Welcome to IMC 2018 International Mycological Congress
Ascospores- and conidia-specific differential gene expression analysis in Aspergillus nidulans
- H. Park
- M. Lee
- J. Yu
- K. Han
In the homothallic model filamentous fungus Aspergillus nidulans, developmental process and spore formation is environmentally and genetically regulated. Although asexual spores or conidia differentiation is well-characterized by analyzing important genes for controlling orchestrated developmental pathways, including the brlA gene-mediated conidiophore and conidia morphogenesis, only a few genes such as nsdD, nsdC and veA have been elucidated for playing important roles in sexual development and ascospore formation. Unlike conidia, however, physiological and genetic studies of ascospores are remained to be characterized. To know more about the ascospores biology as well as conidia, we performed RNA-seq analysis from from A. nidulans conidia and ascospores RNA samples. Comparative analysis of transcription profiles of conidia and ascospores revealed many genes that are expressed differentially in both spores. Detailed investigation of the differentially expressed genes is in progress.
A genomic-level recombination study of familial Aspergillus flavus
- G. Moore
- B. Mack
- S. Beltz
- M. Gilbert
Aspergillus flavus is an agriculturally-significant, filamentous micro-fungus that has the potential to contaminate food and feed crops with toxic secondary metabolites such as aflatoxin (AF) and cyclopiazonic acid (CPA). Once considered an asexual organism, recent discoveries within the last decade have shown A. flavus is capable of overcoming heterokaryon incompatibility to undergo meiotic recombination through sexual out-crossing. Recombination in the aflatoxin gene cluster has been reported, but at the genomic level the impact of recombination has not been studied. Therefore, the goals of this study are: 1) to elucidate the heterokaryon incompatibility loci that give rise to VCG designation; 2) to determine the level of overall recombination impacting a single generation of F1 progeny as well as identify any hotspots for recombination; and 3) to elucidate the impacts recombination can have on chemotype diversity. We paired a toxigenic (AF+/CPA+) MAT1-1 A. flavus strain with an atoxigenic (AF-/CPA-) MAT1-2 A. flavus strain tagged with green fluorescent protein. Ten F1 progenies (five fluorescent and five non-fluorescent) were randomly selected from single-ascospore colonies and broadly examined for evidence of recombination, as well as inheritance of AF and/or CPA production. We observed progenies that had inherited one or both of the examined mycotoxins (AF and/or CPA) from the MAT1-1 parent, but had inherited the MAT1-2 gene from the atoxigenic parent. Other progenies had inherited the mating-type and atoxigenic profile from the MAT1-2 parent, but did not inherit the eGFP gene that facilitates fluorescence. Traditional vegetative compatibility group (VCG) testing of the progenies with one another, as well as with the parent strains, revealed four of the progeny strains to be a unique VCG. We then sequenced the genomes of this familial population for examination of genetic changes and patterns of inheritance at the genomic level. This project is ongoing and will involve results from genomic-level investigations upon presentation at IMC11.
Aspergillus flavus ecm33, a GPI-anchored protein-encoding gene, plays a role in vegetative growth, production of conidia and sclerotia, and resistance to calcofluor white
- P. Chang
- Q. Zhang
- L. Scharfenstein
- B. Mack
- A. Yoshimi
- K. Miyazawa
- K. Abe
Many glycosylphosphatidylinositol-anchored proteins (GPI-APs) of fungi are membrane enzymes, organization components, and extracellular matrix adhesins. We analyzed eight Aspergillus flavus transcriptome sets for the GPI-AP gene family and identified that AFLA_040110, AFLA_063860 and AFLA_113120 were among the top two highly expressed genes in seven of the eight sets. Disruption of the former two genes did not drastically affect A. flavus growth and development. In contrast, disruption of AFLA_113120, an orthologue of Saccharomyces cerevisiae ECM33, decreased vegetative growth and conidiation, promoted sclerotial production and altered conidial pigmentation. The developmental defects were remediated by incubation under constant light. The A. flavus ecm33 null mutant showed decreased sensitivity to congo red at low concentrations (25-50 µg/mL) but had increased sensitivity to calcofluor white at high concentrations (250-500 µg/mL). Analyses of cell wall carbohydrates indicated that the α-glucan content was decreased but the contents of chitin and ß-glucan were increased in the mutant strain. A. flavus Ecm33 is critical for proper cell wall composition and plays an important role in normal fungal growth and development.
Climate change effect on ochratoxin A production and gene expression in Aspergillus carbonarius
- G. Perrone
- A. Gallo
- L. Piemontese
- D. Magistà
- M. Solfrizzo
- C. Cervini
AbstractOchratoxin A (OTA) is a potent pentaketide nephrotoxin diffusely distributed in food and feed products (grains, legumes, coffee, dried fruits, meat derived products, beer and wine); it is also carcinogenic, neurotoxic, teratogenic and immunotoxic. This mycotoxin is produced by species belonging to the genus Aspergillus and Penicillium. OTA is the primary mycotoxin risk in wine and dried vine fruits, its maximum level is regulated by law. Several studies focused on Aspergillus section Nigri, due to their role as causative agents of black rot of grapes, and subsequently as cause of ochratoxin A contamination. In particular, Aspergillus carbonarius has been identified as the major cause of contamination in grape berries. This contamination is strongly related to climatic conditions, geographical regions, grape varieties, damage by insects, growing season; in particular great variations may occur from one year to another. So, climate represents an important key-factor in the agro-ecosystem, influencing fungal colonization and ochratoxin A production in grapes. Climate change is expected to have a profound effect on our landscape worldwide, and also to have an important impact on sustainable food production system. Recent studies have reported how the climate change may affect mycotoxins production in the fields and the relevant risk on economically important crops.
In this regard, the interacting effect of water stress (aw 0.99-0.93) and different day/night climate conditions simulating nowadays (18-31 and 15-28°C) and climate change scenarios (18-34 and 20-37 °C) in high OTA risk area of southern Italy during the ripening season, were studied. Mycelial growth rate, OTA production and molecular expression of key genes (PKS, NRPS, Hal, p450, bZIP) of OTA biosynthetic cluster by A. carbonarius ITEM 5010 were measured. Our results showed that, in water stress conditions (0.93 aw), no OTA production was observed and, except at 20-37°C, the growth rate was slower compared to 0.99 aw. A significantly higher amount of OTA was observed at 0.99 aw and 18-34°C climate change scenario. Gene expression, monitored by quantitative real time RT-PCR, gave evidence of the high expression levels of OTA biosynthetic genes in this condition, in particular NRPS and Hal genes were strongly expressed. These preliminary and new results on A. carbonarius in a climate change scenario suggest that a possible slight increase of temperature may lead to higher OTA contamination and to a possible expansion of the risk area in the Mediterranean basin.
Biosynthesis, characterization and anti-lung cancer activity of fullerene nanoparticles using an endophytic fungal extract of Penicillium simplicissimum
- G. Melappa
- V. Anil
- N. Mn
- A. Bs
- D. Popli
In the present work, PsFNPs were prepared using a simple bioreduction method. It is economically and environmentally safe. Methanol extract of endophytic fungi, P. simplicissimum are used as bioreducing agent in the study. The formation of fullerene nanoparticles was confirmed by UV-visible spectrophometer and characterization of nanoparticles by using FTIR, SEM, XRD and EDS. The P. simplicissimum extracts exhibited biologically important phytochemicals viz., alkaloids, tannins, saponins, carbohydrates, flavonoids, terpenoids, phenol and anthraquinones. The PsFNPs have shown significant anticancer activity on lung cancer cell line H1975 through cytotoxicity, increasing caspase-3,7, 8 and 9 activity and reduced expression of COX-2 activity significantly.
Thioredoxins and their allies in the foodborne yeast Debaryomyces hansenii
- H. Hallen-Adams
- N. Banjara
- M. Wilson
Debaryomyces hansenii (Debaryomycetaceae, Saccharomycetales) is a halophilic yeast commonly isolated from cheeses and fermented meats, to which it may be added as a culture organism. A close relative of human commensal and pathogenic Candida yeasts, D. hansenii does not itself grow at 37 C but multiple strains show killer activity against Candida albicans and C. tropicalis. In our efforts to identify the killer protein(s) we isolated and purified a single protein of approximately 11 kDa from cell-free culture supernatant using ultrafiltration and successive anion exchange and size exclusion chromatography, and identified the protein as a predicted thioredoxin by tandem mass spectrometry and comparison with the D. hansenii genome. We have expressed the gene in E. coli and are currently working to understand its mode of action.
Studies on Cordyceps guangdongensis, an edible and medicinal fungus with broad application prospects
- T. Li
- Q. Lin
- W. Yan
- C. Zhang
- W. Deng
Cordyceps guangdongensis T. H. Li, Q. Y. Lin & B. Song (Cordycipitaceae) was discovered in southern China and formally described in 2008. Biological characteristics, artificial cultivation, safety assessments, bioactive constituents and medicinal properties of the fungus were systematically studied by the authors in recent years. Through a series of tests for optimum conditions, biological studies showed that C. guangdongensis fruitbodies could be domesticated as a cultivated fungus, and with suitable substrates, pH value, temperature, water content and light density. In the study of safety assessment, bacterial reverse mutation (Ames), bone marrow cell micronucleus, sperm aberration, teratogenic action, acute toxicity and 90 day oral toxicity experiments were conducted; and the analyses of the tests demonstrated that the fruitbody of C. guangdongensis did not have any mutagenic, clastogenic nor genotoxic effects. The maximal tolerance dose（MTD of the biomass in rats was greater than 20 g/kg bw. The no-observed-adverse-effect-level (NOAEL) was 5.33 g/kg bw according to the 90 day oral toxicity analysis. Therefore, the fruitbody of C. guangdongensis is considered safe for long term consumption. In 2013, it became the second novel food of Cordyceps approved by the Ministry of Public Health of China. In the study of physiological function evaluation, a series of animal tests were carried out. The researches indicated that the fruitbody of C. guangdongensis had antioxidant activities, anti-viral activity against the avian influenza virus H9N2, longevity-increasing activities, anti-fatigue effects, curative effects on chronic renal failure, and anti-inflammatory effects. Moreover, this fungus has rich bioactive compounds including cordycepic acid, adenosine, polysaccharides, and so on, many of them are similar to those in Cordyceps sinensis (Berk.) Sacc. The above results show that C. guangdongensis has a great potentiality for application in food and medical industries. Recently the whole genome of C. guangdongensis was also studied, being sequenced and assembled with Illumina and PacBio sequencing technology. The generated genome is 29.05 Mb in size, comprises 9 scaffolds with an average GC content of 57.01%, and is predicted to contain a total of 9150 protein-coding genes. The genome project provided a beneficial source of molecular information, and will lay a better foundation for elucidating the functional genes and exploring more bioactive components for further studies and application. This work was supported by Natural Science Foundation of Guangdong Province, China (2017GD1351), the Science and Technology Planning Project of Guangdong Province, China (No. 2015A030302052; 2016A030303035), and the Science and Technology Planning Project of Guangzhou, China (No. 201504291620324).
Cordyceps biodiversity and industrialization of Cordyceps militaris
- T. Wen
- F. Long
- Y. Xiao
- L. Zha
- J. Kang
- K. Hyde
Cordyceps sensu lato create tremendous economic value due to their medicinal, biological and nutritional importance. Hence, it is necessary to have further study on classification and identification of this taxa. Cordyceps militaris was approved as a functional food and Traditional Chinese Medicine owing to its significant pharmacological activity. cordycepin is one of the most important bioactive compounds produced by Cordyceps. However, large scale industrial production of cordycepin is still problematic. The objective of this study is classification of Cordyceps s.l. and optimization of large-scale production conditions for C. militaris and cordycepin. Furthrmore, this study helps in industrialization of C. militaris. More than 50 species of Cordyceps s.l. have been identified from China, Thailand and Russia based on morphology and multi-gene phylogenetic analyses. Among them, 22 are new to the science. Cordyceps militaris has been used as a common mushroom in China and the market demand for artificial C. militaris has increased continuously. In the industrial application works, (1) the Liquid Static Culture: cordycepin production by three strains of C. militaris; using different working volumes and bioreactors. The best cordycepin production; 3005.83 mg/L was obtained in 5 L-flasks, containing 2 L medium, total cordycepin content reached 6011.66 mg/flask. The utilization ratio of adenine reached 91%; this the highest amount recorded in a single fermenter. (2) Large-Scale Culture Conditions: after using single factor design, Plackett-Burman design, and central composite design. Under the optimization culture conditions, a maximum production of cordycepin was 2008.48 mg/L for 700 mL working volume in the 1000 mL glass jars and total content of cordycepin reached 1405.94 mg/bottle. (3) Solid-state fermentation for fruit body and cordycepin production: the optimization strategies in solid medium culture lead to a fruit body yield increased 67.96% (about 1.73 g/bottle) and cordycepin yield in fruit body increased 55.36% (0.87%). Larger particle size of rice in the medium offers better fruit body growth, and the cordycepin production prefers smaller particle size. (4) Solid-state fermentation only for cordycepin production: medium components glucose, peptone, adenine and histidine have been examined. The levels of variables for CCD experiments were selected according to the above results of the One-factor-at-a-time method; maximum response of 18.92 mg/g cordycepin at levels of glucose 26.25 g/L, peptone 26.25 g/L, adenine 7.50 g/L, and histidine 4.50 g/L as optimized medium components. This is the first report for improving the cordycepin production by using additives in this method. Isolates from colony sector mutation could be used for screening high-yield strains in cordycepin production and colony colour is one of the markers to detect fruit body and cordycepin production. (5)The separation and purification process of cordycepin: this optimization strategy leads to crystal of 60.45 g with ratio of pure 98.05%, and the recovery rate is 43%. Our production line capacity is about 200 kg/year. This study enriches the biodiversity of Cordyceps s.l. This method provides an effective way for increasing the C. militaris fruit body and cordycepin production at a large scale in order to improve industrial applications.
New insights into Nowakowskiella genus (Chytridiomycota)
- G. Jerônimo Alves
- A. Jesus
- C. Pires Zottarelli
The genus Nowakowskiella was proposed by J. Schröt. in 1893 to include the type species Nowakowskiella elegans, previously named as Cladochytrium elegans Nowak.. In the following years, several species were described and currently this genus contains 18 legitimate species, which are morphologically recognized by the production of polycentric thalli with non-septate swellings besides operculated zoosporangia. Although this genus represents one of the most numerous in number of species within Cladochytriales, the most recent species, Nowakowskiella keratinophila Hassan & Batko, was described in 1988 and since then this group have not been undergoing modifications except by the proposition of the family Nowakowskiellaceae in 2009. Considering that, the aim of this study is to introduce a new species, Nowakowskiella sp. nov., clarify the phylogenetic positioning of Nowakowskiella elongata Karling and pointed another potential new species of Nowakowskiella. Besides that, we will include Nowakowskiella multispora Karling and Nowakowskiella ramosa Butler for the first time in phylogenetic reconstructions. The strains were isolated during two studies developed in aquatic ecosystems (streams and reservoirs) located in different fragments of Atlantic rainforest at Sao Paulo State, Brazil. Their SSU and LSU regions of rDNA were amplified with specific primers and a concatenate tree was build using Garli software package. The new species, Nowakowskiella sp. nov., is characterized by production of operculated zoosporangia with a prominent apophysis and crenulated resting spores. The second taxa, Nowakowskiella sp.1, could also represent a new species which produce apophysis in both sides of zoosporangia and a small tube during the zoospores releasing, however, we were not able to observe the resting spore production to confirm this supposition. Our Maximum Likelihood analysis showed that Nowakowskiella ramosa is sister group of the type species (N. elegans) and Nowakowskiella sp. nov. is related to our potential new species (Nowakowskiella sp.1). Nowakowskiella elongata belongs to other group outside of Nowakowskiellaceae and it is related to Nephrochytrium sp. JEL125, but seems to represent a new genus. These results bring important information about the taxonomy and molecular relationship inside of Nowakowskiella genus besides to contribute to increase the knowledge about the phylogenetic data of South America isolates.Financial support: FAPESP/CAPES/CNPq
Diversity within a single Peronosporaceae sample and implications for phylogenetic studies.
- E. Wallace
- C. Salgado-Salazar
- N. Leblanc
- J. Crouch
Downy mildews are plant diseases caused by a diverse group of diploid organisms in the Oomycota (Peronosporaceae). Over 700 species cause downy mildew diseases. Some of these organisms are thought to affect a wide diversity of plants, but most well-described downy mildew species are delimited by host genus. Downy mildews typically produce foliar lesions but some species are also reported to cause stunting, mottling, and in extreme cases, complete defoliation. The obligate biotrophic nature of downy mildew organisms creates difficulties in maintaining strains of these pathogens for research. This is particularly true for newly emergent or under-studied species where ideal environmental conditions and propagation protocols have not been established. As a result, single-spored isolates are rarely used in molecular phylogenetic studies. Common practices for generating DNA sequences from downy mildew samples involve performing PCR on genomic DNA extracted from sporulating pathogen tissue scraped from the leaf surface, or from excised disease lesions. When nucleotide sequences are generated directly from amplicons via Sanger sequencing technology, forward and reverse sequences are used to generate a consensus sequence, and any conflicting peak calls and double peaks in the chromatogram are indicated with IUPAC nucleotide ambiguity codes. However, by eliminating steps to ensure a single isolate is evaluated in a phylogeny, conclusions may not fully capture diversity of the population in one sample, nor the potential for heterozygosity of nuclear markers. In the current study, diversity within individual samples of Peronospora infecting Monarda didyma and Hyaloperonospora infecting Cleome sp. plants was evaluated using two approaches. DNA was extracted from sporulating leaf lesions, and amplicons of ITS rDNA and cox2 mitochondrial DNA markers were cloned to produce haplotypes, and 7 to 10 inserts were bi-directionally sequenced per sample. Amplicons from the same two markers were also sequenced to a high depth of coverage using next generation sequencing on an Illumina MiSeq to evaluate the level of diversity within individual samples. The resultant data demonstrated that the level of diversity within individual downy mildew samples has the potential to be much greater than what is traditionally captured using direct Sanger sequencing of potentially heterogeneous amplicons. These unaccounted for variant sequences may have substantial impacts on phylogenetic analyses and should be considered when making taxonomic decisions.
The lignin degrading systems of Polyporales
- C. Faulds
- M. Daou
- D. Navarro
- S. Raouche
- M. Rosso
The Polyporales order of Basidiomycetes contains most of the known wood-rot fungi, effective degraders of lignocellulosic biomass. While many studies have previously examined examples of these fungi during growth on lignocellulose, measured the mineralization of labelled lignin and mapped the secretion of many carbohydrate- and lignin-active enzyme activities, little is known about their ability to grow directly on a pure lignin substrate and the enzymes directly induced by this polyaromatic material. Previously it had been considered that fungi could not grow well on lignin alone.
Eleven species belonging to the Polyporaceae (10 strains) and Hypocreaceae (1 strain) families were obtained from the Centre International de Ressources Microbiennes-Champignons Filamenteux (CIRM-CF) culture collection (https://www6.inra.fr/cirm/Champignons-Filamenteux). Their selection was based on the availability of a sequenced genome and prior knowledge relating to growth on lignosulphonate. The fungal strains were screened for their ability to grow on both agar plates and liquid media containing a commercial alkaline lignin from grasses, and for the production of ligninolytic activities, such as laccase and heme peroxidase. The strains showed differences in their growth rates in the presence of lignin, especially in liquid cultures, and also differences in the secretion of laccase and/or lignin peroxidase activities. Proteomic analysis of the secretomes produced by five of these eleven fungal strains during growth on the alkaline lignin will be presented and the different enzymatic mechanisms employed by filamentous fungi for the deconstruction of lignocellulosic biomasses, and in particular lignin, will be discussed.
Presence and abundance of nitrogen metabolism genes explain fungal community shifts and ecosystem-carbon dynamics in nitrogen enrichment field experiments
- A. Romero-Olivares
- S. Frey
- N. Dunkirk
- A. Pringle
Nitrogen (N) deposition in terrestrial ecosystems is a strong driver of ecosystem-level carbon (C) dynamics. In field experiments, simulated N deposition results in accumulation of soil carbon, an increase in recalcitrant compounds, and a decrease in soil respiration. Because rates of N deposition are increasing globally due to anthropogenic activities, we must understand how ecosystem C dynamics will change in response. Fungi are key drivers of ecosystem C cycling and are highly susceptible to soil N enrichment. In addition, N-induced soil C accumulation is related to disruptions in organic matter decomposition due to disturbances to the fungal community. Simulated N deposition reduces fungal biomass, alters the activities of microbial extracellular enzymes, and restructures the fungal community. However, little is known about the evolutionary adaptation of fungi under N deposition that shapes ecosystem C dynamics. Our objective was to analyze genomes of fungi that have been reported in N amended soil to search for presence and abundance of genes associated with N metabolism. We hypothesized that fungi in N enriched plots would have higher abundance of N metabolism genes, compared to fungi observed in control soils, but a lower abundance of genes associated with decomposition of recalcitrant carbon. We predicted this tradeoff because higher abundance of N metabolism genes in fungi may be selected for and facilitate fungal survival at higher soil N concentrations at the expense of genes associated with an ability to decompose energetically expensive recalcitrant carbon compounds. To this end, we searched for genes associated with ammonium transporters, nitrate transporters, and amino acid permeases. We also searched for genes associated with carbon metabolism to gain a comprehensive understanding of the ecosystem-level observations of the fungal community and of the carbon dynamics under elevated N depositions. We found that on average, fungi from N amended plots had significantly more genes for uptake of organic N, as well as more genes to break down cellulose. Moreover, a higher percentage of fungi in N amended plots had more than one gene for uptake of inorganic N in comparison to fungi from control plots. Similarly, a higher percentage of fungi from N treatments had more than one gene for breakdown of labile carbon in comparison to controls. We also detected a shift in the taxonomical distribution of fungi. The order Agaricales, Polyporales, and Russulales decreased in response to N; whereas, Hypocreales, Pleosporales, and Pezizales increased. Concomitantly, we detected an increase in functional guilds associated to plant pathogens, an overall increase of saprotrophs, and a decrease of ectomycorrhizal fungi in response to N. Finally, we analyzed specific decomposition traits, such as white, soft or brown rot, and found that rot fungi are present under both control and N enriched conditions. However, there was a slight increase in abundance of soft and white rot in response to N. We provide genome- and functional-level evidence for the response of fungi to simulated N deposition and suggest that N may serve as a selective force on fungal communities, driving changes in C dynamics at the ecosystem-level.
Differential gene expression linked to fungal trophic switches (symbiotrophism and saprotrophism) using the moss Dicranum scoparium and its associated fungi
- K. Chen
- H. Liao
- H. Korotkin
- S. Wu
- P. Matheny
- F. Lutzoni
While the plant holobiont concept is becoming more widely accepted, interactions between mosses and fungi remain scarcely explored. Yet, moss-fungi model systems offer advantages that are complementary to other plant-fungi experimental systems. Our goal is to understand moss-fungal interactions by integrating environmental samples and laboratory co-culture experiments. Many naturally occurring mosses have stratified growth forms with a photosynthetic layer, a senescent layer, and a decomposing layer. Therefore, these mosses offer a unique opportunity to study shifts in fungal communities and functions along a senescence gradient, simultaneously, i.e., without the confounding effect of time. We examined fungal communities along the senescence gradient of a perennial moss Dicranum scoparium using metatranscriptomic data and a culture-based endophyte isolation approach. By integrating the fungal rRNA sequences generated by metatranscriptomics (i.e., as a proxy for fungal activity) and culture-based approaches, we assigned fungi into three types of association categories: 1) High activity in photosynthetic tissues, 2) High activity in decomposing tissues and 3) Low activity throughout the gametophyte but frequently detected using a culture-based method. Using these three categories, seven fungal strains representing distinct fungal lineages (four Ascomycota, two Basidiomycota, one Mortierellomycotina) were selected to establish fungus-plant re-synthesis experiments. We included both the axenic living and dead moss gametophytes for the re-syntheses. The former enabled us to investigate plant-fungus interactions; the latter allowed us to examine fungal functional switches when inhabiting living vs. dead plant tissues. For every fungus-plant pair, growth rates were monitored for two months after fungal mycelia reached the mosses. Our results showed that various fungal structures were produced on the inoculated moss hosts. However, none of these tested strains caused significant moss growth losses. Subsequently, we selected three fungi potentially representing different ecological guilds and we obtained metatranscriptomes for these three moss-fungal pairs. The three fungal strains selected were: 1) a Coniochaeta strain closely related to fungal endophytes from other plant lineages, 2) an unknown strain from the class Leotiomycetes closely related to ericoid mycorrhizal fungi, and 3) a Rickenella fibula strain producing fruit-body directly on D. scoparium. Based on our metatranscriptomic data, mosses inoculated with fungi had genes related to plant defense (e.g. leucine rich repeat receptor, chitinase) and nutrient transportation (e.g. phosphate transporter) upregulated. We also compared GO (Gene Ontology) categories enriched for fungi growing in living plant tissues versus in dead plant tissues. The former scenario was enriched for genes related to carbohydrate transport, while the latter was enriched for genes responsible for oxidation-reduction process, suggesting functional switches of fungi between symbiotrophy and saprotrophy. Our study sheds light on the importance of understudied plant-fungal systems, and provides evidence for functional lability of individual fungal strains in response to host senescence.
A metatranscriptomic key to open the black box of fungal process in forest soil
- F. Barbi
- U. Menzel
- B. Lindahl
Boreal forests represent a large carbon sink, indeed this ecosystem represent 32% of the global carbon store on Earth. Due to the intensive human activity (e.g. forestry, clear-cutting, land use change, fertilization) occurring today in a vast majority of boreal forest, it is essential to advance our understanding of soil processes, to enable informed future policy decisions about forest management and ensure a maintained, and preferably increased, carbon sink. Soil microbial communities play a central role in regulation of soil organic matter dynamics in forest ecosystems and are therefore subject to particular attention. In boreal forest soils, fungi are the main microbial group in term of biomass, and mycorrhizal fungi, living in symbiosis with plant roots, are particularly important in regulating carbon sequestration. New innovative molecular techniques (metatranscriptomics) allow us to open the black box of microbial process in soils, by obtaining massive data of expressed genes from entire microbial communities, and investigate how microorganisms regulate soil organic matter dynamics, directly in the ecosystem. The objective of this study is to develop an approach to highlight fungal functional traits related to soil fungal community ecology. Specifically, to evaluate the mechanisms relative to carbon storage under variation in ecosystem fertility and production. In this purpose, the role of fungal community in carbon transformation, during decomposition and microbial metabolism, is assessing by analyzing expression of genes involved in the production of enzymes implicated in organic matter degradation, stress tolerance and intracellular CO2 release, at the ecosystem level. The studied site is a native boreal forest presenting a clear split between an N-poor low productive and an N-rich high productive plot. RNAs have been extracted from soil of 16 plots and rRNAs have been removed for a massive Illumina HiSeq sequencing (more than 100 million reads per sample). To decrease the calculation time during bioinformatic analysis and focus on relevant ecological questions, we develop a targeted markers assembly using the HMMER software. Publically available reference sequence databases (e.g. CAZy or JGI) are used to guide transcript filtering and identification of expressing organisms. This study demonstrate the efficiency of the developed approach, to select specific gene family relative to functional traits, from a raw metatranscriptomic dataset. Moreover, the primary results reveal differences in fungal lifestyle strategies, according to forest fertility and productivity, identified by using expressed genes as marker for fungal process such as decomposition (eg. lignocelluolytic enzymes), biomass production (eg. 1,3-β-glucan synthase), stress tolerance (eg. melanin synthesis) and respiration. To conclude, the purpose of this pioneer approach is to provide novel insights about the interplay between microbial traits, such as decomposer capacity and metabolic efficiency, and ecosystem level processes, such as carbon sequestration, in order to increase the predictive capacity of ecosystem models.
Chitin synthase inhibitors from soil fungi
- G. Vidyasagar
The fungi are ubiquitous group of Eukaryotes that occur in multiplicity of natural and manmade environment. Isolating microorganisms from the environment is the first step in screening for natural products such as, secondary metabolites and antibiotics. Fungal population in soil usually lies numerically between bacteria and actinomycetes. Therefore, the present studies aimed to exploit the available soil fungal flora of the Hyderabad Karnataka Region (HKR) of India for the production of novel antifungal molecules, which could inhibit chitin synthase activity in fungal cell wall. Chitin could be the target molecules used in the development of antifungal drugs as it is an important component of fungal cell wall and absent in human cell. A total of 53 fungal strains were isolated from various soil samples collected from different locations using PDA medium and screened them for antimicrobial activity against 12 strains of Candida. Among the fungal isolates, two isolates namely, VSGUF1 and VSGUF 2 were found inhibitory. The extracellular and intracellular crude extracts showed a maximum of 17mm inhibition zone against C. albicans1637. Antifungal susceptibility assay of VSGUF1 and VSGUF 2 performed in RPMI 1640 medium against different strains of Candida after 36 h showed MIC90 at 128µg/ml and 32µg/ml against C. glabarata, respectively. In vitro chitin synthase activity indicated 100% inhibition of Candida albicans ATCC 24433 in intracellular crude extract of VSGUF1 at 64 µg/ml. The biomolecules produced by VSGUF1 and VSGUF2 are the potent molecules could be used in the development of new antifungal drug for Candida infections. Further studies are in progress.
Bioprospecting for chitinases among bacteria in Puerto Rico
- B. Sang-Laboy
- B. Serrano-Torres
- L. Cruz-Flores
- A. Healion-Del Valle
- J. Perez-Jimenez
Chitinases are enzymes responsible for modification of chitin. A variety of chitinases has been isolated from marine bacteria, as many of them live close to chitin-containing invertebrates. Chitinases have been proposed as antifungal alternative since it distorts the chitin-containing cell wall of the fungi. Our objective has been to determine the chitinolytic activity of bacteria capable of degrading organic pollutants or expressing antibacterial activity. A collection of bacterial strains recently isolated from diverse ecosystems in Puerto Rico was subjected to chitinolytic assays. These strains have demonstrated capabilities to degrade xylene or inhibits bacteria growth. Twenty of them were cultivated in mineral media containing chitin (~0.2% w/v) as sole carbon sole. Incubation proceeded at room temperature on an orbital shaker (130 rpm). After four days, minor turbidity was noted on the liquid enrichment. An aliquot (~100 ml) was spun to concentrate cells for microscopic examination. Gram staining resulted on three Gram-negative rods and twenty-one Gram-positive (eleventh rods and nine coccus) among the putative chitin-degrading bacteria. Among them, strains of Alcaligenes faecalis, Pseudomonas sp., and Achromobacter sp. were represented. Sequencing of their 16S rDNA and specific antifungal assays are in progress. This project contributes additional diverse strains with potential for fungal control based on chitinolytic activity.
Reduction of proteolytic activity in filamentous fungal protein production hosts
- W. De Bonte
- S. Segers-Klaubauf
- A. Ram
- P. Punt
AbstractFungal host strains such as Aspergillus niger, Aspergillus sojae and Trichoderma reesei are used for the production of a wide variety of industrially relevant enzymes. About 80% of all industrial enzymes are derived from filamentous fungi, making these organisms also the hosts of choice for the production of new enzymes and proteins. The presence of unwanted proteolytic activity in filamentous fungi is a major bottleneck for (heterologous) protein production. Despite the accumulation of data by all kinds of -omics techniques, the regulation of protease genes in fungi remains largely unexplored. Dutch DNA Biotech is working on several approaches to tackle this problem in industrial fungal strains. All these approaches are focused on identifying regulatory genes specific for the pathways underlying protease production. Based on strain engineering, using both classical screening and targeted molecular approaches, regulatory mutants are being developed. The research presented here is focussed on three methods to find relevant factors that are involved in the regulation of proteolytic genes. The first method is a positive screenings method for protease mutants, we named SUI selection. SUI selection is a growth based screening method and is much more efficient than activity based screening methods. The second method is the screening of a transcription factor knock out library in Aspergillus niger and Neurospora crassa for mutants with a lower proteolytic activity. The third method involves a multicopy suppressor approach based on strains carrying multiple copies of the major A. niger protease regulator gene, prtT.
Xylariaceae volatiles and their applications in agriculture
- C. Krill
- R. Mann
- D. Auer
- J. Edwards
- S. Rochfort
- S. Mattner
- I. Porter
- G. Spangenberg
The Xylariaceae is one of the largest families within the fungal kingdom. The majority of the 800 identified species are commonly classified as saprobes, with many isolated as endophytes. The family is one of the best studied fungal taxa based on morphological, molecular and chemotaxonomic data. Members of the Xylariaceae are recognised for producing a wide variety of secondary metabolites such as pigments, volatiles and biocidal compounds, many of which have demonstrated bioactivity against important agricultural pests and pathogens. Current agricultural practices rely heavily on the use of agrochemicals for crop protection before and after harvest, however their sustainability is constantly under question. Bioactive secondary metabolites from Xylariaceae fungi represent promising candidates as novel “green” agrochemicals, that are (1) beneficial in breaking current and emergent resistances, (2) less prone to induce toxic side-effects on human and animal health, (3) and are more environmentally sustainable. Using Gas Chromatography – Mass Spectrometry, we profiled the volatolome of various native Australian fungal isolates of the Xylariaceae genera Nodulisporium (Hypoxylon), Muscodor, Daldinia and Xylaria. We identified a number of VOCs and investigated their biocidal properties, synergistic effects and modes of action against common pre- and postharvest pests and pathogens. Here, we present an overview of common and species specific Xylariaceae volatiles, their properties, mode of action, as well as their potential application, environmental fate and use as stored grain disinfestants, soil fumigants and food-grade disinfectants.
Chitinolytic efficacy and secretion of cell wall degrading enzymes from Trichoderma spp. in response to phyto-pathological fungi
- D. Khatri
- D. Tiwari
- H. Bariya
Chitinolytic activity and major antifungal enzyme secretion form Trichoderma spp. was studied. Soil samples were collected from different environmental niche of North Gujarat Region, India and 12 different species of Trichoderma were obtained and identified. Among 12 isolates, 4 isolates were identified as T. harzianum, 5 isolates were identified as T. viride and remaining 3 isolates were as of T. hamantum. These isolates were identified by using species specific primers amplification by PCR. All identified isolates were screened for chitinase activity using colloidal chitin derived from commercial chitin on the media supplemented with bromocresol purple. According to results of chitinase activity screening assay, T. viride was found to be more potential isolate for chitinase production. From biocontrol assay by using duel culture method, T. viride was found to be more potent antagonist against fungal plant pathogens like A. niger, F. oxysporum and S. rolfsii. T. viride was selected for further study of biocontrol potential and production of cell wall degrading enzymes. T. viride was inoculated in media containing basal media and mycelia of fungal pathogens for cell wall degrading enzyme production. It was found that T. viride secrete three major cell wall degrading enzymes i.e. chitinase, protease and β-Glucanase. Optimum production of all three enzymes was found at 96 hr incubation. Details of antifungal protein secretion are mentioned in this paper.
The Thkel1 gene of Trichoderma harzianum plays a key role in its ability to colonize the roots of Arabidopsis thaliana
- J. Poveda
- A. Alonso
- R. Hermosa
- E. Monte
- C. Nicolás
Trichoderma is a genus of filamentous fungi widely studied and used as a biocontrol agent in agriculture due to different mechanisms of action, such as its ability to parasitize and inhibit the growth of phytopathogenic fungi, promote plant growth and improve the response of the plant against both biotic and abiotic stresses. The Thkel1 gene of T. harzianum encodes a protein with kelch domains involved in protein- protein interactions. Expression of Thkel1 in Arabidopsis thaliana enhanced plant tolerance to salt and osmotic stresses, accompanied by an increase in β-glucosidase activity. The aim of the present work was to analyze the role of this gene in Trichoderma-plant root interaction, using Trichoderma transformants silenced in Thkel1 and Arabidopsis plants that overexpressed this gene. We observed that root colonization ability of Arabidopsis wild type plants was dramatically reduced in Trichoderma silenced transformants whereas this ability was restored in Thkel1 overexpressing plants. Similar results were observed in canola (Brassica napus) plants. On the other hand, Thkel1 gene was not relevant in Trichoderma root colonization of tomato plants. These results suggest that the Thkel1 gene can play a crucial role in root colonization of Brassicaceae plants.
Role of fungal volatiles produced by Trichoderma on plant growth
- S. Ojha
- J. Dunbar
- C. Kuske
- A. Porras-Alfaro
Microbial volatile organic compounds (VOCs) play important roles in plants influencing their physiology and development. Trichoderma is well known for the production of signal molecules that influence the growth of plants and other fungi. The objective of this research was to compare the effect of different Trichoderma species obtained from soil and surface sterilized roots. Fungi were isolated across the USA and Trichoderma strains were identified using ITS rRNA, and tested on the dominant arid grass, Bouteloua gracilis (blue grama) in a closed chamber experiment. Volatiles emitted by different species of Trichoderma exhibited a wide range of effects on plant growth and development. Trichoderma gamsii (CK71) and Trichoderma saturi (CK1108) showed the greatest growth promoting abilities in B. gracilis, with a significant increase on seed germination, plant size, and root development compared to the controls. Trichoderma strains were also tested in direct contact germination experiments. The association of the fungi with plant roots was analyzed using microscopy. B. gracilis seeds inoculated with Trichoderma strains showed an increased root length and proliferation of lateral roots compared to the controls. Microscopy examination of stained roots revealed small round fungal-like structures in cortex and intercellular hyphal growth. Trichoderma high abundance in soils across multiple ecosystems as demonstrated by Illumina sequencing and culturing methods showed important ecological functions of these fungi as regulators of plant growth through multiple mechanisms. Future research will be conducted to evaluate factors that influence Trichoderma-plant interactions using different growth conditions (e.g. temperature and media).
Isolation and metabarcoding of aquatic mycodiversity in lakes along a humic substance gradient
- C. Baschien
Christiane Baschien1, Andrey Yurkov1, Josephina Barkowski1, Sixing Huang1, Boyke Bunk1, Jörg Overman1
1Leibniz-Institute DSMZ German Collection of Microorganisms and Cell Cultures, Inhoffenstrasse 7B, 38124 Braunschweig, Germany.
Emails1: Christiane.Baschien@dsmz.de; Andrey.Yurkov@dsmz.de; Jop16@dsmz.de; Sixing.Huang@dsmz.de; Boyke.Bunk@dsmz.de; Joerg.Overmann@dsmz.de
Fungi in aquatic environments degrade organic matter and thus transfer nutrients to other trophic levels. This phylogenetically heterogeneous group of fungi is widespread and well adapted to life in aquatic habitats. However, biodiversity assessments and living fungal cultures from lakes are both scarce. We investigated the fungal diversity in three different lakes which cover a gradient in organic carbon (OC) content and quality. Cultivation and meta-genomic barcoding were employed to study freshwater fungi in littoral water, fine particulate organic matter (FPOM), sediment and coarse organic matter (CPOM) such as leaves and wood. Furthermore, isolates were tested for the ability of humic matter degradation and for submerged growth in water. Three isolation methods applied to four substrates collected from three lakes yielded 262 cultures. CPOM showed the highest fungal diversity among analyzed substrates. Compared to other cultivation approaches, multi-well cultivation methods were least successful. The taxonomic diversity was dominated by ascomycete species, most of which are known as plant pathogens or saprobes. Overall, our study yielded 20 potential new species which were able to grow and sporulate submerged in water. The production of laccases and peroxidases was observed in 25 % of all isolated species. The metabarcoding approach of the natural samples yielded 90,772 ITS2 sequences; the highest numbers were obtained from littoral water samples. The taxonomic assignment of Operational Taxonomic Units (OTU) was made employing several common pipelines and, additionally, manually curated. The final dataset contained 572 OTUs of which 74.5 % were classified on species level. Similar to the cultivation approach, metabarcoding detected many species belonging to Ascomycota and Basidiomycota but also Cryptomycota, Chytridiomycota, Zoopagomycota and Neocallimastigomycotina. The diversity of fungal communities was highly variable among lakes and substrates. Community structure differed along the environmental factors; particularly the quality of carbon (humic or not) and the form of available nitrogen were important factors. Most species were associated with a particular lake and substrate type. About 30 % of the species occurred more frequently across the habitats. Sequences of 90 % of the cultivated isolates were also retrieved by metabarcoding and most occurred in more than one metabarcoding sample. Substrates but not lakes explained the occurrence of cultivated fungi, while metabarcoding suggests the distribution of differing fungal communities according to the type of substrate and lake more clearly.
Metabolism of fungal species isolated from the coast off central Chile: the role of respiration and nutrient assimilation processes
- M. Fuentes
- R. Quiñones
Filamentous marine fungi have recently been identified as a functional component of coastal systems, playing an important role in the degradation of organic matter and hydrolysis of large polymers. However, the ecophysiological and biogeochemical role of these fungi has been scarcely explored. This work seeks to determine the effect of temperature and nutrient availability (such as glucose) on respiration and growth of filamentous fungi, as well as to characterize the use of carbon, nitrogen, phosphorus and sulfur substrates by these organisms. Respiration and growth rates were determined for 5 species of filamentous fungi (Penicillium decumbens, Penicillium chrysogenum, Sarocladium strictum, Fusarium fujikuroi and Fusarium sporotrichioides) isolated from the coastal upwelling zone, so as to examine the effects of temperature and nutrient availability (such as glucose). Growth was monitored via epifluorescence microscopy, ATP concentrations and Optical Density; while oxygen consumption was recorded using a respirometer with “Optodes”. Although responses were species specific, generally respiration and growth increased with temperature and glucose concentration. Growth of P. decumbens, F. sporotrichioides and F. fujikuroi were most favored by when glucose concentrations remained stable. Substrate use profiles for carbon, nitrogen, phosphorus and sulfur were obtained for three species (P. decumbens, S. strictum and F. fujikuroi). In order to understand their potential impact on the degradation of carbon compounds, their carbon profiles were characterized using Biolog Filamentous Fungi (FF) MicroPlates. These species were found to be versatile, with a large capacity (57.2 % of total) for using a wide range of carbon sources, principally carbohydrates (monosaccharides, disaccharides, oligosaccharides and polysaccharides), but also amino acids (0.99 %), suggesting the use of metabolic pathways, such as glycolysis /gluconeogenesis. These species also displayed high indices of substrate use complementary to nitrogen, phosphorus and sulfur, where organic components accounted for greater hyphal growth. Here, L-amino acids, amines and nucleotides/nucleosides were preferential sources of nitrogen, suggesting the use of pathways involved in the amino acids and/or purine metabolism. Other compounds, such as urea, allantoin and uric acid produced moderate growth. The ribonucleotides adenosine and guanosine were the main substrate for phosphorus use; and cysteine and methionine were the main sources of sulfur for growth in these species. Moreover, growth was observed with several inorganic substrate sources, such as nitrate, nitrite, thiophosphates, tetrathionate, in the three species. Considering the active heterotrophic role of filamentous fungi, their importance in the degradation of organic matter and their participation in important biogeochemical cycles in the ocean, we suggest the inclusion of the mycoplanktonic community as an integral component of the microbial community in the coastal ocean, to be included in microbial conceptual models of degradation of organic matter and biogeochemical cycles given their use of a wide variety of organic and inorganic carbon, nitrogen, phosphorus and sulfur compounds.
Fungi diversity and enzyme activity associated with sailfin sandfish egg masses in Korea
- M. Park
- Y. Lim
The aggregation of decaying egg masses of sailfin sandfish along the mid-east coast of Korea is a major environmental problem with concurrent negative economic consequences. In an effort to ameliorate decaying egg masses, we investigated the diversity and community structure of fungi from egg masses and tested for their cellulase and protease activity. A total of 1,108 strains were identified based on morphology and multigene analyses, and found to represent 184 fungal species. Paradendryphiella salina was the most dominant species, followed by Penicillium crustosum and Penicillium aurantioviolaceum. The fungal community displayed a significant degree of variation relative to both egg mass color and locality. Over 50 % of species detected in this study exhibited both cellulase and protease activity. This study suggests that fungi play an important role in nutrient recycling at intertidal zones and thus may have potential industrial applications that can help resolve the environmental problems associated with egg mass aggregation.
Analysis of coral-associated fungal and microbial communities in Fiji using high throughput amplicon sequencing
- R. Longley
- O. Alian
- G. Bennucci
- X. Pochon
- V. Bonito
- G. Bonito
Coral reefs are biological hot-spots on Earth and provide important habitats for a wide variety of marine species. For this reason, sections of many reefs have been the targets of conservation projects that aim to protect coral and other species that make up the reef ecosystem. Microbial communities make up an integral part of a healthy reef ecosystem, and therefore must be considered when assessing the success of conservation efforts. To this end, corals were sampled from four different genera: Porites, Montipora, Acropora, and Sinularia. Sampling occurred in two adjacent shallow reef areas: 1) a no-take marine protected area with relatively low disturbance, and 2) a fished area with relatively high disturbance from fishing pressure and watershed discharge. These samples were then analyzed for communities of Fungi, Bacteria, and the endosymbiotic dinoflagellate, Symbiodinium. Microbial communities were targeted using primers specific to the Fungal ITS-1 region, the Bacterial 16s region, and the Symbiodinium ITS-2 rDNA region. Amplicons from these primers were then tagged with barcodes and pooled into libraries for sequencing on an Illumina’ Miseq platform. Sequence data were then run through a non-biased bioinformatic pipeline and the unoise algorithim in usearch to cluster OTUs for each group by a single nucleotide difference in sequence. The initial analysis on the entire sample set for Fungi recovered a broad diversity of species dominated by Ascomycota (65%) and Basidiomycota (33%). The Ascomycetes were dominated by Cladosporiaceae (15%) and Aspergillacea (10%); while the Basidiomycetes were predominantly Malasseziaceae (45%) and Nectriaceae (15%). The bacterial communities were predominantly Cyanobacteria (51%), Proteobacteria (37%), and Bacteroidetes (8%). Within the bacterial communities, the Cyanobacteria were dominated by Ulvophyceae (84%), Proteobacteria were dominated by Endozoicimonaceae (36%), Bacteroidetes were most highly represented by Chitinophagacae (30%). Symbiodinium communities were dominated by clade C (96%), but clades D (3%), as well as A and G were also present at low levels. This preliminary data on the sample set shows diverse microbial communities and lays the framework for comparing the microbiomes of corals in protected and unprotected areas.
Marine Fungi from India: Range of Substrata
- A. Tuwar
The present paper deals with distribution and substratum range of 217 species of marine fungi (14 Labyrinthulomycetes, 4 Chytridiomycetes, 4 Oomycetes, 143 Ascomycetes, 3 Basidiomycetes and 46 Mitosporic / Asexual fungi) reported so far from the marine waters of India. These fungi were reported as parasites / saprophytes on Animal substrates (16 sp.), saprophytic on intertidal and deep sea sediments (7 sp.), on intertidal woody debris (131 sp.), on decaying algae (17 sp.), saprophytic on salt marsh plants (3 sp.), saprophytic on sea grasses (4 sp.), saprophytic on woody debris of mangroves from intertidal region (165 sp.). Ascospores and conidia of 27 species were recorded in foam samples from sandy beaches. Fungal species (165 sp.) recorded on mangrove substrata forms the largest group after intertidal woody debris (131 sp.). It also shows that most of the fungal species have been recorded from the West coast (154 sp.) after the East coast (152 sp.), Andaman-Nocobar Islands (66 sp.) and Lakshadweep Islands (55 sp.). Maximum number of marine fungi were encountered along the coast of Tamil Nadu state (106 sp.) and followed by Karnataka (100 sp.), Maharashtra (95 sp.), Goa (98 sp.), Kerala (93 sp.), Andhra Pradesh (73 sp.), Gujarat (70 sp.), West Bengal (69 sp.), Orissa (54 sp.), Pondecherry-Mahe (46 sp.), Daman (17 sp.), Diu Island (14 sp.) and Pondecherry (12 sp.). This data will be useful in the compilation of marine fungal biodiversity of India. The taxonomy, morphology and ecology of these fungi are discussed.
Structure of halophilic fungi within hypersaline environments
- J. Jimenez
- A. Tidwell
- K. Palmer
- M. Binggeli
- G. Zahn
Very little work has been performed to thoroughly inventory halophilic fungi within hypersaline environments. Although it is known that halophilic fungi live within hypersaline environments, this study will use the Great Salt Lake in Utah due to it’s extreme salinity gradient to create an inventory of species. By using ITS amplicon sequencing of fifty water column and sediment samples, this study is determining how salinity shapes fungal community structures and examining whether or not there is a “tipping point” in which salinity is the main limit on fungal diversity. These results will be beneficial to understanding the ecosystem dynamics of The Great Salt Lake.
Marine filamentous fungi from Sweden
- S. Tibell
- L. Tibell
- K. Pang
- G. Jones
Marine filamentous fungi have been little studied in Sweden, which is remarkable given the depth and width of mycological studies in the country since the time of Elias Fries. A summary of historical records along with numerous additions is given in a commented list of the marine filamentous fungi so far recorded from Sweden. New records for the country are based on morphological identification of species mainly from marine wood, most of them originating from the Swedish west coast. Identifications have been obtained by morphological studies, where spore structure frequently is a diagnostic character, and also by cultivation of the fungus followed by DNA isolation and amplification, mostly of the ITS region and comparisons with sequences in GenBank. A total of 67 filamentous marine fungi were recognized as occurring in Sweden based on a critical assessment of historical records, and additions contributed during field-work for the past two years. This is a substantial increase of earlier assessments (31 by Henningsson in 1974; 31 species of marine ascomycetes by Eriksson in 2014; and an additional seven by Tibell in 2016). Thirteen species are recorded as new to Sweden, whereas fifteen old records, some of which only identified to genus, were for different reasons not accepted in the present list. The number of species listed is a rather modest and this certainly reflects the fact that marine fungal diversity is poorly known and that the species so far described only represent a small fraction of their diversity. Most of the records were obtained from marine wood undoubtedly leaving a rich diversity occurring on marine algae and plants unaccounted for. The study is part of a project for assessing the diversity of marine fungi of Sweden supported by The Swedish Species Initiative.
Fungi are abundant and uncharacterized members of Arctic marine ecosystems
- B. Hassett
- R. Gradinger
AbstractFungi are among the most diverse groups of organisms in the world that interface essential nutrient cycling and carbon degradation process in the biosphere. The oceans cover 70% of the world’s surface; however, the unknown contributions of fungi to ecosystem processes remains a gap in marine ecology. We used molecular sequencing data generated from the Arctic Ocean to assess the diversity, regional abundances, and functional gene potential of marine fungi. We found the Arctic Ocean is comprised of a diverse fungal community that is predominated by the Chytridiomycota. Phylogenetic analysis of 28S rRNA clone data from sea ice revealed a previously undescribed clade of Chytridiomycota that branches sister to the taxonomic order Lobulomycetales. This clade was detected across the entire Arctic Ocean, underscoring the wide-distribution of Arctic marine fungi, especially the Chytridiomycota. To supplement these analyses, we used a GeoChip microarray to screen for known functional genes involved in biogeochemical cycling. We detected a suit of catalytic genes allied to the fungi that interface nitrogen cycling and carbon degradation. Ultimately, out data continue to help establish an ecological paradigm for marine fungi that remain to be fully integrated into marine sciences.
After the Fire: Fungi and wildfires in the Great Smoky Mountains National Park
- K. Hughes
- P. Matheny
- A. Case
- R. Swenie
- A. Miller
- T. Iturriaga
- D. Raudabaugh
The effects of wildfires on fungi are not well documented in eastern deciduous forests and likely depend on intensity of the fire. After the November 2016 wildfires in the Great Smoky Mountains National Park (GSMNP), we undertook a survey of above-ground fungi fruiting in lightly burned, moderately burned and heavily burned areas over a one-year period (January of 2017 through December of 2017). Consistent with studies in the western United States, a number of fire-response fungi were collected. Many had not previously been documented in the GSMNP including Pyronema omphalodes, Anthracobia (4 putative species), Pholiota highlandensis, Morchella exuberans, Peziza echinospora, Rhizina undulata and Geopyxis carbonaria. An unexpected mass fruiting of Hygrocybe conica was observed in high intensity fire zones in mid-summer. Germinating pine seedlings in high intensity fire zones formed mycorrhizal associations very early in development consisting of a single species, often Telephora/Tomentella but by mid-summer, many developing pine seedlings had more than one mycorrhizal species. Forty-eight new GSMNP taxa were identified. At least five are new to science.
Compositional and functional responses of soil and litter fungal communities to prescribed fires in pine savannas of North America
- T. Semenova
- J. Hopkins
- P. Hansen
- W. Platt
- B. Sikes
Wildfires threaten many ecosystems, but frequent fires are essential to sustain and renew pine savannas ecosystems. In these threatened ecosystems, repeated fires suppress hardwoods and result in one of the most biodiverse plant communities on Earth, filled with fire-adapted plants. Longleaf pines that dominate these communities, for example, have bark adaptations to resist fire and produce flammable needles that increase the probability and severity of future fires. In contrast, very little research has explored fire effects on the fungal communities that are both biodiverse and important decomposers of the fuels that pines and other plants produce. In a system adapted to recurrent fire, we know almost nothing about how a new fire might shift fungal communities, and in particular if it eliminates or promotes particular taxa or functional groups of fungi, some of which may be relevant to fuels. Given frequent prescribed fires, are fungal communities relatively immune to a new fire? Do any fire effects on fungi lessen from litter to soils, as soils insulate fungi from heating? To assess these questions, we used DNA-metabarcoding (Illumina MiSeq or ITS2 region) to assess and compare communities of soil and litter fungi following fires in a well-preserved North American pine savanna (The Wade Tract, Thomasville, GA, USA). We compared communities from 56 pairs of plots either experimentally burned or left unburned for at least 1 year. Within each plot we also estimated fungal abundance using digital droplet PCR, collected several different metrics of soil properties, and measured microbial decomposition in a parallel experiment. Despite all sites historically experiencing frequent fires, recent fire strongly altered fungal community composition and reduced fungal biomass. Shifts were stronger in litter (25% of variation in communities explained by fire) compared to soils (10% explained by fire) as expected given pine litter’s concentration of flammable compounds (resin) and the greater insulation for soil fungi. Burning strongly reduced the number of saprotrophic species, which paralleled slower litter decomposition in burned plots. Fire also effected specific taxonomic groups of fungi; Coniochaetales increased, but Ostropales, Botryopshaeriales and Lophiostoma (Pleosporales) strongly declined in species richness between burned and unburned plots. Most fungal lineages, however, showed no richness response to fire, including orders with the highest number of species - Pleosporales (22% of fungal OTUs), Agaricales (10%) and Chaetothyriales (7%). Across most taxa, regardless of lineage, OTU’s occurred in either burned or unburned areas, with very few taxa present in both. Our data reveal a fire-adapted fungal diversity that can respond rapidly to fire. Rather than being a trait of a specific fungal lineage, fire is present for individual taxa across nearly all lineages, possibly as repeated adaptations to recurrent fires in pine savannas. Future work will address fire-induced changes in the expression of fungal genes involved in heat resistance and decomposition, to provide better understanding of fungal functional responses to prescribed burning in pine savannas.
Increase in the outdoor levels of fungal spores in San Juan, Puerto Rico in the aftermath of Hurricanes Irma and Maria
- B. Bolanos Rosero
AbstractThe effect of hurricanes on the calendars of fungal spores and pollens have not been studied. Hurricanes Irma and Maria hit Puerto Rico on September 6 and 20, 2017 respectively and allowed us to determine their impact on the San Juan, PR fungal spore and pollen calendars. In San Juan, PR peaks of fungal spores occur during April and May and from September through November. In contrast, tree pollens, the most abundant in PR, are present all year long but at lower levels during the summer (June-August). We use the Burkard air sample to take daily air samples on a glass slide to count and identify the fungal spores and pollens a week after Maria hit PR, just at the beginning of the fungal spore season. We observed a significant decrease in the levels of fungal spores and the 2017 fungal spore’s season was severely affected. Nevertheless, the fungal spores rapidly rebound reaching unexpected record high levels during January through March 2018 and the April and May season is the highest on record. Several days of very high fungal spores levels (red alerts) and very rapid release of fungal spores were observed. This could be due to the accumulation of biomass for the fungal growth and an unusual rainy season at the beginning of 2018. In contrast to the fungal spores, and due to the devastation of the vegetation after the hurricanes, the tree-pollen levels reached very low levels during the beginning of 2018. Hurricanes may be an important factor in the increase of outdoor fungal spores. As we described that high levels of fungal spores are significant triggers of asthma and allergies in PR, we will expect an increase in the use of asthma and respiratory medical services for 2018 in the aftermath of hurricanes Irma and Maria.
Different fire severities result in distinct soil fungal community trajectories.
- S. Fox
- A. Jumpponen
- C. Reazin
- J. Smith
- A. Cowan
Wildfires burn large areas of forested land annually, and they are projected to increase in frequency and intensity. These wildfires are not uniform; rather, they burn in patches that vary in severity. We compared experimental fires of different severities mimicking landscape mosaics created by a wildfire with patches analogous to whole log combustion within a background burn. The primary aim was to improve our understanding of soil fungal community trajectories following varying fire severities. We established ten pairs of plots in the Pringle Falls Experimental Forest in Oregon, USA. For each pair, one plot served as a background control (low severity burn), whereas another included logs piled in 1.5m x 8m x 1m structure for intense whole log combustion (high severity burn). The soils were sampled from 0-10cm depth within each plot before the burn, one weeks after the burn, as well as 2 and 4 years after the burn. DNA was extracted from these soil samples, the ITS2 barcode region of the ribosomal RNA gene PCR-amplified, and amplicons Illumina MiSeq sequenced to compare community richness, diversity, and composition among the severity treatments and over time. The data show that the fungal communities rapidly change in response to fire, and the recovery time depends on the fire severity. Following a high severity fire, the fungal communities follow trajectories distinct from those in low severity fires, although neither has fully recovered to communities resembling pre-fire conditions. Fire events, especially high severity fires have lasting impacts on the above ground system. Our study indicates that similar lasting impacts also happen to the fungal communities in the soil.
Mycobiomes of boreal forest soils: Unraveling patterns in fungal communities following disturbances
- J. Rodriguez Ramos
- J. Cale
- S. Simard
- J. Cahill
- J. Karst
- N. Erbilgin
Boreal forest soils support a vast array of fungal communities that play important roles in forest composition and regeneration, plant nutrient uptake, carbon sequestration, and biogeochemical cycles. Lodgepole pine (Pinus contorta var. latifolia), a dominant and economically important forest tree species in Alberta, depends on communities of ectomycorrhizal fungi for successful establishment and survival through increased access to nutrients and water in exchange of photosynthates. These symbioses are critical in forests affected by a variety of tree-killing disturbances where forest regeneration is impacted by negative disturbance-soil fungal feedbacks. While the responses of many specific fungal taxonomic groups to individual biotic and abiotic disturbances have been well characterized, how the cumulative effects of successive events impact overall soil fungal diversity is not known. In this DNA-metabarcoding study, we analyze soil fungal communities in lodgepole pine-dominated forests following several forest disturbances: mountain pine beetle (Dendroctonus ponderosae) outbreak, clearcut harvesting, wildfire, and clearcut harvesting of previously beetle-killed stands. Utilizing two genetic markers, ITS1 and SSU, in combination with next-generation sequencing, Illumina MiSeq, we unravel alpha- and beta-diversity of fungal functional groups within forest stands and among disturbance types, respectively. Results on how individual and cumulative forest disturbances shape patterns in fungal community composition and structure will be discussed under the hypothesis that both community traits will vary with disturbance types as well as single vs. multiple disturbances.
Effects of prescribed burning on wood-decay fungi in the forests of northwest Arkansas
- N. Alshammari
Prescribed burning is a widely used management technique in the forests of Northwest Arkansas, but it is not known to what extent it can affect the biodiversity of wood-decay fungi. The present study was carried out to characterize the different species of wood-decay fungi present in burned (BPR) and unburned (UPR) areas of Pea Ridge National Military Park (PRMP) and an unburned area of Devil’s Den state park (DDP). In order to do this, we extracted genomic DNA from 140 specimens of wood-decay fungi collected from these three study areas. This was done using the Promega DNA isolation kit. The Internal transcribed Spacer (ITS) region of fungal ribosomal DNA was amplified using ITS1 and ITS4 primers and sent for Sanger sequencing after quality checking of amplicons by means of 1% agarose gel electrophoresis. Altogether, 110 out of 140 sequences that passed quality checking were further used for identification of species of fungi by nucleotide BLAST searching against the NCBI database. From all study areas, 61 different species of fungi species were identified, with 30, 23, and 28 different species present on DDP, UPR, and BPR, respectively. Only six species were common between the two forests areas (PRMP and DDP) and only four between BPR and UPR of PRMP, indicating that an appreciable difference appears to exist for burned and unburned areas. The relative abundance of Stereum ostrea voucher She2067 was highest in BPR (24%) as compared to the other study areas (UPR, 17% and DDP, 3%). The present study is ongoing and will be continued during the 2018 field season.
Fire and alternate ecosystem states of tallgrass prairie: how do soil fungal communities respond?
- L. Mino
- A. Jumpponen
- L. Zeglin
- K. Ismert
- C. Reazin
The encroachment of woody species is among the greatest threats to tallgrass prairie ecosystems. Management choices including the suppression of fire facilitate a transition from grassland to shrub- and/or woodland, resulting in a shift to an alternate ecosystem state. The woody encroached state affects ecosystem productivity and biodiversity, renders pastureland less suitable for grazing, and alters key ecosystem functions above and below ground. Frequent, recurring fire acts as an attractor for the non-encroached grassland state, but restoration of woody encroached states using fire have been unsuccessful. Soil fungi are critical in determining plant community structure; however, little research exists on potential differences between soil microbial communities associated with these two alternate ecosystem states. To improve our understanding of soil microbial community composition and dynamics in response to fire, we dissected fungal and bacterial communities before and after introduction of fire in both encroached and non-encroached states in a tallgrass prairie ecosystem and describe soil microbial responses on a high temporal resolution scale. We characterize fungal and bacterial abundance and community composition using qPCR and Illumina MiSeq (16S and ITS), as well as their functional responses using extracellular enzyme assays and soil microbial respiration. These data are amended with responses in nutrient dynamics including total concentrations of carbon, nitrogen and phosphorus as well as pH and inorganic nitrogen. Mycorrhizae and other symbiotic microbes have the potential to hinder or facilitate the rapid transition of grasslands to woodland ecosystem states. Understanding compositional and functional responses of these communities to fire likely offers insights into conservation of remaining grasslands and restoration of encroached woodlands.
Western Ghats of India: A cradle of fungal consortium
- R. Dubey
India is one of the countries blessed with rich biodiversity. The Western Ghats and Eastern Himalaya are a treasure trove of biological diversity in India. The Western Ghats are one of the eight ‘hottest hot spots’ of biodiversity and is known to possess, the most diverse, weird habitats of the globe and a high levels of endemism in respect to plant, animal and microbial groups. About one-third of the world mycota so far described is known from India. About 5% of Indian fungi are endemic to Western ghats. Therefore, such bio diverse regions need to be protected & regulated in a manner to conserve its uniqueness. The mycoflora and their dynamics consequently became a subject of interesting study in this area and therefore systematic study on taxonomic diversity of micro fungi was undertaken with an objective to explore and characterise the diversity of Floristic microfungi from Western Ghats of India. The Fungi were diagnosed down to species level based on conventional parameters, detailed microscopic features and SEM studies . The present study is the result of the 19 extensive and systematic field fungal sample collection trips made to different geographical areas of Western Ghats of India such as grassland plateaus, deciduous forest, semi-deciduous forests, moist deciduous forests, semi-evergreen forests, subtropical hill forests, scrub jungles and cultivated plantations during the period from 2010 to 2018. This multipronged effort resulted in the collection of 2025 diseased samples with identification of 744 isolates of micro fungi, which were assignable to 267 fungal genera, 523 species and 04 varieties, infecting 342 sp. of host plants. All the fungi documented during the studies are grouped under Phyla- Ascomycota and Basidiomycota. The present study area forms the type locality of two new genera Sheathnema indicum Dubey and Moonnambeth, 2014 and Sawantomyces indica Dubey and Moonnambeth, 2013; eleven new species Custingophora ratnagiriensis Dubey & Moonambeth, 2013; Goosiomyces bambusicola Dubey & Moonambeth 2014; Kamalomyces mahabaleshwarensis Dubey & Moonambeth, 2013; Periconia chandoliensis Dubey, 2017; Solicorynespora matheransis Dubey & Moonambeth, 2014; Sporidesmium biligiriensis 2015; Stigmina koyanensis Dubey & Sengupta, 2016; Tharoopama livistonae Dubey & Moonambeth, 2013; Tripospermum melghatensis Dubey and Sengupta, 2016, Vermiculariopsiella papaya Dubey & Moonnambeth 2014 ; Zygosporium cocos Dubey, 2014 and Zygosporium dilleni Dubey, 2014. In addition to this 39 fungal taxa were new additions to Fungi of India and 121 fungal taxa were found to be new to Western Ghats. Some fungi was encountered after a period of 35 years or more viz. Conidiocarpus betle T. Bose, Asterina woodfordiae V.P. Sahni, Cercospora blumeicola S. Das; Cercospora careyae T. S. & K. Ramakrishnan, Meliola diospyri Yates Syd. & P. Syd, from India. Helicomina costi M.A. Salam & P.N. Rao was recorded after a period of 65 years from India. 71 % of the total fungal isolates forms new host records from India. In conclusion, this research investigation presents an overview of fungal diversity existing in the Western Ghats of India and also made here to unravel the cryptic fungal consortium of this region.
Comparative study of wood rotting Fungi from two different forests in Mizoram,India
- J. Vabeikhokhei
- J. Zothanzama
- Z. Chawngthu
- L. Hmar
A three years investigation was carried out to study the diversity of wood rotting fungi from two different forest stands, Hmuifang forest and Tanhril forest of Mizoram, northeast India. A total of 45 species were identified from both the study sites. It was observed that a total of 21 species were common to both the forests whereas 19 species were found only found in the Hmuifang forest and 5 species only in the Tanhril forest. Auricularia auricula-judae, A. polytricha, Coprinus dessimentus, Cyathus sp., Daldinia concentrica, Fistulina hepatica, Hexagonia tenuis, Lentinus badius, Marasmius sp., Microporus affinis, M. xanthopus, Mycena sp., Schizophyllum commune, Stereum hirsutum, S. rugosum, Tremella fuciformis, T. mesenterica, Trametes hirsutum, T. trogii, Xylaria hypoxylon, X. longipes are the species/genera common to both study sites. Microporus xanthopus represents the most abundant species in both the study sites.
Brazilian fungal diversity represented by DNA markers generated over 20 years
- N. Menolli Jr
- M. Sánchez-García
The relatively recent molecular techniques using fungal DNA barcoding (ITS) and other markers have been key to identifying and exploring the biodiversity of different geographic areas, and show even more useful to explore megadiverse countries. Here, we provide an overview of the fungal diversity in Brazil based on the main DNA markers of phylogenetic importance generated since 1998, when the first ITS sequence from a Brazilian sample was submitted to GenBank. We retrieved fungal sequences of ITS, nLSU, nSSU, tef1, β-tubulin, RPB1, RPB2, actin, chitin synthase, and ATP6 from GenBank using different field keywords that indicated their origin in Brazil. A Maximum Likelihood phylogeny based on LSU illustrates the main Brazilian taxa grouped in orders. We obtained a total of 19,564 sequences. ITS is the most representative marker with 57.2%, followed by LSU (14.6%), tef1 (11.5%), β-tubulin (8.7%), RPB2 (3.1%), SSU (2.5%), RPB1 (1.2%), actin (0.7%), chitin synthase (0.3%), and ATP6 (0.2%). Based on all the sequences, there are representatives of Ascomycota (48 orders), Basidiomycota (26 orders), Blastocladiomycota (Allomyces arbusculus), Chytridiomycota (4 orders), Microsporidia (3 spp.), Mucoromycota (7 orders), Zoopagomycota (3 orders), and the incertae sedis taxon Olpidium bornovanus. Among the 11,187 ITS sequences, 70.1% are samples of Ascomycota, 18.6% Basidiomycota, 10.2% unclassified taxa, 1.1% Mucoromycota, 2 sequences of O. bornovanus, 1 sequence of Blastocladiomycota, and 1 sequence of Chytridiomycota (Batrachochytrium dendrobatidis). Based on ITS using a cut-off of 98%, all the fungal sequences comprise 3,036 OTUs, Ascomycota 2,088 OTUs, Basidiomycota 670 OTUs, and Mucoromycota 69 OTUs. Hypocrelales is the order of Ascomycota with more sequences retrieved (1,286 ITS seq.) and also the most diverse order (263 OUTs). In Basidiomycota, Pucciniales has the largest number of ITS sequences (615 seq.) but they represent only 9 OTUs. Agaricales is the most diverse (202 OTUs) and the second most sampled order in Basidiomycota (437 ITS seq.). Among the most sampled genera in Ascomycota (> 50 ITS seq.), the following are the most diverse: Phyllosticta (467 seq., 109 OTUs), Penicillium (199 seq., 95 OTUs), Diaporthe (257 seq., 90 OTUs), Candida (536 seq., 83 OTUs), Fusarium (449 seq., 80 OTUs), Colletotrichum (969 seq., 76 OTUs), Phomopsis (149 seq., 69 OTUs), Aspergillus (204 seq., 63 OTUs), Xylaria (95 seq., 52 OTUs), and Trichoderma (289 seq., 44 OTUs). In Basidiomycota, although Phakopsora (444 seq.) and Puccinia (168 seq.) are the most sampled genera for ITS, their diversity represents only 3 and 4 OUTs, respectively. Rhizoctonia (90 seq., 38 OTUs), Pluteus (56 seq., 29 OTUs) and Cora (70 seq., 24 OTUs) are the most diverse genera among the most sampled Basidiomycota for ITS. Previous data based mainly on morphological studies and bibliographical records pointed out 5,264 fungal species recorded from Brazil with predominance of Basidiomycota (2,741 spp.) and Ascomycota (1,881 spp.). The discrepancy of that number of species and the OTUs found in this study suggests that Basidiomycota has been well studied with regard to morphological diversity, while Ascomycota has been better investigated for their molecular composition and mainly for taxa of clinical and agronomic importance.
Insight into the diversity of soil fungi in Changbai Mountain by high-throughput sequencing
- G. Lyu
- D. Shi
- Z. Zhao
- H. Yang
- D. Su
- X. Sun
- F. Guan
- X. Shang
AbstractSoil fungi are an important group of microorganisms in forest ecosystem, they play significant roles in cycling of organic compounds and can affect the underground and upground ecosystems. In contrast to soil bacteria, soil fungi have been poorly investigated and understood in forest ecosytem. The fast development of molecular technologies offers a powerful method to access more functional information on soil fungal diversity. We applied the technique of Illumina Miseq High-Throughput Sequencing to investigate the soil fungal diversity and community structures in the northern slope of Changbai Mountain, Jiling Province, China, which is characterized with an evident vertical vegetation distribution pattern along the altitude. The metagenome sequence analysis was conducted by targeting ITS1f-ITS2 fragments for 80 soil samples collected in the four characteristic forest vegetation belts ranging from the root of 700m to the top of 2600m in altitude, it shows a huge abundance of soil fungi in Changbai Mountain forest. Totally 2,294,552 rDNA fragments of reads are grouped into 25,282 operational taxonomic units (OUTs), they are ascribed to 1056 species, 622 genera, 195 families, 87 orders, 24 classes and 5 phyla of fungi. Among which 182 genera are of Basidiomycota (48.72%), 411 genera of Ascomycota (31.67%), 13 genera of Zygomycota (10.21%), 13 genera of Chytridiomycota (0.27%), 3 genera of Glomeromycota (0.04%), and the left 9.09% are unknown taxa. The species of Basidiomycota are the predominant occupiers in the forest soil of the mountain, especially the genera of Laccaria (6.17%), Inocybe (5.54%), Hygrocybe (3.06%), and Russula (2.37%) of Agricales are found to be rich. While the genera of Mortierella (6.73%) and Inocybe are the most widely distributed in all sampling sites in the mountain. The soil fungal richness evidently tends to decrease from the root to the top of the mountain, and the fungal compositions vary in the four characteristic vegetation belts of the mountain. The novel profile of soil fungi in the mountain uncovered by means of metagenome technique could not be paralled by conventional culture-based fungal research methods.
Fungal and bacterial diversity in soils beneath native and introduced plants in Fiji, South Pacific
- G. Bonito
- G. Benucci
- V. Bonito
The Fiji Islands are an archipelago of more than 330 islands located in the tropics of the South Pacific Ocean, and offer a unique opportunity for the study of fungal and microbial biogeography and dispersal. Here we present the first molecular characterization of fungal and bacterial communities in soils from different habitats within the largest Fijian island, Viti Levu. Soil samples were collected from under native vegetation in maritime, forest, stream, grassland, and casuarina dominated habitats, as well as from agricultural sites of sugarcane, cassava, pine, and mahogany cultivation. Fungal and bacterial communities were analyzed using high-throughput MiSeq amplicon sequencing of ITS, LSU and 16S rRNA genes. We found lower richness of fungi and bacteria under single tree species habitats than under native forest and grassland habitats. ITS and LSU were congruent in β-diversity patterns. Fungal communities were dominated by Ascomycota (~57-64 % of relative abundance), followed by Basidiomycota (~20-23%) and Mucoromycota (~10%) according ITS region, or Chytridiomycota (~9%) according LSU region. Indicator species analysis found Cenococcum, Wilcoxina and Rhizopogon statistically associated to Pinus caribaea, and were likely co-introduced with the host. Entoloma was statistically associated with the grassland soils, and Fusarium and Lecythophora with soils under cassava. Bacterial communities were dominated by Proteobacteria (~25%), Acidobacteria (~19%) and Actinobacteria (~17%). Observed richness varied from 65 (Casuarina) to 404 OTUs (cassava) for Fungi according ITS region, and from 1268 (Pinus) to 2931 OTUs (cassava) for Bacteria and Archaea. This preliminary survey provides important baseline data on fungal and bacterial diversity and biogeography in the Fiji Islands.
The mycobiome of Karee Malformation Disease symptoms on Searsia lancea (karee) trees in South Africa
- M. Mqeku
- E. Casson
- R. Tonjock Kinge
- M. Gryzenhout
Searsia lancea (karee) is a common native tree in South Africa. A new disease called Karee Malformation Disease (KMD) consists of malformations of mostly vegetative and floral tissues. The cause of these malformations is still unknown. Using Illumina-based environmental sequencing, the aim of this study was to compare the mycobiomes found in diseased and healthy floral and vegetative tissues of Searsia lancea. Previous studies indicated that the two communities differed vastly in number and diversity of species. Minibarcodes using the ITS regions of the ribosomal operon confirmed results from previous studies. The notable differences found between the healthy and malformed tissues confirmed a succession of the fungal communities. As consistent with the latent pathogenic life cycle of many pathogens, genera such as Alternaria, Botryosphaeria and Valsa were present in all tissues but no dominant fungal group that could be the cause of the disease was detected. It is clear that the malformations greatly changed the fungal communities that would normally be present in an unaffected tree and such disease symptoms thus present a niche of their own within the tree.
Yeast diversity in Neotropical Savannah soils of the Quadrilátero Ferrífero, Minas Gerais, Brazil
- G. Moreira
- H. Vale
The Quadrilátero Ferrífero occupies an approximate area of 7000 km2 in the central-southeast portion of the State of Minas Gerais and is considered one of the regions of greater floristic diversity of South America inserted in the transition zone of the two Brazilian hotspots: Atlantic Forest and Neotropical Savannah. This region is recognized for its 'special biological importance' due to the occurrence of phytophysiognomies with plant species restricted to the region, as a consequence of the peculiar characteristics of soils that are ferruginous, acidic and of low fertility. It is a unique environment in the state with a great diversity of microorganisms still unexplored, mainly regarding the diversity of fungi in the soil. Yeasts are single-celled fungi and in the soil participate in important ecological processes. The objective of this work was to describe the yeast diversity and to compare the composition of the communities in ecosystems of the Quadrilátero Ferrífero under different seasonal seasons. The yeast diversity was analyzed in a total of 40 soil samples from two ecosystems (Cerrado “latu senso” and Campo Rupestre) in two seasonal seasons (dry and rainy). Soil samples were characterized by their physical and chemical properties and were grouped by Principal Component Analysis (PCA). Yeast diversity was assessed by culture technique and isolated species were identified by sequencing the D1/D2 region of the 26S rRNA gene. A total of 64 yeast isolates were recovered and identified in 20 species belonging to 10 genera. The Ascomycota Phylum (75%) predominated over the Filo Basidiomycota (25%). Candida melibiosica, Meyerozyma guilliermondii and Cryptococcus laurentii were the dominant species. In the Cerrado area, only one species was shared among the evaluated seasons, five were detected only in the dry season and only one species in the rainy season. In the Campo Rupestre area, two species were detected in both seasons, five only in the dry season and four species only in the rainy season. Four species were shared between the two analyzed ecosystems. The environmental variables explained 76% of the data variation, grouping the samples by ecosystem, but there was no separation between seasonal season. The soil texture was positively correlated with the Campo Rupestre samples (dry and rainy), while the organic matter content and soil acidity were correlated with the Cerrado (dry and rainy) soil samples. In conclusion, the ecosystems analyzed did not show differences in soil attributes, but showed differences in the yeast diversity between the areas and between the seasonal seasons.
Preliminary study of macrofungal diversity in three Private Reserves of San Pedro (Misiones province, Argentina)
- N. Ramírez
- D. Lining
- N. Niveiro
- A. Michlig
- O. Popoff
The Atlantic Forest is an ecoregions complex that extends from the northeast of Brazil, by the coastal mountain ranges to Rio Grande do Sul. In the south, it enters to the east of Paraguay and northeast of Argentina, forming the Interior Atlantic Forest or the Paranaense forest. Although it occupies less than 1% of the planet's surface, it contains 7% of the known species on earth. “Juntos por la Selva” is an initiative of a small group of people in order to protect a fragment of the Paranaense forest. This is how, in 2011, the Itaovy, La Coral, and Yacutoro Private Reserves were created. They are located in Argentina, in the center of Misiones Province, in the Guaraní Department, just 15 km from San Pedro city. They conserve about 500 Ha of forest with an important number of plants and animals species. In order to document the macrofungi within these protected areas, 2 samplings were performed during the 2016 - 2017 period. A total of 190 samples were collected in different environments of the reserves. The material was dried, kept in freezer for a week, and deposited as reference in the CTES herbarium. The morphological analysis was made based on observations of macroscopic and microscopical characters with stereoscopic and optical microscopes. The species found were identified consulting specific literature. The Basidiomycota was the most diverse group and from which a larger number of specimens was obtained, representing 93% of the collections, belonging most of them to the Agaricales, Polyporales, and Hymenochaetales. The remaining species belong to Ascomycota and Myxomycota, representing 7% of the fungi of the reserve. The presence of common and widely distributed genera, such as Schyzophyllum, Fuscoporia, Pycnoporus, Trametes, Hexagonia, Marasmius, Mycena, was observed, as well as rare species that have been registered only few times (e.g. Ascopolyporus polychrous, Marasmiellus volvatus) and others that has been cited only in the original description (e.g. Mycena moconensis). A list of species is presented, with comments about its habitat and distribution, together with a catalog of photographs to illustrate the species. It is expected to move forward with the determination of the collected species and to continue with the samplings at favorable seasons for the development of basidiomata in order to achieve a more complte inventory. It is highlighted that although the reserves harbor a restricted fragment of forest, they protect a high diversity of fungi, being an important region for its conservation.
Revisiting the secotioid and gasteroid Cortinarius species from Patagonia
- E. Nouhra
- F. Kuhar
- C. Truong
- A. Mujic
- R. Healy
- M. Smith
The diversity of secotioid taxa within Cortinarius in the Nothofagaceae forests of Patagonia has drawn attention of mycologists during the last century. In the Patagonian region of Argentina and Chile Cortinarius is among the most diverse and abundant genera of ectomycorrhizal fungi with at least 240 species from the Andean mountains. Secotioid and gasteroid forms were until recently considered primarily within Thaxterogaster, resulting in a confusing intrageneric classification system. Moser and Horak suggested that Thaxterogaster was nested within Cortinarius. The modern molecular analysis of Peintner et al. investigated the multiple origins of sequestrate taxa related to Cortinarius and consequently synonymized Thaxterogaster to Cortinarius. Subsequent molecular phylogenies have resolved the polyphyletic nature of Thaxterogaster and other “cortinarioid” taxa within Cortinarius but have also highlighted the fact that most sequestrate Patagonian taxa lack molecular data. Original descriptions of these fungi are available mostly in German and Spanish and the interpretations of morphological structures are outdated considering the current state of knowledge about spore morphology and ontogeny. For example, verrucae on spores were illustrated as globose structures whereas SEM shows that they are complex conical structures that are sometimes interconnected by reticula or sub-reticula. External walls or episporia were sometimes pictured in original descriptions but our analyses suggest that these may have been optical illusions due to non-DIC microscopy. Recently, the incorrect interpretation of this episporium in the “cortinariod” fungi was found to be a misleading character. Despite recent advances in Cortinarius systematics, the current classification, diversity and ecology of secotioid and hypogeous “cortinarioid” fungi in the Nothofagaceae forests of southern South America remains unclear. The objective of this study is to update descriptions with diagnostic characters, including color photos of basidiomata, SEM images of spores, and ITS sequence data to clarify the biodiversity of these fungi in Patagonia. Original descriptions of secotioid and gasteroid taxa were also revised and translated to English. Our analyses based on SEM and ITS rDNA resolves at least 15 species with names that need to be considered as synonyms. The use of these tools combined with an extensive database of described species also facilitated the recognition of several new and undescribed Patagonian species. Analysis of spore ultrastructure across many specimens clearly shows that sequestrate species of Cortinarius always lack a perisporium. It also indicates that there is a transition process in shape and ornamentation that occurs in taxa as they switch from ballistosporic to statimosporic spore dispersal.
Study on tropical and subtropical Marasmius: Four new species of the sect. Spinulossi
- N. Niveiro
- N. Ramírez
- A. Michlig
- E. Albertó
- V. Antonín
Marasmius is a large genus with worldwide distribution with more than 500 species, which are characterized by their generally small to medium-sized basidiomata, often membranaceous pileus, and for the ability to be revivescent when are rehydrated. This last feature makes them tolerate conditions of seasonal drought or high temperatures, what allows them to be diverse and abundant in tropical and subtropical forests. Marasmius comprises many sections and subsections (e.g. Globulares, Marasmius, Neosessiles, Sicci,). However, recently studies tested the monophyly of the sections traditionally proposed by Singer and confirm that they are highly homoplasic. A small group of species of Marasmius, characterized by having setae in the pileus and stipe surface, and even in the hymenophore (e.g. M. actinopus, M. jalapensis, M. coharens), belong to section Spinulosi. The species of Marasmius with setae are not common, being better known from southeastern Asia and Neotropical region. The aim of this study is to propose four new species and to present a worldwide key of Marasmius sect. Spinulosi. We studied specimens identified as M. jalapensis, M. spiculosus, M. echinulatus, and M. flammans deposited in CTES, K, LIL, NY and XAL mycological collections, including type specimens. For microscopic characters, a light microscopy (LM) and a scanning electron microscopy (SEM) were used. We discovered two species never described before from northern Argentina and we segregated other two species from the M. jalapensis concept based on type material analyses. Marasmius sp. 1, resembling M. chrysoblepharis, is characterized by its yellowish-orange pileus, with a sulcate-striate margin, entirely pilose orange-brown stipe, setiform caulocystidia with a tapering and thick-walled apex and bacilliform to fusiform large spores. Marasmius sp. 2 has characters between M. trichotus and M. ciliatus, but differs in its large setiform cystidia on the pileus and stipe surface, the absence of broom cells in the stipitipellis and spores size. Both species are collected in northern Argentina. Marasmius sp. 3 and Marasmius sp. 4 are segregated from the M. jalapensis concept. Marasmius sp. 3 differs by its narrower spores and two cheilocystidia types and it is distributed in the tropical and subtropical regions of South America. Marasmius sp. 4 is restricted to northern Africa and differs mainly by its smaller spores. Marasmius jalapensis is confined to the mesophilic mountain forests in Mexico. A key of the tropical and subtropical species of Marasmius sect. Spinulosi is presented. In conclusion, based on the morphological, biogeographic and phylogenetic characteristics, we propose these four species as new for science.
Fomes fomentarius lineages throughout the world
- K. Náplavová
- J. Ján Gáper
- S. Gáperová
Currently, the existence of three distinct ITS lineages/sublineages among Fomes fomentarius isolates has been established. The sublineage A1 occurs only in North America, whereas the other two lineages/sublineages have a wider distribution: the sublineage A2 and the lineage B occur in Europe and Asia. This study represents the first description of limited sympatry between the Southern and Northern phylogeographical groups. The line passes through Central-western Europe, Central Europe, and Central-western Asia. A clear correlation was observed between lineage (sublineage) and host range. The North American sublineage A1 follows the geographical distribution of its main hosts: North American birches Betula spp. and Fagus grandifolia. The two Eurasian lineages/sublineages, sublineage A2 and lineage B, have different host species preferences (Acer negundo, Alnus, Betula, and Picea, vs. Abies, Acer platanoides, Aesculus, Platanus, Prunus, Salix, and Tilia). European beech (Fagus sylvatica) is only host of both Eurasian lineages/sublineages.
A revision of the genus Graphis (lichenized ascomycete) in Colombia
- K. Motta
- K. Amortegui
- B. Moncada
- R. Lücking
Graphis is the type genus of the family Graphidaceae. It contains nearly 400 species and is considered the lichen genus with the highest number of species in the Neotropics, and the number of species described within the genus has grown steadily in the last 15 years. Based on the 2015 Catalogue of the Plants and Lichens of Colombia, 67 species of the genus are registered for Colombia, compared to 115 for Costa Rica. Since Colombia is twenty times larger than Costa Rica and has a broad array of ecosystems to harbor a diverse lichen biota, the number of species of Graphis is expected to be substantially higher that in Costa Rica. We studied collections of Graphis deposited in the herbarium of the District University in Bogotá (UDBC-Cryptogamic Section), which harbors about 700 specimens of this genus from different regions of Colombia, in order to obtain a first assessment of the true richness of Graphis in the country. As a result of this study 65 new records of Graphis for the country are presented and six new species are described, increasing the total to 138 species known from Colombia. Additionally, the first key to species of the genusGraphis in Colombia was elaborated, as well as a rapid color guide following the models of the Field Museum, Chicago.
Three new white sporulating species of Aspergillus sect. Terrei
- Z. Jurjevic
- V. Hubka
- F. Sklenář
- S. Peterson
The objective of our study was to perform morphological and molecular analyses on a number of white sporulating Aspergillus strains from section Terrei in order to confirm any undescribed species. The strains were isolated from clinical material, soil and from the built environment (residential home, hospital) by active air and swab sampling. Cultures were grown on Czapek’s yeast autolysate agar (CYA) (25 °C and 37 °C), malt extract agar (MEA), potato dextrose agar (PDA), Czapek’s agar with 20% sucrose (CY20S), dichloran 18% glycerol agar (DG18), and oatmeal agar (OA) (25 °C) for 7 days in darkness. Cultures were then described by morphological, physiological (maximum growth temperature) and microscopic analysis followed by multilocus DNA sequencing of four unlinked genetic loci. Based on morphological analysis and molecular confirmation, the unknown isolates were described as new species in the Aspergillus section Terrei. Historically, members of Aspergillus sect. Terrei have been isolated from soil, indoor environments, various food and feeds, and have been associated with many health issues of humans and animals. Also, some species have been known to produce a wide range of exometabolites. Future studies are required to determine any pathogenic roles of these new species and exometabolite production.
Current status of the genus Entoloma in Ecuador based on the QCAM Fungarium collections
- A. Barili
- M. Ordoñez
The genus Entoloma is very rich and complex, with close to 2,500 described species divided in 36 sections, subgenera and subsections. A megadiverse country like Ecuador potentially harbors a large number of Entoloma species. The QCAM Fungarium at Pontifical Catholic University of Ecuador in Quito currently houses over 7400 fungal specimens, a number that has almost tripled in the past six years. It is the largest macrofungi collection of Ecuador, and therefore, where most information related to the genus status can be found. Of the 76 Entoloma collections made since 1983, 35 specimens have been identified to 12 different species and 8 aff. open nomenclature designations. Forty-one specimens have yet to be identified to species. Some important findings include the holotype of Alboleptonia sulcata T.J. Baroni & Lodge (1998), currently Entoloma sulcatum, and six new species. The new species were found after morphological and molecular analyses of a limited number of specimens chosen at random from the QCAM collection in 2017. These new species are listed here with tentative names until they are formally described and published: E. yanaumense, E. astroasprellum, and E. yanacolor that belong to the subgenus Leptonia (Fr.) Noordel, section Cyanula (Romagn.) Noordel; Entoloma squamosum within the subgenus Trichopilus (Romagn.) Noordel; Entoloma crinipellis, a very close species to Pouzarella ferreri T.J. Baroni, Perd.-Sánch. & S.A. Cantrell (2008), and E. umbellatum which belongs to the subgenus Inocephalus, section Staurospora Largent & Thiers (1972). Almost all Entoloma species in the QCAM collection are represented by only one specimen, except E. austroasprellum with two collections, and E. serrulatum, with three. This limited amount of data shows not only the high diversity of the genus in Ecuador, but also the need to devote specific studies to catalogue and discover new species of Entoloma in tropical and subtropical areas.
The Rhodocybe/Clitopilus clade (Entolomataceae, Agaricomyetes) in the Dominican Republic: a new genus, new species and first reports for Hispaniola
- T. Baroni
- C. Angelini
- S. Bergemann
- D. Lodge
- S. Cantrell
- T. Curtis
AbstractThis report serves as the first ever to outline the diversity of the Rhodocybe/Clitopilus clade for the Dominican Republic. Based on collections made during several expeditions exploring for basidiomycete macrofungi over a span of 20+ years, we now have a reasonable understanding of the biodiversity of these saprotrophic fungi in the Dominican Republic. Diverse ecological habitats were sampled during these studies, ranging from tropical seashore vegetation to the endemic pine covered high peaks in the central mountainous regions. Based on morphological and phylogenetic analyses, we document one new genus, three new species, one new variety, one new combination and four first reports for members of the Rhodocybe/Clitopilus clade on the island of Hispaniola. Images of the new taxa and the three gene phylogenetic data to support these taxonomic conclusions will be presented. A broader biogeographic analysis of the Rhodocybe/Clitopilus clade around the Caribbean region will also be considered.
Use of hyphal image analysis and machine learning to classify Mucoromycota soil fungal isolates
- J. Liber
- E. Gall
- N. Vande Pol
- D. Silvia
- G. Bonito
Fungi belonging to the Mucoromycota are abundant in soil communities globally; these fungi are also important industrially for the production of lipids, and some isolates are known to harbor endobacteria. Isolation and identification of fungal strains from soil is an intensive process involving culturing, DNA extraction, PCR, and sequencing. Classification of filamentous fungi is typically dependent on reproductive structures; however, reproductive structures are not always present when isolates are grown in culture. Image classification of hyphae using machine learning algorithms offers a method to streamline prospecting for novel fungal strains. Micrographs of hyphae obtained while isolates were growing in Petri dishes were used to avoid additional sample preparation. We wrote a Python 3.6 script, in which images were converted to grayscale, then fast Fourier transformation was applied to detect distinctive patterns in hyphae. Taxonomic labels were assigned to images based on ITS sequences. A Random Forest Classifier object from the Scikit-learn library was trained using a subset of images and validated on a separate set. Images of Mucoromycota strains were identified with a weighted F-score exceeding 92%. Our method can effectively be used to classify fungal isolates using only hyphal imagery for accelerated identification. Future research will include improving accuracy and specificity to a wider range of taxonomic ranks and diversity. Image classification is a promising tool to aid in the prospecting fungal strains by helping to reduce the number of samples requiring sequencing and intensive culturing.
Updating the taxonomy of the genera Aspergillus and Penicillium in South Africa
- C. Visagie
South Africa is a well-known biodiversity hotspot with high endemicity. This is also true for Penicillium, with 29 of 61 isolated species described as new from a 2012 survey from the fynbos biome in the Western Cape. Much of our local knowledge are solely based on morphology. However, morphological interpretations are very difficult, unreliable and inconsistent considering the large number of accepted species (378 Aspergillus and 427 Penicillium). This makes morphological identifications impossible for many species. Recent taxonomic changes, combined with their diverse nature and economic importance necessitates the modernization and updating of local Aspergillus and Penicillium knowledge. The PPRI collection of the National Collection of Fungi has ~1000 accessions of Aspergillus and Penicillium. These represents the best resource for obtaining base line knowledge on what species occur in the country. Additional strains are also regularly isolated during diagnostic work done at the ARC-Plant Health and Protection. This project has the goal of (1) placing PPRI strains into morphogroups and then (2) sequencing the secondary identification markers for representatives from each group. Sequences will be compared to a curated database and identifications updated. For new/rare species, additional genes will be sequenced (ITS, BenA, CaM and RPB2). This presentation will be focused on recent developments in the taxonomy of Aspergillus and Penicillium, and how we can apply taxonomy to benefit the scientific community with special focus on future identifications based on culture dependent and independent techniques.
Arthropod infecting fungi in the genus Cordyceps sensu lato (Hvpocreales, Ascomycota) and related genera from Australian tropical rainforests
- S. Abell
- T. Marney
- Y. Tan
- E. Aitken
- R. Shivas
Our collections have shown that Australian tropical rainforests are rich in arthropod infecting fungi, especially on ants, flies, spiders and scales. The aim of our Australian Biological Resources Study project was to systematically catalogue the Australian arthropod infecting fungi in the genus Cordyceps sensu lato (Hypocreales, Ascomycota) and related genera, from tropical and sub-tropical rainforests. Specifically, we investigated the systematics of Australian Hypocrella (Clavicipitaceae) on scale insects; Cordyceps sensu stricto (Cordycipitaceae) on ants and flies; Ophiocordyceps (Ophiocordycipitaceae) on ants; and Akanthomyces, Gibellula and Hirsutella that are mostly spider pathogens. Specimens held in Australian mycological herbaria were examined alongside collections made during field trips in the Wet Tropics World Heritage Area (WTWHA) and Cape York Peninsula over a three year period, 2014 - 2016. We revised the taxonomy based on multigene (ITS, SSU and LSU) DNA sequence phylogenies, fungal morphology and arthropod/host plant associations. We yielded 991 specimens of entomopathogenic fungi, including 337 living cultures. Of these DNA sequence data has been obtained from 138 specimens. Those specimens represented 112 species in 35 genera. The genera most commonly encountered were, Akanthomyces, Aschersonia, Beauveria, Gibellula, Hirsutella, Hymenostilbe, Isaria, Lecanicillium, Metarhizium, Ophiocordyceps, Paeciliomyces and Torrubiella. Many of the species identified in these genera either represent species complexes and/or novel taxa that have not been recorded previously in Australia. One new genus, Hyweljonesia, and four new species H. queenslandica, Ophiocordyceps norreniae, O. dawkinsii and O. oecophyllae have been described so far. Fact sheets including high resolution images and descriptions have been completed for 40 target species. Collectively, this information will form the basis for an online, interactive key of Australian entomopathogenic fungi using Lucid software that is currently under development. Taxonomic knowledge about Australian arthropod infecting fungi has increased substantially from this project. This will underpin future systematics and ecological studies on plant-arthropod-fungi interactions, e.g. the role of fungi in insect morbidity in rainforests, as well as the potential of arthropod infecting fungi as biocontrol agents for agricultural insect pests.
Molecular identification of Aureobasidium and Rhodotorula from spore trap air samples
- J. McLoud
- E. Levetin
Aureobasidium and Rhodotorula are both allergenic yeasts that have been previously identified in viable air samples; however, the daily exposures to these aeroallergens are not known. Though viable sampling is a reliable method to distinguish which yeasts are airborne and the immediate concentrations, the samples are collected for a short period of time (1-2 minutes). Consequently, the daily exposure to airborne Aureobasidium and Rhodotorula are not known, because yeasts are not morphologically distinct in nonviable (24 hour) spore trap air samples. The objective of our study is to determine the frequency and concentrations of Aureobasidium and Rhodotorula in daily air samples by using genus-specific DNA targets for molecular identification. Air samples were collected at The University of Tulsa (Tulsa, Oklahoma) on the roof of a building, 12 m above the ground, using a Burkard 7-day nonviable spore trap sampler (Burkard Manufacturing, Co. Ltd., Rickmansworth, Hertfordshire, England). Samples were collected from 22 July to 22 November 2017. DNA was extracted from daily samples and quantified with genus-specific TaqMan assays. The Aureobasidium assay hybridized to a region of the fatty acid elongase gene and the Rhodotorula assay hybridized to the ITS1 region of the ribosomal RNA operon. The daily concentrations (cells per cubic meter) of Aureobasidium and Rhodotorula were calculated using a standard curve of known yeast cell concentrations. Real-Time PCR indicated the frequency of Aureobasidium was 70% and Rhodotorula was 46%. Aureobasidium had a higher daily concentration when compared to Rhodotorula with maximum concentrations of 25 cells/m3 and 7 cells/m3 respectively. In conclusion, the daily exposure to allergenic Aureobasidium and Rhodotorula occur frequently, but at low daily concentrations. More sample collection is warranted before annual trends or predictive meteorological variables may be identified.
Molecular approach to clarify taxonomy and ecology of Puccinia species on Gramineae and Cyperaceae in Jilin, China
- J. Ji
- Z. Li
- Y. Li
- M. Kakishima
Jilin Province is located in the northeast of China and geographically divided into three main areas: an eastern mountainous area, a western dry plain area and the rolling hilly area between them. The natural vegetation consists of prairie grasses in the western plains and broad-leaved deciduous forests mainly consisting of species of Betula, Populus and Quercus in the hilly and mountainous areas. These areas are rich in vegetation and other suitable environmental conditions to predict a proliferation of rust fungi, especially in the Changbai mountain ranges located at the border of North Korea that harbor conserved natural forests. However, the inventory and ecology of rust fungi have not been sufficiently investigated in Jilin Province. Therefore, we surveyed rust fungi in several locations in Jilin Province from 2013 to 2017 and collected about 1000 specimens. Among them, about 300 specimens were Puccinia species on Gramineae and Cyperaceae which were dominantly growing in Jilin Province. We also collected about 50 specimens which are hypothesized to represent the spermogonial and aecial stages of these Puccinia species. However, identifications of these specimens are very difficult because of morphological similarity among specimens and difficulties of host identification. Although many are suspected to be heteromacrocyclic species the connections among different stages (spermogonial, aecial, uredinial and telial stages) cannot be confirmed because of difficulties of inoculation experiments and also difficulties of obtaining plants from conservation areas. For resolving these problems molecular analyses were applied. ITS and 28S regions of rDNA from specimens were amplified and sequenced. After constructing phylogenetic trees by the ML and BS methods about 35 clades were detected. These clades were suspected as species based on morphological observations and host relations. Species of these clades were identified and life cycle connections among stages were clarified based on the species reported in China although many cryptic species were found among these clades.
Cryptic speciation within the Puccinia hieracii and P. calcitrapae species complexes
- L. George
- N. Stringer
- J. Roden
- A. Chater
- D. Evans
- M. Woodcock
- S. Tvedt
- G. Griffith
There has been an long-standing inconsistency within the scientific literature regarding the nomenclature and taxonomic identification of the autoecious macrocyclic rust fungus species Puccinia hieracii s.l. and Puccinia calcitrapae s.l. within the order Pucciniales. Some concepts of the species consider that this species infects hosts within two distinct sub-tribes of Asteraceae, Cichoriae and Cardueae, whereas others separate the complex into several distinct host specific taxa. The aim of this study was to undertake phylogenetic and morphological analyses of representatives of the various taxa in order to generate a consistent taxonomic arrangement. DNA barcoding of the ITS2 and LSU (28S; V1 and V2 domains) of the rRNA locus was undertaken from specimens obtained from diverse hosts across the UK. The rusts present on hosts belonging to different genera (Hieracium, Leontodon, Scorzoneroides and Taraxacum) were genetically distinct from each other. However, quantitative morphological analyses using both light and scanning electron microscopy could only distinguish a few of these taxa based on features of urediniospores and teliospores, for instance spine density and the presence of a spineless tonsure region. These analyses also identified a new clade of P. hieracii infecting Scorzonderoides autumnalis and also a distinct variety of P. jaceae infecting Centaurea nigra.
Molecular bio-markers and phenotypic characterization as a means of determining genetic diversity within Aspergillus flavus isolates
- M. Rafudeen
- S. Okoth
- A. Mitema
- S. Rafudeen
Toxigenic Aspergillus species produce mycotoxins that are carcinogenic, hepatotoxic and teratogenic immunosuppressing agents in both human and animals. Kenya frequently experiences outbreaks of aflatoxicosis with the worst occurring in 2004, which resulted in 215 deaths. We examined the possible reasons for these frequent aflatoxicosis outbreaks in Kenya by studying A. flavus diversity, phenotypes and mycotoxin profiles across various agricultural regions. Using diagonal transect random sampling, maize kernels were collected from Makueni, Homa Bay, Nandi, and Kisumu counties. Out of 37 isolates, nitrate non-utilizing auxotrophs complementation test revealed 20 vegetative compatibility groups. We designated these groups by the prefix ʻʻKVCGʼʼ, where ʻʻKʼʼ represented Kenya and consequently assigned numbers 1 to 20 based on our findings. KVCG14 and KVCG15 had highest distribution frequency (n = 13; 10.8 %). The distribution of the L, S and S/L-morphotypes across the regions were 57 % (n = 21); 7 % (n = 3) and 36 % (n = 13) respectively. Furthermore, a unique isolate (KSM015) was identified that had characteristics of S-morphotype, but produced both aflatoxins B and G. Coconut agar medium (CAM) assay, TLC and HPLC analyses confirmed the presence or absence of aflatoxins in selected toxigenic and atoxigenic isolates. Diversity Index (H) analyses ranged from 0.11 (Nandi samples) to 0.32 (Kisumu samples). Heterokaryon compatibility ranged from 33 % (for the Makueni samples, n = 3) to 67 % (Nandi samples, n = 6). To our knowledge, this is the first reported findings for A. flavus diversity and distribution in Nandi, Homa Bay and Kisumu counties and may assist current and future researchers in the selection of biocontrol strategies to mitigate aflatoxin contamination as has been researched in Makueni and neighbouring counties.
Regional variation of endophyte community diversity in Dalea purpurea (purple prairie-clover)
- M. Demers
- G. May
Fungal endophytes are found living asymptomatically in the tissues of most plants, and some endophytes may provide benefits to their hosts. The factors affecting the assembly of endophyte communities are not well understood, since the results of current studies differ regarding the relative importance of host species and genotype, abiotic factors, and random sampling of inocula from the environment. To understand the ecological factors affecting the diversity and taxonomic composition of endophyte communities, we sampled foliar endophytes from sixteen populations of purple prairie-clover (Dalea purpurea) in remnant prairies in four regions in Minnesota: northwest, west-central, southwest and southeast. We compared the beta diversity among these communities and found strong clustering in the northwest compared the other regions, indicating that the northwest communities were more similar in composition to each other than to other sites. The genetic results are consistent with our observations of morphological traits such as pigmentation because cultures sampled from the northwest were consistently more similar than cultures sampled from other regions. Since distances between sites in the northwest are comparable to or greater than distances between sites within other regions, abiotic conditions particular to these northwest sites may be responsible for the similarity of their endophyte communities.
Characterization of root fungal endophytes of native and invasive Phragmites australis along a salinity gradient
- M. Gonzalez Mateu
- A. Baldwin
- J. Maul
- S. Yarwood
In the last 150 years an invasive lineage of the wetland plant Phragmites australis has spread aggressively throughout many areas in the United States. Some of the problems associated with the rapid expansion of Phragmites are changes in wetland hydrology, biogeochemistry, reduction of wildlife habitat, and the loss of biodiversity, including a native lineage of the same species Phragmites australis subsp. americanus. Studying the microbiome associated with invasive and native species can lead to new insights into invasive species success, because host microbiome associations can greatly influence plant performance. The objectives of our study were to characterize the fungal endophyte communities associated with native and invasive Phragmites, determine the prevalence of dark septate endophytes (DSE) during the growing season, and examine the role of salinity in fungal root colonization. We identified three sites along a salinity gradient in the Choptank River, an estuary of the Chesapeake Bay (MD, USA), and collected roots from contiguous stands of native and invasive Phragmites every two weeks from June to October. We used microscopy to determine percent colonization of DSE, and Illumina sequencing of the ITS1 region to characterize the root endophyte communities of each lineage. DSE colonization did not vary during the growing season, but the invasive lineage was consistently more colonized than the native. Fungal colonization of invasive Phragmites also increased with salinity. All identified, sequenced OTUs matched the phylum Ascomycota, and the endophyte communities differed between lineages and among sites. In conclusion, invasive and native Phragmites have distinct root endophyte communities that vary across a salinity gradient, and might play a role in aiding the spread of the invasive lineage into higher salinity sites.
Effect of the leaf developmental stage on the chemical and fungal endophytic composition in wild Rubiaceae.
- H. Castillo Gonzalez
- M. Steward
- P. Juárez
- E. Escudero
- J. Slot
- A. Alonso
- P. Chaverri
Plants host a complex internal microbiome from which endophytic fungi (EF) represent an important component. This highly diverse group is assumed to have profound impacts on plants, therefore, much attention is now being paid to understand the interactions and relationship between endophyte colonization and leaf traits. Plant-associated environments are highly dynamic, constantly exposed to factors that can affect the structure and composition of colonizing species. In this study we aim to determine the effect of the leaf developmental stage (young vs mature) on the EF biodiversity, leaf chemistry and metabolites bioactivity as facets of the relationship between endophytes and their host. We hypothesize that the prevalence of antifungal secondary compounds is higher in young leaves which makes them more chemically protected than mature leaves, and such defenses may limit endophytic colonization. The sampling took place in the tropical forest of Golfito, southeast Costa Rica, where plants belonging to the Rubiaceae family were collected and then processed to eliminate epiphytic and environmental contamination. Fungal diversity was assessed using metabarcoding by amplification of the ITS4 and ITS5 regions and library sequencing was completed by Ion Torrent technology. Data was then analyzed using Geneious and USEARCH. For the metabolomic assessment, whole leaves were lyophilized and then ground for quantification of phenolics and other untargeted metabolites. The powder was extracted, derived, and then analyzed using LC-MS and GC-MS workflows. High-throughput sequencing identified most operational taxonomical units (OTUs) belonging primarily to the Ascomycota phylum. Pleosporales and Capnodiales were the orders contributing the most species to the endophytic assemblages. The total colonization frequency and species richness of endophytic fungi were higher in mature leaves than in juvenile, meaning, the structure of fungal communities differed significantly by developmental stages of leaf. For secondary metabolites a T-test between young and mature leaves showed little to no significant changes in constitutive concentrations of most compounds through the juvenile-mature stages, but instead showed greater metabolic differences between families of plants sampled. These results suggest that neither phenolics, nor other chemical compounds vary sufficiently with leaf age to be identified as causal agents of the changes in endophyte richness at the leaf stage. Moreover, in tropical forests, leaves of different developmental stages differ in duration of exposure to environmental fungi, which could be influencing these results, along with other structural and chemical properties that change during the leaf’s life cycle. In conclusion, it is imperative to continue efforts to understand the degree to which apparent patterns of host colonization are dictated by the host, the endophyte or other ecological mechanisms.
Fusarium prevalence on Vanilla from different cultivation systems using culture-based methods
- T. Torres Cruz
- M. González-Chávez
- R. Carrillo-González
- A. Porras-Alfaro
Vanilla is widely cultivated for its economic importance for the production of natural vanillin. This crop has been extensively studied in different countries but little research has been conducted at its center of origin regarding fungal associations. Our goal was to determine fungal diversity in plants from different agrosystems. Sampling was conducted in agrosystems located at Totonacapan (Puebla and Veracruz, Mexico). Samples were collected from four species of Vanilla (V. planifolia, V. pompona, V. insignis, V. rayada) on traditional and shade house systems that used dead, alive and concrete tutors. Fungal isolates were obtained from surface-sterilized plant material (i.e. terrestrial and adventitious roots, pelotons, and leaves) and using baiting techniques for soil, humus, litter and vanilla support tree cortex. Isolates were then identified using ITS nrDNA sequencing. Culturing yielded a total of 382 isolates, with 47% identified as Fusarium mainly recovered from roots (140 isolates). Other commonly cultured genera include Trichoderma (6%), Penicillium (5%), and Aspergillus (4%). Further characterization of representative Fusarium isolates will be performed using the TEF-1α and RPB2 regions. This study reveals the presence of fungi on Vanilla from Mexico and reports a high dominance of Fusarium in a culture-based study in V. planifolia.
Diversity and temporal dynamics of endophytic fungi in leaves, and roots and associated soil of Coffea arabica in Panama
- L. Mejia
- L. Ramirez-Camejo
- E. López-Vdovenko
- J. Vinda
- K. Avila
- M. Vargas
- K. Saltonstall
Endophytic fungal communities contain species that can confer their host plants with benefits such as tolerance to pathogens and pests. Understanding how these communities assemble, their temporal dynamics, and the factors that affect them is key for developing strategies for using applications of these fungi as protectors of plant crops. In this work, we used classic techniques of microbiology and next generation sequencing (NGS) of amplicons of the ITS1 region to identify endophytic fungi that make up the microbiome of leaves and roots of three coffee varieties, Coffea arabica (Geisha, Catuai and Typica). We compare these fungal communities with those in soil associated to the sampled roots, to generate information on the temporal dynamics of these communities and possible factors that influence them. Our results suggest a high diversity of endophytic fungi associated with Coffea arabica in Panama, with > 9,000 fungal OTUs and community composition more influenced by plant organ or substrate and source locality than the genetics of the studied varieties. Additionally, we found variation in the relative abundance of taxa over time (one sample per month, per substrate, for six months, for each of 45 sampled trees) and isolated fungi that inhibit the growth of coffee pathogens. We will discuss these results in the context of their relevance for the development of endophytic fungi as plant protectors, incorporating data on agronomic management, plant genetics, disease incidence and climate. This juxtaposition of classical and NGS techniques provides robust and valuable information that can be used to develop optimized regimes of application of endophytic fungi as plant protectors.
Investigations into the diversity and function of Mortierella fungi with plants and bacteria
- A. Vazquez
- A. Desirò
- G. Bonito
Plants are host to thousands of fungi and bacteria, which constitute the plant microbiome. Some of these microbes appear to contribute to plant development, growth and resilience to stress. Mortierella is an early-diverging lineage of fungi that are common in soils and rhizospheres of diverse plants. Many Mortierella species host intracellular bacteria including Burkholderia- and Mollicutes/Mycoplasma-related endobacteria. The function of Mortierella and its endobacteria in the plant microbiome remain unknown. The goals of this research are two-fold: (1) assess the diversity of Mortierella and its endobacteria; (2) determine the impact of different Mortierella species on plant growth and their resilience to drought stress. We first isolated Mortierella from Puerto Rican and US soils. To identify the fungi, ITS rDNA regions was amplified and Sanger sequenced. NCBI BLAST queries and phylogenetic analysis conducted. Isolates were screened for the presence of endobacteria using 16S rDNA primers and PCR, and identified by sequencing amplicons. Endobacteria were cleared from their host with antibiotics. To assess impacts of fungi and their endobacteria on plant growth isolates were inoculated onto Raphanus raphanistrum (radish) and Phaseolus vulgaris (bush bean). Above- and below-ground biomass was weighed and measured. At week 5 plants were exposed to severe drought and their photosynthetic efficiency (Phi2) was monitored. In this research, 11 new isolates of Mortierella were obtained. Endobacteria were detected in two isolates (18%). Both new Mortierella fungi and endobacteria detected, form part of possible new clades within its phylogeny. Only Mortierella humiliss PMI1414 strain appeared to improve the growth of radish bulb biomass and Phi2 measurements in bush bean plants. Additional studies are needed to assess impact of endobacteria on plant-fungal interactions. Response variables additional to Phi2 and dry biomass ought to be measured in future studies.
Characterization of novel endophytic Pleosporales fungus from grasses
- K. Burnett
- M. Romero-Jiménez
- A. Porras-Alfaro
Dark septate fungi (DSF) are commonly found in semi-arid regions of Southwest America. Grasses benefit from symbiotic DSF that are well adapted to survive stressful environmental conditions such as UV radiation, drought and heat. A vast diversity of DSF belong to the Pleosporales order, though studied, impact on plants are poorly understood. The objective of this study is to evaluate the effect of a novel Pleosporales fungus on plant growth. Fungal specimens were isolated on MEA with antibiotics from sterile roots. Using the ITS rRNA region isolates were identified to the order level of Pleosporales with low similarity to other described fungal genera. Six isolates were chosen for morphological characterization of the fungus by growing them on multiple media. Direct contact bioassay were conducted on Bouteloua gracilis and B. dactyloides to evaluate the effect of fungal isolates on plant growth. LSU sequences show evidence of a potential novel fungus closely related to Didymocrea found in the internal tissue of the South China Sea sponges Clathrina luteoculcitella and Holoxea sp. Additionally, the morphology of the isolates varies between media (MEA, Emerson, PDA, Czapek dox, soil agar) in terms of conidia production . Sexual structures were not observed. Isolates showed a range of effects on plant growth showing signs of pathogenicity to stimulation of root growth. Fungal colonization was observed in plant roots as hyphae and spores. Additional bioassays and sequencing will be conducted with the six isolates to gain a better understanding of their function and taxonomic placement.
Arbuscular Mycorrhizal and dark septate endophytic fungal association in finger millet (Eleusine coracana) varieties in Southern India)
- N. Kandhasamy
- T. Muthukumar
We investigated the extent on root morphology in finger millet varieties cultivated in southern India. Root samples of cultivated finger millet varieties were collected from Agricultural field GKVK campus at Bangalore. All the 17 varieties were root morphology charactersitics. They are characterized by intercellular and intracellular hyphal and arbuscular coils. Moreover, our results suggest that AM and DSE fungal association were significant among the 16 varieties of finger millet roots. We also found spores of 4 species of AM fungi were associated with finger millet varieties and recorded in different rhizospheres. It suggests that AM and DSE fungal association were significant with finger millet varieties that showed strong symbiotic association found between mycorrhizal fungi and finger millet varieties.
Arbuscular mycorrhizal fungi suppression of Fusarium verticillioides in maize
- O. Olowe
The protection of plants from pathogenic organism results to better performances of their growth and yield characters. Hence, the efficacy of Glomus clarum and G. deserticola against Fusarium verticillioides (AKR 05, ILR 06 and ERW 05 strains) on maize T2L COMP.4 was investigated. Glomus clarum and G. deserticola were inoculated separately at concentrations of 10 g (20 spores), 20 g (48 spores) and 30 g (72 spores) per 8 kg of soil at 4 wk after planting (WAP) with a control. In addition, spore suspension (1.0 × 106 spores/mL) of the pathogen Fusarium verticillioides was also inoculated at 8 WAP while the effects of these treatments were observed based on the plant’s morphological, biomass and yield traits. Effect of F. verticillioides on plant height and shoot weight was significantly reduced by 20 g (48 spores) and 30 g (72 spores) treatments of G. clarum and G. deserticola, also the treatment at 10 g (20 spores) had biocontrol effect on the husk cover while the percentage ear rot severity ranged from 3.0% to 22.2% across the treatments. Therefore, this result established biocontrol potential of the tested arbuscular mycorrhizal against Fusarium verticillioides at 30 g (72 spores) concentration.
Effects of arbuscular mycorrhizal fungi on growth of Medicago sativa in acidic soil
- F. Li
- Y. Li
- T. Duan
Acidic soils are harsh environments for plants. Arbuscular mycorrhizal (AM) fungi play an important role in protecting plant growth against such stresses as acidic soil. To understands the relationship between AM fungi colonisation and soil acidity to evaluate the possibility that AM fungi facilitate the existence of plants on acidic soils, the effects of arbuscular mycorrhizal (AM) fungi Claroideoglomus etunicatum, Rhizophagus intraradices and the mix of the two AM fungi on the growth of alfalfa (Medicago sativa) were assessed at three of soil pH by growing plants in a greenhouse experiments, with and without AM fungi inoculation, individually in pots. The different acid growth medium were established with H2SO4 to pH 3.0, 5.0 and 6.58. The results showed that the C. etunicatum, R.intraradices individual and the mix of the two AMF increased plants dry weight and P uptake at all acid treatments compared with un-inoculation of AM fungi treatment (P<0.05). Plants shoot height, leaf numbers, shoot dry weight, root dry weight and total dry weight were averagely increased by 214.77%, 312.67%, 105.39%, 95.46% and 101.85%, respectively by the mix of two AMF compared with un-inoculated treatments. Acid decreased the total dry weight, chlorophyll content, superoxide dismutase (SOD) and peroxidase (POD) activity, of which the above mentioned index were 17.59%, 21.94%, 29.51% and 51.26% lower at pH 3 than that at pH 6.58. The inoculation of AM fungi averagely increased SOD and POD activity of plant by 108.7% and 49.68%, respectively, and decreased malondialdehyde (MDA) of plants dramatically compared to un-inoculated plants (P<0.05).
Arbuscular mycorrhizal fungi alter plant interspecific interaction under the addition of nitrogen
- H. Feng
- A. Bahadur
- Z. Jin
- X. Long
- S. Jiang
- D. Li
- Z. Cheng
- Y. Zhang
- J. Wang
- Q. Zhang
- J. Pan
- Y. Liu
Arbuscular mycorrhizal fungi (AMF) form linkage with plants and ubiquitous in agricultural ecosystems. AMF are acknowledged to contribute to plant nitrogen (N) uptake, but it is critical to understand how N-addition affects AMF and plant interspecific interaction. However, AMF inoculation response to N-addition for plant competitive interaction are still unclear. Thus, we studied the competitive interaction between mycorrhizal and non-mycorrhizal individuals of Vicia faba, Hordeum vulgare, and Brassica napus species differing in both biomass allocation and mycotrophic degree. Two nitrogen fertilizer treatments (N0 and N15 g N m-2 yr-1) were used to originate nutritional differences across the three plant species. Species-specific variation in mycotrophy revealed evident differences in root/shoot biomass allocation in B. napus, higher mycorrhizal dependency in V. faba, and higher aggressivity of H. vulgare with B. napus (host vs non host specie) in AMF inoculation under N0 treatment. This pattern was supported by our study where solitary B. napus displayed pronounced investments into root and shoot growth rather than in competition. In addition, V. faba indicated as a best competitor and obtained greater biomass across the treatments for the relative yield total. This is predominantly vital for sustainable agriculture for food security because dealing for higher AMF abundance and function may lessen or eliminate incentives for environmentally and costly problematic nitrogen.
Host genotype and plant phenological growth stage are important drivers of root-associated mycobiome
- K. Loit
- L. Soonvald
- A. Astover
- L. Tedersoo
The importance of microbial communities in plant nutrition and health is repeatedly noticed. Microorganisms are ubiquitous in the ecosystem; they interact positively or negatively with plant roots in the rhizosphere or with above-ground plant parts. Fungal assemblage changes in the soil are affected by many factors. It’s known that different plant species host specific microbial communities when grown on the same soil, and are able to suppress pathogenic organisms in the rhizosphere. In this study, the indigenous fungal pathogen and symbiotic arbuscular mycorrhizal fungal (AMF) community were studied in conventionally treated field soil by Illumina MISeq sequencing of ITS region. In total 315 soil and root samples were collected on different plant phenological growth stages of 21 potato cultivars. The interactions, possible pathogen suppression ability and the variation of mycobiome structure were determined. The results showed the variable richness of AMF and pathogenic fungi throughout the growing season. The study indicated that potato cultivar and plant growth stage were essential factors that altered pathogenic fungal community composition. In contrast, AMF community was not influenced either by cultivar nor plant growth stage. Despite the applied conventional management regimes, plant roots were highly colonised by pathogenic fungi. Compared to the pathogens, the abundance of AMF in the plant roots was relatively low and insufficient to suppress pathogenic organisms.
Arbuscular mycorrhizal fungi of areas in different stages of natural regeneration in a tropical dry forest
- J. Veras
- I. Escobar
- J. Silva
- D. Silva
- I. Silva
- V. Santos
- F. Oehl
- L. Maia
Considering the importance of conservation of Caatinga, one of the most diverse arid biomes in the world, and the role played by the arbuscular mycorrhizal fungi (AMF) that provide to their hosts an adaptive strategy for surviving in the stressful conditions of semiarid environments, the aim of this study was to assess the impact of forest age on the FMA assemblages in the National Park of Catimbau, Pernambuco, Brazil. Soil samples were collected in July/2016 in four areas, one with natural vegetation and three in process of regeneration, each of them with 3 subareas: (A) area with native vegetation of Caatinga with ages estimated to be over 100 years old; (B) area in initial stage of natural regeneration with ages of 4, 6 and 10 years; (C) intermediate area in stage of natural regeneration with 17, 23 and 30 years; (D) area in late stage of regeneration that are 37, 40 and 45 years old. We compared the species richness of the community of spores, composition, abundance, and similarity among areas under different successional stages. Fifty-two species of AMF were identified, representing 18 genera and 11 families. Acaulospora and Glomus were the most recorded genera, with 25% and 16% of the taxa, respectively. In general, G. macrocarpum was the most abundant species with >78% of relative frequency. The highest FMA richness was recorded in the area of initial regeneration stage with 42 species, followed by the intermediate recovery area (31 species), the area of natural vegetation (28 species) and the area under late regeneration (24 species). Most of the taxa were exclusive (42.30%): 13 for the area of initial regeneration; four for the intermediate regeneration area; three for the native vegetation area and two for the late regeneration area. Members of Paraglomerales were only recorded in areas of initial regeneration. The results showed that forest age has a significant effect on FMA community; however, the age of the areas is not the main modulator in the succession of FMA assemblage, with an interdependent succession occurring, where the ecological succession of fungi occurs independently of the succession of the plant. While the complexity of the plant community occurs with increasing age, that of the FMA community tends to decrease with the age of the areas.
The diversity and distribution pattern of arbuscular mycorrhizal fungi in Taiwan conifer ecosystem
- W. Lin
- T. Lin
- L. Lin
- H. Cheng
- P. Wang
Arbuscular mycorrhizal (AM) fungi influence plant diversity and productivity. To elucidate their biodiversity patterns is fundamental to understand their community assembly mechanism and ecosystem functions. Using morphological identification and high-through put sequencing, we investigated distribution patterns and ecology of AM fungal diversity to address the relative effects of altitude and soil properties on their diversity and community compositions in Chamaecyparis formosensis forests. Classical method analysis revealed 26 AM fungal species including Acaulospora, Glomus, Entrophospora, Sclerocystis, Cetraspora, Diversispora, Paraglomus, Racocetra and Scutellospora along three altitudinal transects. Number of total AM fungal species and spore density were not correlated with the altitude of the study sites. The composition of the AM fungal community was significantly different at each location and altitude. Changes of soil properties across an altitudinal gradient played an important role in shaping AM fungal diversity and community. Species richness was negatively correlated with total organic carbon. Total organic carbon also contributed remarkably to the variations of AM fungal communities. The dominated species, Acaulospora laevis, A. morrowiae and Sclerocystis rubiformis were found at about all elevations and locations, and their spore densities were not correlated with the altitude of the study site. Some taxa were more restricted in particular elevation and their spore density showed a correlation with the altitude. For example, spore density of A. koskei increased with increasing altitude. Primary result of molecular analysis showed that Acaulopora, Rhizophagus and Sclerocystis was the dominant AM fungal genus associated with C. formosensis roots.
Ecological networks between arbuscular mycorrhizal fungi and plant hosts following restoration in a tropical forest
- C. Egan
- S. Swift
- N. Hynson
A fundamental goal during ecological restoration is to return communities to comparable diversity as their natural counterparts. Prior studies of ecological restoration have tended to have an aboveground bias that neglects changes in diversity belowground following restoration efforts. This is an issue because often restoration efforts are unsuccessful, due to either biotic and/or abiotic constraints, both of which can be mitigated by soil microbial organisms. Multiple studies have also shown that soil-borne microbes, especially arbuscular mycorrhizal (AM) fungi, are important drivers of plant community development, accentuating the importance of examining changes in AM fungal diversity during ecological restoration. Here, expand our understanding of belowground community responses to ecological restoration by examining changes in AM fungal community structure following ecological restoration efforts in a degraded Hawaiian tropical forest. For this study, both intact and restored sites were sampled within the Hakalau Forest National Wildlife Refuge on the Island of Hawai`i, where ecosystem recovery attempts have been ongoing since 1985. Within sites we sampled the roots and soil of seven AM fungal plant hosts that were found in both intact and restored patches, and characterized AM fungal diversity using Illumina sequencing. In addition to examining general AM fungal community composition patterns, we also examined interaction networks between plants and AM fungi to determine how network architecture differs between remnant and restored forest patches. We predicted that communities would be significantly influenced by patch type, and that interaction networks between plants and AM fungi would vary in robustness between remnant and restored patches, where networks in restored patches would be significantly less complex, making them more susceptible to perturbation than intact forest networks. Furthermore we predict that a few species of AM fungi will be dominant within both patch types, where taxa with ruderal life history strategies would be dominant within restored patches, while taxa with competitive life history strategies would be dominant within intact patches.
Expanding the known diversity of North American fetid Russula
- P. Avis
- M. Barajas
AbstractRussula is a highly diverse genus of ectomycorrhizal fungus. Over 700 species of this genus are known making up about 10% of the known ECM fungi in world. One especially intriguing group of Russula are the members of subsection Foetentineae, commonly referred by some as the fetid Russula since their brown to light yellow capped sporocarps regularly have unpleasant smells and tastes. Their ecological attributes include the ability to be “dominant” (i.e. numerically abundant) in temperate, boreal and tropical ecosystems worldwide, to persist in high nitrogen environments, and to associate with mycoheterotrophic plants. Despite these ecological attributes, much of the diversity of this group remains unknown and/or fully documented. In this presentation, we will provide a thorough overview of the molecular diversity (using ITS and RPB based phylogenies) of fetid Russula in North America and place this overview within a context of the current understanding of subsection Foetentineae globally. We will also highlight a set morphological and molecular characteristics of two groups of North American fetids, Russula garyensis and R. amerorecondita currently under consideration as species new to science.
Diversity and phylogeny of Russula subgenus Compactae
- R. De Lange
- A. Verbeken
Within the genus Russula, one of the major ectomycorrhizal fungal genera in all ecosystems worldwide, R. subg. Compactae is a large subgenus, mainly characterised by the presence of lamellulae which are absent in the rest of the genus. The fruitbodies are rather big, firm and compact with a short and thick stipe and a white, yellowish or brown cap. The context is often browning or blackening, sometimes reddening. The subgenus consists of three main groups: R. sect. Compactae, R. sect. Lactarioides and the oldest group R. sect. Archaeinae. Especially the first two groups are characterized by a high diversity, with several species complexes and undescribed species. In a first part of this project, we aim to make a general phylogeny of Russula to get a clear placement of each of these groups. Hence, we will test the hypothesis that R. subg. Compactae is monophyletic, placed at the base of the Russula phylogeny. The preliminar data suggest that our hypothesis about the monophyly of R. subg. Compactae will be rejected, but the position of the different sections is still unclear. The second objective of this project is to delimit species within R. subg. Compactae in Europe. We aim to unravel the species complexes and describe new species through phylogenetic and morphological analysis. As it is thought that ecology might play an important role in speciation, we also focus on mycorrhizal host associations. An analysis was done on the existing data and a phylogenetic tree was created. Based on this tree we assume the existence of at least 15 undescribed species within R. subg. Compactae in Europe in our current sequence dataset. This dataset contains sequences mostly from Europe, some from Brazil, Martinique, Thailand, North-America and Africa.
Molecular phylogenetic analysis reveals new and noteworthy Lactarius spp. in different subgenera from Pakistan
- Z. Ullah
- S. Jabeen
- M. Faisal
- H. Ahmad
- A. Khalid
Lactarius is a large genus of agaric fungi in Russulaceae (Basidiomycota, Agaricales) with more than 600 species, commonly known as milk-caps. The members of the genus are characterized by the milky fluid they exude when cut or damaged. They are ectomycorrhizal and form symbiosis with trees species belonging to different families. Due to these ectomycorrhizal associations, Lactarius is one of the dominant agaric genus in many ecosystems, from the boreal forests to the temperate ones, subtropical woodlands and tropical low lands in South-East Asia. The estimated number of species in the genus is up to 700. From Pakistan, six species have been reported to date. These species have been reported on the basis morphological characters. The molecular method of characterization of these species spilt the genus into several subgenera.The objective of our study is to document fungal flora of pakistan on the basis of molecualr phylogenetic analysis and morpho-anatomical characterization. Genomic DNA was extracted using 2% CTAB method. The polymerase chain reaction (PCR) was carried out using the fungal-specific ITS1F primer and the eukaryotic ITS4 primer to amplify the nuclear ribosomal internal transcribed spacer region. For 28S region amplification, LR0R and LR5 primers were used. For anatomical studies, tissues from pileus, stipe and gills were mounted in 5% KOH and observed under microscope (LABOMED, Labo America, Inc. USA). During this investigation, a total of seven collections have been examined, out of these, four Lactarius species have been identified which fall in all subgenera of Lactarius well supported by morphological characters and molecular phylogenetic analysis based on ITS sequences. Lactarius sp. ST-01, ST-04 and ST-05 fall in subgenus Piperitus, Lactarius sp. SH-01 falls in subgenus Russularia and Lactarius sp. ST-10 and ST-26 falls in subgenus Plinthogalus. All these species found to be distinct morphologically and phylogenetic data based on ITS region confirmed their identity. Lactarius scrobiculatus has been identified on the basis of morphological characters and its molecular phylogenetic analysis supported its systematics in subgenus Piperitus. The work on these taxa is in progress, more collections are being examined which will lead to the provision of nomenclature to the previously undescribed species.
Filling the gaps: completing the Lactifluus phylogeny by assessment of its Neotropical biodiversity and building a bRIDge between taxonomists and environmental ecologists.
- L. Delgat
- A. Verbeken
The Research Group Mycology from Ghent University has a large expertise in the Russulaceae family, a family of important ectomycorrhizal fungi. Traditionally the two main groups were russulas and milkcaps, but since 2008 we know that milkcaps are paraphyletic and divide them over the genera Lactarius, Lactifluus and to a lesser extent Multifurca. There are some striking differences between Lactarius and Lactifluus concerning their distribution, phylogeny and evolutionary history. Lactifluus has a mainly tropical distribution and is well-studied in tropical Africa and SouthEast Asia. However, little is known about its diversity in the Neotropics because for a long time ectomycorrhizal fungi were wrongfully assumed absent in most South American ecosystems, leaving one big gap left in our world-wide phylogeny. Since 2015 we have been working to assemble a dataset based on herbarium specimens, specimens collected by collaborators and specimens collected during expeditions in Martinique, French Guiana and Brazil. Sequence data indicates the presence of at least 58 Lactifluus species in the Neotropics, so it is clear that there is in fact a large diversity of Lactifluus species in the Neotropics contrary to previous expectations. These neotropical species occur in three out of four subgenera and they form entirely new clades/sections. So far the Lesser Antilles have been studied in detail. This area was already well studied morphologically before and our phylogenetic study revealed a total of 10 species in the Lesser Antilles, of which 3 new species. A new identification key to the species was constructed. The completed worldwide phylogeny will provide a much-needed taxonomical framework for environmental ecologists. In function of this, a well-documented database is being established with historical and recent data of Neotropical Lactifluus specimens, which will in the future be extended to Russulaceae worldwide. The database will be called bRIDge (Russulaceae Identification Database) and is built with BioloMICS Software for biological data management, identification, classification and statistics. In the database the records are linked to photographs, microscopical illustrations, ecological data and sequences. Environmental ecologists will be able to consult this database to interpret (part of) their datasets.
Survival and growth of western white pine (Pinus monticola) is not impeded by association with Armillaria altimontana in northern Idaho
- J. Hanna
- M. Warwell
- G. McDonald
- M. Kim
- B. Lalande
- J. Stewart
- N. Klopfenstein
Armillaria solidipes is well known as a cause of root disease on diverse conifers in areas of inland, western North America, where A. altimontana also frequently co-occurs. In contrast, impacts of A. altimontana on tree health are not well known. At the Priest River Experimental Forest in northern Idaho, USA, a provenance planting of western white pine (Pinus monticola) was established in 1971 in a 0.8-ha plot to examine growth and survival. In 1987 (16 years post-planting), measurements, inspections, and sampling were conducted on 1215 living/recently dead trees to determine potential influences of A. solidipes and A. altimontana on growth and survival of western white pine. Of these trees, 48% were associated with A. solidipes and/or A. altimontana. Somatic pairing tests were used to condense the Armillaria isolates into unique genets and translation elongation factor 1-α sequencing was used for species identification. Results demonstrated that the plot contained two genets of A. altimontana comprising ca. 83% of the isolates and five genets of A. solidipes comprising ca. 17% of the isolates. As expected, A. solidipes was associated with decreased tree growth and survival. In contrast, A. altimontana was not associated with increased tree mortality or decreased tree growth, which suggests that A. altimontana was not harmful to western white pine within this northern Idaho planting. Maps of Armillaria distribution within the plot indicate that A. solidipes was uncommon in areas dominated by A. altimontana. Furthermore, the wide spatial span of individual Armillaria genets suggest that this site has been occupied by both Armillaria spp. for >250 years. Interactions between these two Armillaria species appear critical to understanding Armillaria root disease in this region.
Armillaria mexicana, a recently described species from Mexico
- R. Elías-Román
- R. Medel-Ortiz
- D. Alvarado-Rosales
- J. Hanna
- A. Ross-Davis
- M. Kim
- N. Klopfenstein
Armillaria mexicana (Agaricales, Physalacriaceae), a recently characterized species from Mexico (Elías-Román et al. 2018), was described on the basis of morphology and phylogenetic analyses that show it is clearly distinct from previously reported species in North, Central, and South America. Morphological characteristics of A. mexicana include the absence of fibulae in the basidioma, abundant cheilocystidia, and ellipsoidal, hyaline basidiospores that appear smooth with light microscopy, but slightly to moderately rugulose with scanning electron microscopy. Macro-morphological characters, such as annulus structure, pileus coloration, stipe coloration, and other structures, are useful to distinguish A. mexicana from other Armillaria spp. Furthermore, phylogenetic analyses of the nuc rDNA internal transcribed spacers (ITS1-5.8S-ITS2 = ITS), 28S D-domain, 3′ end of 28S-Intergenic spacer 1, and translation elongation factor 1-α (TEF1) clearly show that A. mexicana sequences are quite distinct from sequences of other Armillaria species. Interestingly, sequences of ITS sequences of the A. mexicana ex-type culture reveal an ITS1 of 1,299 bp and ITS2 of 582 bp, which represent the longest ITS regions reported thus far in fungi. Phylogenetic analysis based on TEF1-based phylogenetic analyses place A. mexicana within a well-separated, monophyletic clade basal to the polyphyletic A. mellea complex, which may represent the earliest evolving lineage of Armillaria in the Northern Hemisphere. Presently, A. mexicana is known to be highly virulent on planted peach (Prunus persica) and other tree species in central Mexico.
Improving the management of Armillaria in gardens
- E. Beal
- J. Drakulic
- G. Clover
- M. Cromey
Honey fungus (Armillaria spp.) is one of the most common diseases troubling gardeners in the UK. Seven species are found in the UK but only three were found during our four-year garden survey (2004-7): A. mellea (83.1%); A. gallica (15.8%); and A. ostoyae (1.1%). Honey fungus has a large host range, causing death and decline of trees, shrubs and non-woody plants such as bulbs and vegetables. Because of its broad host range, garden choices are restricted if susceptible hosts are to be avoided when replanting. Gardeners want to know the best ways to manage and control the disease. Currently management and control options for gardeners are costly, often difficult to perform in gardens and labour intensive, and thus they are often unsuccessful. Our research aims to improve understanding of the disease and increase the success of Armillaria management in gardens. We are engaged in projects to examine (1) a new approach to disease prevention, (2) the importance of species-specific diagnosis for gardeners, and (3) the efficacy of current management protocols. Firstly, the potential of Trichoderma endophytes to improve host resilience and promote growth during plant establishment was studied. The introduction of suitable isolates would enable gardeners to replant in Armillaria-infected beds. Endophytes sampled from root systems of susceptible hosts within infection foci at RHS Garden Wisley, were cultured, identified and screened for beneficial effects on common garden plants (Rosmarinus officinalis and Thymus vulgaris). Isolation of T. atrobrunneum occurred most frequently, and showed improved host growth and in vitro inhibition of A. mellea. Secondly, the interactions of different Armillaria species was studied. We hypothesised that the less virulent but highly rhizomorphic species Armillaria gallica can occupy the same niche and prevent invasion from the aggressive species A. mellea. The implication being that gardens with A. gallica could be ‘protected’ from more pathogenic species. Privet plants were inoculated twice with infected hazel stick inoculum. The first inoculation was either a control (sterile hazel stick) or with A. gallica. The second inoculation (3 or 6 months later) was either a control or with A. mellea. Plant vigour and mortality is being measured over time, and isolations and molecular analysis will be used to determine the Armillaria species colonising host tissues at harvest. Our final research direction concerns the growth and survival of rhizomorphs once severed from their food source. Present advice to gardeners is to remove as much of an Armillaria-infected plant as possible and cultivate the soil. However, it’s virtually impossible to remove all small fragments, including rhizomorphs, so information on survival and growth of severed rhizomorphs would be beneficial to validate our advice to gardeners. In order to test this, rhizomorphs of A. mellea and A. gallica were cut from their food source and their growth and viability in compost and loam were examined compared to rhizomorphs of the same length still attached to a woody substrate. Following the completion of these projects we aim to use a combination of information from the results to create a suitable Armillaria management program.
Pathogenicity of a parasitic macrofungus in the Southern regions of Tanzania
- D. Tibuhwa
- M. Sijaona
- S. Tibell
The Southern regions of Tanzania are highly vulnerable to food insecurity, and a collapse of the agroforest industry and poverty due to main cash crop failure is threatening. This is caused by a pathogenic macrofungus. The fast expanding outbreak has devastating consequences since the fungus attacks and causes wilting of cashew trees, which is the main cash crop in the region, cassava which is the main staple food, eucalypts and some other trees. This work has investigated the pathogenicity of the fungus by isolating its germ plasm and re-inoculating it to uninfected plant cultivars. The re-inoculated cultivars showed the same disease symptoms as those of the wilting plants. DNA of the pathogenic macrofungus was re-isolated from the plant cultivars which showed symptoms of being infected. Anatomical study of the infested plants from the field showed that the fungus is present in the plant vascular tissues as evidenced by the presence of mycelium and extra-cellular polysaccharides in the xylem vessels. Excessive growth of the mycelium in these vessels most probably interferes with the translocation of nutrients by blocking the flow in phloem and xylem vascular bundles which leads to plant stress, then chlorosis, wilting and ultimately to death. The spread of this pathogenic fungus has to be tackled immediately and with great concern since the Southern regions are the main producers of Cashew in Tanzania, and the population depends heavily on the cassava as their staple food and both of these crops are heavily affected.
Pathogens or Associates? Leptographium and Grosmannia blue-stain fungi associated with dying loblolly pine in Georgia
- M. Buland
- B. Barnes
- K. Klepzig
- K. Gandhi
- C. Villari
Leptographium and Grosmannia spp. (order Ophiostomatales) and their root-feeding beetle vectors (e.g., Hylastes, Hylobius, and Pachylobius spp.) have been reported from areas of loblolly pine (Pinus taeda - the most commercially important tree species in the southeastern United States) dieback in recent decades. However, species composition of the fungal complex associated with these beetles, and the frequency of association between individual fungal and vector species is currently unknown, with unclear implications for forest management. Our research objectives are to: 1) determine the phenology of loblolly pine root-feeding beetles in Georgia loblolly pine stands; 2) assess the diversity and composition of the Ophiostomatoid fungal complex associated with these root-feeding taxa; and 3) analyze whether pathogen pressure or vector species composition varies in stands with various management practices and across multiple seasons. Root-feeding beetles were live-trapped around girdled loblolly pine trees at two sites in central Georgia with differing management histories [regularly prescribe burned (every 2-3 years) versus unburned (at least for 5 years)] and across seasons (spring, summer, and fall). A subset of collected beetles from each target species was used to isolate and identify associated Ophiostomatoid fungi based on morphological and molecular features. During 2017, we collected H. porculus, H. salebrosus, H. tenuis, H. pales and P. picivorus beetles with notable variation in numbers of H. pales, the most abundant beetle species. Total number of beetles was higher in the burned than unburned sites, suggesting possible attraction of these root-feeding beetles to the burned areas. Three Leptographium and Grosmannia species with varying reported degrees of virulence (i.e., L. procerum, G. alacris, and G. huntii) have been identified via sequencing of the β-tubulin gene, with the potential for other species to be identified. Species-specific PCR primers will be developed for each fungal species identified via sequencing and fungal DNA extracted directly from the integument of the adult beetles to determine fungal species composition and incidence per beetle species. Determination of the species composition of the fungal complex associated with loblolly pine root-feeding beetles in Georgia is a crucial first step to determining their role, if any, in affecting tree and stand health.
Genetic and genomic approaches toward understanding the biology of the Korean oak wilt fungus (Raffaelea quercus-mongolicae)
- M. Kim
- J. Stewart
- J. Ibarra Caballero
- N. Klopfenstein
Raffaelea quercus-mongolicae is associated with oak wilt disease in Korea. To date, this ambrosia beetle-vectored fungus has only been found in South Korea, and it is phylogenetically distinct from R. quercivora, which causes a similar oak wilt disease in Japan, and other Raffaelea spp. When this fungus was discovered on a dead Mongolian oak (Quercus mongolica) in 2004, the disease epiphytotic was centered around Seoul and the adjacent Gyeonggi Province; however, it has continued to spread southwards since then. Despite continued expansion of the disease and associated major impacts on forest ecosystems, little genetic and genomic information of R. quercus-mongolicae available for understanding its biology, evaluating pathways of spread, and developing improved disease prediction and management methods. The objectives of this study were to assess genetic diversity and population structure of the Korean oak wilt fungus, R. quercus-mongolicae, using Restriction-site-Associated-DNA sequencing (RAD-seq); sequence the whole genome of R. quercus-mongolicae; and analyze the transcriptome (expressed genes) of R. quercus-mongolicae by RNA sequencing and reference genome-based assembly. Fifty-four isolates of R. quercus-mongolicae were collected from five regions of South Korea. RAD-tag libraries and DNA sequencing were conducted at Floragenex, Inc. (Eugene, OR, USA). The draft genome of R. quercus-mongolicae (KACC44405) was sequenced by Illumina NextSeq and MiSeq systems for paired-end reads and HiSeq2000 for mate-pair reads. Total RNA was extracted from R. quercus-mongolicae (KACC44405) grown under in vitro conditions on two artificial media [potato dextrose agar (PDA) and water agar (WA)]. The mRNA was sequenced using Illumina HiSeq™2000 with a read length of 101 bp. Trimmed sequences were mapped to reference genome. Sequencing the RAD-tag libraries generated 143,696,855 reads using Illumina HiSeq. In total, 179 SNPs were identified among 2,639 RAD loci across the nuclear genome of the 54 R. quercus-mongolicae isolates (0.00080 SNPs per bp). Overall low expected heterozygosity and no apparent population structure were found among South Korean populations R. quercus-mongolicae, which supports the hypothesis that this ambrosia beetle-vectored fungus was introduced to South Korea. The genomic sequence of R. quercus-mongolicae (KACC44405; 27-Mb), along with other Raffaelea spp., will provide valuable resources for comparative genomic analyses and identifying genes that contribute to potential pathogenic relationships between the fungus and host, and potential symbiotic relationships between the fungus and insect vector. After mapping with a reference genome, 7,739 transcripts were identified as the R. quercus-mongolicae transcriptome dataset. The predicted gene products are associated with diverse functions, such as production of ATP for growth, recovery under stressed conditions, fluidity of cell membrane, maintenance of cell membrane, and regulation of fungal virulence. The transcripts that differed significantly in expression levels between PDA and WA media were associated with localization within subcellular components, hydrolase activity, and intrinsic membrane components.
Diversity of potential emerging pathogens of conifers in Colorado, USA
- J. Ata
- K. Otto
- J. Stewart
Conifer foliage possess dynamic communities of microorganisms that may have either beneficial or parasitic relationships with its host plant. The increasing prevalence of disease emergence factors like climate change and globalization can favor the pathogenicity of introduced fungal species and/or enhance virulence of pathogens within the needle community structure. The lack of distinct morphological and molecular information on these conifer foliage pathogens make it also challenging to identify them and recommend appropriate control measures. This ongoing study initially surveyed the health condition of pine forests in Colorado and evaluated the diversity of pathogens in needles of selected conifer host species. Five conifer species (i.e. limber, ponderosa, lodgepole, bristlecone and Himalayan pine) which were recorded to exhibit disease symptoms were sampled from various regions in Colorado, USA. Morphological and genetic characteristics were used to identify pathogenic and non-pathogenic fungal isolates. Known pathogens were assessed based on symptoms, life history, and its distribution and spread. Disease symptoms of host species included needle cast, blight, and red bands. Black fruiting bodies were recorded in some needles of limber and ponderosa. In general, the most common pathogenic species isolated included Sydowia polyspora, Lophodermium sp., Rhizosphaera sp., and Cytospora sp. Most of these pathogenic species were associated with necrotic diseases. S. polyspora were found in three host species (limber, ponderosa, and Himalayan pine) while Lophodermium and Rhizosphaera were commonly observed only in limber. Known pathogens of several angiosperms such as Alternaria alternata and Phaeomoniella effusa were also isolated from lodgepole and limber. Results of the study are relevant for the development of molecular tools for pathogen identification and disease risk assessment. Further studies on interactions of pathogens within host species and its impacts on disease development are also recommended.
Microsatellite characterization of Penicillium digitatum, causal agent of green mold of citrus
- E. Varady
- M. Kellerman
- P. Fourie
- G. Vidalakis
- G. Douhan
Penicillium digitatum is one of the most important post harvest pathogens of citrus on a global scale causing significant annual losses due to fruit rot. However, little is known about the diversity of P. digitatum populations both within the field and subsequently after processing where significant post harvest treatments for control could influence the population dynamics. The genome of P. digitatum was recently sequenced, providing an opportunity to determine the microsatellite distribution within P. digitatum to develop markers that could be valuable tools for studying the population biology of this pathogen. In the analyses, mono, di, tri, tetra, penta, and hexanucleotide microsatellites within the genome of P. digitatum were restricted to 12 repeats for mononucleotides and above and the rest were restricted to 5 repeats and above. A total of 3,134 microsatellite loci were detected; 66.73, 23.23, 8.23, 1.24, 0.16, and 0.77% were detected as mono, di, tri, tetra, penta, and hexanucleotide repeats, respectively. As consistent with other ascomycte fungi, the genome size of P. digitatum does not seem to correlate with the density of microsatellite loci. However, significantly longer motifs of mono, di, and tetranucleotide repeats were identified in P. digitatum compared to 10 other published ascomycete species with repeats of over 800, 300, and 900 motifs found, respectively. One isolate from southern California and 5 additional isolates from other countries (‘global isolates’) were used to initially screen microsatellite markers utilizing the genome to find candidate loci and Primer3 to design primers. Twelve additional isolates, referred to as the ‘local isolates’, were also collected from the University of California Riverside citrus collection and were subsequently used to screen the primers that sequenced well and were polymorphic based on the global isolates. Thirty-six primers were screened but nine trinucleotide loci and one hexanucleotide locus were chosen as robust markers. These loci yielded 2 to 3 alleles within the global isolates and 2 to 7 alleles in the local isolates. These markers will be useful to study population genetic differentiation, migration, genetic drift, mutation rates, and other ecological and evolutionary processes that shape P. digitatum populations.