for the infection outcome, many of them are uncharacterized and the mechanisms behind their evolution remain largely unexplored. We analyzed the population genetic diversity for AvrStb6 and Avr3D1, the first avirulence genes cloned and functionally validated from the important wheat pathogen Zymoseptoria tritici, using 142 strains sampled from four naturally infected wheat fields growing on three continents. Both Avr genes were found in every Z. tritici strain, but orthologs were only detected in the most closely related sister species. This suggests that these genes are likely to have important non-redundant functions and probably emerged in Zymoseptoria very recently. Paralogs of both Avr genes were present in all Z. tritici isolates, suggesting that they belong to multigene families of candidate effectors that have expanded recently through gene duplications. Intragenic recombination appears to have affected the diversity of AvrStb6, while Avr3D1 is located in a highly plastic genomic region, in which independent transposable element insertions occurred in the global sample of Z. tritici strains. The Avr sequences exhibited strong evidence for non-neutral evolution, including a large number of non-synonymous mutations, with significant positive, diversifying selection operating on many of the codons. Despite the high sequence polymorphism, features considered essential for effector function, including signal peptides and the cysteine residues, were highly conserved, even among homologs found in the sister species, supporting the conservation of these effectors in evolution and their crucial role in the life history of Zymoseptoria species.

Abstract

Rice blast disease caused by a filamentous fungus, Magnaporthe oryzae, has been one of the most devastating of all cereal diseases worldwide. It is estimated that each year rice blast causes harvest losses of 10-30% of the global rice yield and leading to serious epidemics throughout rice-growing regions of the world including Viet Nam. The interaction between rice plant, Oryza sativa and rice blast fungus, Magnaporthe oryzae was activated by the interaction between protein products of the resistant gene of rice and the avirulence gene of fungus. The resistant gene can protect rice from infection of rice blast fungus. However, the resistant genes are usually broken down several years due to the evolution of new fungal races. Thus, the finding of rice blast isolates containing avirulence Avr-Pita1 gene and analyzing sequence variation of this gene are necessary for further research in reducing rice blast infection. In this study, avirulence Avr-Pita1 gene of 25 rice blast isolates collected from the northern, middle and southern of Viet Nam were amplified and analyzed. The phylogenetic trees were constructed using Neighbor-Joining and Maximum likelihood methods in MEGA 6.0 program. The result showed that 18 rice blast isolates have Avr-Pita1 gene and most of them were collected from the middle and southern of Viet Nam. The result of phylogenetic analysis and polymorphism analysis also showed the diversity of nucleotide sequence of Avr-Pita1 gene among 18 rice blast isolates and differences between Avr-Pita1 of Vietnam isolates and other countries.

Abstract

Coltricia and Coltricialla are a monophyletic clade in the Hymenochaetales, which includes more than 66 species, but monophyly on each genera is still controversial. Main morphological difference between both genera is that Coltricia has smooth spores and Coltriciella has ornamented ones. Even when both genera were traditionally considered saprotrophs, several species have been reported with ectomycorrhizal habit. In Mexico five species of Coltricia and three of Coltriciella are known, and one of those is only known from Mexico (Coltriciella sonorensis). At the present work, we inferred a phylogeny for the known species of both genus, with emphasis in Mexican and neotropical taxa, including recently discovered species not previously included in a phylogeny, and one new species from Mexico. Some of the Mexican species of Coltriciella were recovered in a monophyletic clade, next with species from North American and Caribe. One of the Mexican species was recovered as sister group with one of the Coltriciella ecomychorrhizal sequences from Africa, raising doubt regarding its ecological habit. We performed a preliminary study on the evolution of the ectomycorrhizal habit in the Coltricia-Coltriciella clade. Using BayesTraits v3 we estimate the evolutionary rates of gainings and loosings of the ectomycorrhizal condition, and we inferred the ancestral condition for selected nodes. Our result indicate at least seven independent origins of the ectomycorrhizal condition in the clade, as well as equal rate of gainings and loses. Previous reports had documented a dual ecological habit on some species of allegedly saprophytic Agaricomycetes (i.e. species in Phellinus), and this may be the case for species in the clade like Coltriciella dependens. We consider that our result may be product of the dual nature of several species in the clade, if that were the case, the apparent instability in the habit (equal rate of gains/losses) may be just lack of information regarding the full capabilities of the species in the clade. Probably mycologist need to re-think ecological roles and nutritional modesmycorrhizal habit in the Coltricia-Coltriciella clade. Using BayesTraits v3 we estimate the evolutionary rates of gainings and loosings of the ectomycorrhizal condition, and we inferred the ancestral condition for selected nodes. Our result indicate at least seven independent origins of the ectomycorrhizal condition in the clade, as well as equal rate of gainings and loses. Previous reports had documented a dual ecological habit on some species of allegedly saprophytic Agaricomycetes (i.e. species in Phellinus), and this may be the case for species in the clade like Coltriciella dependens. We consider that our result may be product of the dual nature of several species in the clade, if that were the case, the apparent instability in the habit (equal rate of gains/losses) may be just lack of information regarding the full capabilities of the species in the clade. Probably mycologist need to re-think ecological roles and nutritional modes. If the condition of ectomycorrizal type of nutrition for Coltricia and Coltricialla is present in several specimens of México, the diversity of species will be higher than the known species

Abstract

Xylaria karyophthora was documented as a colonizer of seeds of Greenheart (Chlorocardium spp.), one of Guyana’s most important timber trees. Much is unclear or unknown about this fungus including its life history strategy and this makes our understanding of its transmission, role in disease formation, and strategies for management inadequate. Our previous molecular study of X. karyophthora showed that it was derived from within a clade of wood-inhibiting species, suggesting a possible shift from woody to seed substrates. From this premise, we suggest two conceivable hypotheses for the ecology of this fungus. First, is the foraging ascomycete strategy which theorizes that saprotrophic fungi can present as dormant micro-thalli in healthy plant tissue as a means for dispersing across challenging environments. In this scenario, X. karyophthora possess the ability to move beyond the endophytic state to utilize another substrate, i.e., the woody seed pods of Greenheart. Second, X. karyophthora could represent an emerging infectious organism due to a new host-pathogen interaction that resulted from a recent host jump to Greenheart from another unidentified host. In this presentation, we will examine evidence for both of these hypotheses.

Abstract

Representatives of Mucorales belong to one of the oldest lineages of terrestrial fungi. They probably diverged from other fungal groups already in Precambrian and since then they became ubiquitously distributed worldwide. Together with representatives of Glomeromycota, they probably played crucial role in plant terrestrialization. During their long evolutionary history, they evolved various modes of nutrition. At present, Mucorales representatives are mostly saprotrophic organisms which derive their nourishment from decaying organic matter. However, some animal pathogens (e.g. Saksenaea vasiformis) and phytopathogens (e.g. Choanephora cucurbitarum) are also among this group. Fungi, like all other organisms, may vary in their abilities to use different nutritional compounds (carbon in this study). Their ability to use different compounds available in the substrate can be perceived as one of the main factors shaping the potential for a given fungal taxon to occupy a particular niche. These capacities are shaped by either the permeability of the cell wall or the presence of specific enzymes. Mucorales representatives are commonly called ‘sugar fungi’ as, according to literature, they grow well on media rich in simple organic compounds but are unable to assimilate more complex organic compounds. However, recently some studies showed that it is not true for all Mucorales representatives. Therefore, we hypothesize that carbon assimilation capacities within Mucorales representatives depend on their phylogenetic position. The ability of 74 strains belonging to 63 pathogenic and non-pathogenic species of Mucorales to use 95 different carbon sources was tested using FF Phenotypic Microarray Plates (Biolog^TM). Analysed strains differ significantly in their ability to use varying carbon sources. On average approx. 57 substrates were absorbed per strain and several strains showed capacity to assimilate more complex organic compounds (e.g. cellobiose). Saksenaea oblongispora had the highest capacities to assimilate different carbon sources (approx. 90% of all substrates). However, the ability of the various strains to use the analysed substrates did not show correlation with the evolutionary history of the group. Instead, carbon assimilation profiles are probably shaped by environmental conditions. In the hyphae of strains which had the highest capacities to assimilate different carbon sources, endohyphal bacteria were also detected. The interactions of Mucorales representatives with other microorganisms influencing carbon assimilation capacities is of particular interest and needs further study.

The study was supported by the National Science Centre, Poland under grant No. 2015/17/D/NZ8/00778 and 2017/25/B/NZ8/00473.

Abstract

Sequestrate fungi do not forcibly discharge their spores. They evolved from gilled (or pored) taxa independently in many different lineages. In 1984, Harry Thiers presented the Secotioid Syndrome Hypothesis where the sequestrate state is thought to have evolved as an adaptation to arid climates. Bougher and Lebel hypothesized that there may also be a selection pressure to protect the spore-bearing surface from excessively moist conditions. We test these two hypotheses by correlating the state of being gilled or being sequestrate with numerous BioClim variables in the globally distributed genus Cortinarius. We found that ‘mean diurnal temperature’ and ‘mean maximum temperature in the hottest month’ were significant in estimating the probability of being sequestrate. None of the precipitation variables were significant. A global map of the distribution of sequestrate Cortinarius species shows that sequestrate taxa are absent from the tropics in contrast to their persistence in temperate regions, further supporting the finding that sequestrate taxa are found in habitats with variable temperatures. This study casts doubt on the proposition that moisture is the sole driving force for the evolution of sequestrate taxa.

Abstract

Convergence of truffle-like fruitbodies form other types of mushrooms is one of the major evolutionary tendencies in Ascomycota and Basidiomycota. During our recent intensive field surveys of truffle-like (sequestrate) fungi in Japan, we frequently encountered tiny (up to 3 mm in diam.), subglobose, bright yellow, hypogeous fruitbodies growing in humus in mature Fagaceae forests. Phylogenetic analyses of nuclear ITS and 28S rDNA datasets unexpectedly revealed that this truffle-like fungus is a member of Aspergillus s. str. The multigene analysis suggested that the species fell into Aspergillus subg. Nidulantes and formed one of earlier diverging lineages within the subgenus. We are currently adding other DNA regions to the dataset to obtain a more robust phylogeny. Morphologically, as in the sexual stages of other Aspergillus species, ascospores are produced inside the cleistothecial fruitbody. Ascospores 4–5 μm in diam., ellipsoid to broadly lenticular, colorless, with two equatorial crests. Asci 8-spored, evanescent, uniseriate, cylindrical, which is rare in the genus. Hülle cells ovoid to tubular with irregular inflation, thick walled, surrounding the internal gleba. Peridium composed of loosely interwoven, non-inflated, branched filamentous hyphae. We confirmed germination of the hülle cells on the MNC medium. We further obtained cultures from the fruitbody tissues and successfully induced whitish brown conidiophores with a globose terminal vesicle, typical of the genus Aspergillus, on the MEA medium. Furthermore, we also succeeded to induce fructification of the characteristic yellow hypogeous ascomata after incubation of the culture in a pot stuffed with sterilized humus and brans for about 1 week at 20 ˚C. These findings imply that there still remain many unknown wild species of Aspergillus species that have unique, interesting characteristics in the nature. Further intensive field surveys must be needed to detect such hidden, characteristic taxa.

Abstract

Ceiba pentandra, commonly known as Ceiba or Kapok, is a tree in the Malvaceae grown in tropical regions throughout the world. The Ceiba was designated as the national tree of Guatemala on March 8, 1955. Ceiba trees grow throughout Guatemala and their wood has been used to make canoes, the floss of the fruit is used for stuffing cushions and various parts of the tree have been utilized as medicine. Ceiba trees often mark the center of life in villages, as sites of markets and gathering places. In 2013, one of two Ceiba trees in the botanical garden of the University of San Carlos (USAC) failed to leaf out normally and was declared dead by April 2014. Most of the trunk was removed in 2016. At the same time it was noted that a tree in zone 1 of the city with similar symptoms had been removed, and the trees in the central plaza of the university also exhibited similar symptoms. No symptomatic Ceiba trees were reported from any other area of Guatemala. Fourteen Ceiba trees were surveyed in the city of Guatemala, with 5 of these showing definite lesions or dieback symptoms. Based on a review of the literature, approximately 50 fungi and oomycetes have been reported in association with Ceiba trees throughout the world. None of these organisms have been reported in association with Ceiba trees in Guatemala, although some have been reported on other hosts in Guatemala. In July 2017, samples were taken from the soil surrounding Ceiba trees located on the USAC campus, and trunk samples were taken from the symptomatic trees. The samples were assayed for oomycetes and fungi through baiting and direct plating. The sampling and baiting resulted in 22 total isolates representing multiple organisms. Notably, Phytopythium is reported for the first time from Guatemala. This is also the first record of Albonectria rigidiuscula associated with Ceiba trees in Guatemala. The relative importance of the isolated organisms in the development of the symptoms observed is unknown, but their presence underscores the importance of maintaining the health of urban trees in Guatemala.

Abstract

Studies of Australian and European populations of Erysiphe necator, causing grapevine powdery mildew, have revealed the existence of two genetically differentiated groups, A and B. Group A was usually widespread early in the growing season, and was associated with flag shoots, that is powdery mildew covered shoots emerging from overwintered buds infected in the previous growing season. The two groups were shown to have different temporal distributions: group A was rarely found later in the season, when group B became more prevalent. In Europe, most studies focused on Western European E. necator populations. We sampled grape powdery mildew populations in Hungarian vineyards, in Central-Eastern Europe, to genotype the local populations and to reveal (i) the spatial and temporal dynamics of the local genotypes; and (ii) the genotype(s) responsible for flag shoot symptoms. Samplings were done in two wine regions in May, August and September 2015, to cover the three main stages of host plant growth. Flag shoots were collected in May; in addition, samples were also collected from close vicinity of the original flag shoots in August and September. Following DNA extraction, powdery mildew samples were genotyped based on partial beta tubulin (TUB2) gene sequences. Most sequences were obtained by direct sequencing while some amplicons were sequenced after cloning. About 85% of a total of 183 TUB2 sequences determined in this work showed polymorphism at one or two nucleotide positions, which were revealed by double peaks on chromatograms of direct sequencing. Sequences obtained after cloning of selected PCR products clearly revealed the presence of three different TUB2 genotypes in E. necator populations in Hungary. Two of these were identified as the A and B groups described in earlier studies, while the third one was designated as B2 in this work, based on a single nucleotide polymorphism (SNP) in the 6th exon of TUB2. All flag shoot samples belonged to B and/or B2 groups. Genotype A was present only in 5.5% of the samples and it has always been present in combination with genotype B and/or B2, in August and September. This study revealed three different E. necator genotypes in Hungarian vineyards in 2015. Flag shoot samples represented groups B and/or B2, but not A; this finding provides additional evidence of overwintering of B genotypes in buds, and causing flag shoot symptoms in spring. Our results show that co-infections of the same leaves and/or vines by different genotypes are common. Also, the detection of genotype A later in the growing season indicates that the temporal variation of A and B genotypes may not be as strict as suggested by a number of earlier studies.

This work was funded by the Széchenyi 2020 programme, the European Regional Development Fund and the Hungarian Government (GINOP-2.3.2-15-2016-00061).

Abstract

Strobilurins, belonging to the group of Quinone outside Inhibitors (QoIs), are considered as single-site of action fungicides which inhibit the electron transfer in mitochondria by binding to the cytochrome bc1 enzyme complex. It has repeatedly been shown that a single point mutation in codon 143 of the mitochondrial gene CYTB, which encodes cytochrome b, confers complete resistance to QoI fungicides in many plant pathogenic fungi. However, in some species, such as Puccinia spp., neither QoI resistance nor this mutation, designated as G143A, have been detected so far. This was explained by the presence of an intron in the CYTB gene right after codon 143 in these plant pathogens: it was predicted that a G143A mutation would prevent the splicing of this intron and, thus, the production of functional cytochrome b proteins. Consequently, in these intron-containing species the G143A mutation is considered to be lethal and the risk for QoI resistance is predicted to be low. Guignardia bidwellii (anamorph: Phyllosticta ampelicida), the causal agent of grape black rot, is considered as a CYTB intron-containing species with low risk for the development of QoI resistance in the field. We amplified and cloned CYTB fragments in several G. bidwellii strains, and also in some other Guignardia spp., including authentic strains of G. citricarpa, the causal agent of citrus black spot, and also G. gaultheriae, G. mangiferae and G. aesculi obtained from CBS, to sequence the intron located after codon 143. Surprisingly, no intron was detected in the predicted position in several G. bidwellii strains isolated from different grape varieties in Hungarian vineyards. Also, the intron was not found in either an authentic G. bidwellii strain obtained from LGC ATCC, or the G. aesculi and a G. gaultheriae strains included in this study, while the intron was identified, and sequenced, in all other Guignardia spp. strains examined by us. In vitro fungicide resistance tests did not show a clear correlation between the presence/absence of the intron in Guignardia spp. strains and their sensibility to QoI compounds. This might suggest that other mechanisms may also be involved in their QoI resistance. So far, our results indicate that at least some G. bidwellii strains causing grape black rot could contain the G143A mutation and might be able to develop QoI resistance in this way in the field.
This work was funded by the Széchenyi 2020 programme, the European Regional Development Fund and the Hungarian Government (GINOP-2.3.2-15-2016-00061). Zsolt Bereczky acknowledges the support of a Janos Bolyai Research Fellowship and a grant (NKFIH PD-100724) of the Hungarian Research, Development and Innovation Office.

Abstract

The Botryosphaeriaceae are an important group of trunk disease pathogens that affect wine and fruit industries worldwide. These fungi infect the woody tissues of plants, causing necrosis, cankers, stunted growth and dieback which result in lower yields and can eventually contribute to the death of the plant. Some species display broad host ranges, making it difficult to control these pathogens if they persist in the environment by inhabiting alternative hosts. The threats posed by such reservoirs of trunk disease pathogens to the grapevine industry are unknown. Therefore, this study aimed to survey the diversity of Botryosphaeriaceae species associated with disease symptoms of woody hosts that commonly surround vineyards in the winelands of South Africa. Wood samples were collected from visibly diseased trees and shrubs in the direct vicinity of vineyards and Botryosphaeriaceae strains were isolated. Identifications were made by performing phylogenetic analyses based on ITS and EF1α DNA sequence data. In total, 989 plants from 39 different host species yielded 1674 isolates belonging to 29 different species, eight of which were found to be novel and were described. In addition, 64 of the host/pathogen combinations encountered during this study appear to be novel. Prior to this study a total of 38 different Botryosphaeriaceae species were reported in South Africa. This survey introduces an additional 16 species to this list including the new species. The Botryosphaeriaceae species that were the most frequently encountered during this survey were Diplodia seriata and Neofusicoccum australe. These species are important grapevine trunk disease pathogens and also displayed broad host ranges. This survey clearly shows the prevalence of important grapevine trunk disease pathogens in close proximity to vineyards and lays the groundwork for further studies to investigate the significance of alternative hosts as reservoirs of grapevine trunk disease pathogens.

Abstract

The Botryosphaeriaceae (Botryosphaeriales, Dothideomycetes) represents one of the most important families within Ascomycota, being widely distributed in the non-polar regions of the world. Some species are pathogens of economically important plants, and several are of quarantine concern in China, such as “Botryosphaeria laricina” causing shoot blight or twig dieback of larch, and Diplodia mutila causing cankers on apple as well as black dead arm on grapevines. Of the 23 genera currently accommodated within the Botryosphaeriaceae, 11 have been reported from China, namely Botryosphaeria, Cophinforma, Diplodia, Dothiorella, Lasiodiplodia, Macrophomina, Neodeightonia, Neofusicoccum, Neoscytalidium, Phaeobotryon and Sphaeropsis. Based on the 700 strains of Botryosphaeriaceae isolated from woody hosts in China during this study, the isolation frequency of Lasiodiplodia was 43%, followed by Botryosphaeria (33%), Neofusococcum (10%), and other minor genera (14%). More than 2000 taxa have previously been assigned within Botryosphaeriaceae, while only 123 epithets have been reported from China. Of these 123 epithets, 25 (20%) were first described from China, and 43 have DNA data available. Forty-eight (39%) of the taxa belong to Diplodia, 22 (18%) to Sphaeropsis, 17 (14%) to Botryosphaeria, 13 (11%) to Dothiorella, 12 (10%) to Lasiodiplodia, 4 (3%) to Neofusicoccum, 2 (2%) to Phaeobotryon, 2 (2%) to Neoscytalidium, 1 (1%) to Neodeightonia, Macrophomina and Cophinforma, respectively. Most of the species (65%) have been reported from tropical or subtropical areas, such as Jiangsu (13%) and Guangdong (13%) Provinces, while the northern or western part of China, such as Xinjiang (1%), Qinghai (0%) and Tibet (1%) have been rather poorly studied. Most of the species (>80%) have been reported as plant pathogens. With extended sampling as well as the development of new molecular identification techniques, more taxa are continuously being recovered.

Abstract

Collophorina (syn. Collophora, Leotiomycetes) was described in 2010 from necrotic wood of stone fruit trees in South Africa as a genus forming small aseptate conidia from integrated conidiogenous cells resembling those of the not closely related genus Lecythophora. To date, six species have been described and the genus has been reported from necrotic or symptomless tissue mainly of Prunus spp., but also of Castanea sativa, Acer glabrum var. douglasii and Euphorbia polycaulis in South Africa, Europe, Northwestern United States and Iran. In a survey aiming to reveal the diversity of fungi associated with wood necroses of Prunus trees in Germany as well as from spore traps in vineyards, 84 and 10 isolates, respectively, with reduced conidiogenous cells were obtained. ITS sequence data placed these strains in the genus Collophorina. In a multi-locus molecular phylogenetic analysis (ITS, TEF and GAPDH), 11 species were differentiated. Most isolates studied were identified as C. paarla and C. africana, while further nine species were revealed as new to science and characterized by means of morphological and molecular data.

Abstract

The symptoms of ash dieback caused by the fungus Hymenoscyphus fraxineus include wilting of the foliage followed by dieback of shoots, twigs and branches. Necroses in shoots are assumed to develop after infection through leaf petioles; however, clear evidence of this infection pathway has not yet been provided. Considering the co-occurrence of multiple pathogen genotypes in dead ash petioles, we aimed to obtain a spatial overview of all H. fraxineus genotypes colonizing individual shoots and their corresponding petioles. We labelled selected shoots and corresponding petioles whilst on the tree and reconstructed their position after their natural shedding. This design allowed us to acquire precise information about the infection biology of H. fraxineus and its ability to cross the petiole-shoot junction. Individual genotypes of H. fraxineus were characterized by the analysis of microsatellites using DNA extracted directly from petiole segments or cultures isolated from the segments. We detected 150 different multilocus genotypes in 10 analysed shoots and their corresponding petioles; the highest number of genotypes was eight for a single petiole and three for a single shoot. The genotypes of most shoot lesions were identical to particular genotypes from the proximal segments of petioles, implicating the main pathway of shoot infections. To test whether the amount of colonized substrate or intraspecific competition have an effect on successful infection, genotypes that reached the most proximal end of the petioles were scored for the number of invaded petiole segments and for the number of other H. fraxineus genotypes co-occurring in the segments. However, the extent of colonization of the scored genotypes and intraspecific competition with other H. fraxineus strains did not enhance nor limited pathogen success in entering the shoot. This study confirms for the first time that the majority of ash shoot infections are caused by H. fraxineus strains originating from petioles. Scaling up the counts from petioles to a whole tree suggests that a mature tree must host thousands of H. fraxineus individuals. However, compared to petioles, the number of H. fraxineus genotypes in shoots was much lower, which points to the evolutionary benefit of the petiole representing a strong bottleneck for spread of fungal pathogens.

Abstract

Oaks (Quercus spp.), are some of the most important forest species of the mountainous regions of the northern hemisphere. They are found distributed from southern Canada to Colombia. The center of diversification of the Quercus genus is in Mexico. Of the 531 species described worldwide between 150 to 161 species are found in our country and of them close to 90 are considered to be endemic. Like other forest trees, oaks provide many environmental services that benefit man. Oak trees, like many other plants, can be affected by phytopathogenic fungi. In 2016, foliar spots caused by a fungus not reported in Mexico were observed in Quercus eduardii. The objective of this study was to identify the phytopathogenic fungus responsible for the foliar spots. The description of the symptoms caused by the fungus was made. To be able to identify the fungus, fungal structures were taken from the leaves and were placed between a slide and a cover slips with lactophenol cotton blue. Structures and spores were measured using a Carl Zeiss microscope. The microscope was fitted with an ocular micrometer ruler in one of its eyepieces. The fungus was also isolated and the description of the colonies, that were grown on malt extract agar culture medium, was made. The leaf spots were amphigenous, subcircular to angular-irregular and on average measured 2–13 × 2–9 mm, usually ochraceous brown to medium dark brown, margin indefinite or with a narrow darker brown margin or marginal line. Conidiomata (pycnothyria) borne on a stalk or columella, dimidiate, shield-shaped, black, coalescing or scattered, conidiophores simple; conidia hyaline, I-celled, ovoid, oblong or fusoid. In culture: Colony (after 20 days on malt yeast extract agar, at 22°C ) 80–82 mm diam. Mycelium with a undulated margin white at first, with concentric rings of aerial mycelium, the center was olive green with stripes of brown mycelium, no color change was observed on the underside after 20 days, there was also no sporulation. Mycelium branching, septate, 3.0–5.0 μm diam., hyaline and brown. All the above characteristics agree with the genus Tubakia (Actinopelte Sacc.). The isolated fungus was sent to Dr. Pedro Crous (Westerdijk Fungal Institute, Utrecht, The Netherlands), for molecular identification of the species. In conclusion, the phytopathogenic fungus causing the leaf spots on Qu. eduardii was identified as Tubakia spp.