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Morphological characters with phylogenetic signal in Scleroderma (Basidiomycota)
- L. Guzmán-Dávalos
- M. Sánchez-Jácome
- V. Ramírez-Cruz
- M. Herrera Fonseca
- A. Villalobos-Arámbula
- F. Ramírez-Guillén
- G. Guzmán
Scleroderma, formerly in the artificial group of the Gasteromycetes, is now part of the Boletales. The genus comprises subglobose, ectomycorrhizal mushrooms, with 21 species according to the monograph of Guzmán in 1970 or 30 following the Ainsworth & Bisby’s dictionary, from temperate and tropical regions worldwide. Microscopically, the genus is characterized by its globose thick-walled ornamented basidiospores. The aim of this work was to analyze some morphological characters on the light of a molecular phylogeny, to understand its evolution. Specimens from IBUG and XAL herbaria were macro and micromorphological studied and described. DNA was extracted from herbarium specimens and amplified the ITS rDNA. The purified products were sent to the Sequencing Department, University of Arizona and LaniVeg (CUCBA, UdeG). Sequences were assembled and edited in Chromas and aligned in MacClade. Furthermore, sequences obtained from GenBank were included. Pisolithus arrhizus was selected as outgroup. jModeltest was used to determine the best evolutionary model using the corrected Akaike information criterion. 11 macro and micromorphological characters representing more than 20 taxa were codified and included in the matrix along with 287 DNA bp, and analyzed by Maximum Parsimony and Bayesian Inference. Morphological characters were mapped with Mesquite 3.2. Additionally, ancestral state reconstructions (ASR) were obtained using BayesTrait 1.0. Some of the morphological characters with phylogenetic signal and taxonomic value were the ornamentation of the basidiospores and clamp connections. Three main linages were recognized, corresponding to the traditional classification proposed by Guzmán, as already detected by Phosri et al. in 2009, with three sections: Scleroderma (formerly Aculeatispora), Sclerangium, and Reticulatae (formerly Scleroderma). The synapomorphies for each clade were echinulated basidiospores for sect. Scleroderma, subreticulated for Sclerangium, and reticulated for Reticulatae. Besides, clamp connections are absent in sect. Scleroderma and present in the other two sections.
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.
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.
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.
Testing the ecosystem function of fungi with isotopes: A case study of nitrogen pollution and Russula
- N. Duncritts
- A. Pringle
Understanding how soil fungi use nitrogen will be critical to our understanding of fungal diversity in contexts of N deposition, a driver of global change. Recent studies have shown that when nitrogen is added to forest ecosystems, the fungal community changes and ectomycorrhizal fungal (EMF) abundance declines. However, at an experiment based at the Harvard Forest, at least one species of ectomycorrhizal fungus, Russula vinacea, proliferates in high nitrogen environments, challenging the general finding that EMF species decline when N is added. Stable isotopes of 13C and 15N have consistently revealed differences in the isotopic signatures of EMF and saprotrophic (SAP) fungi. I am using stable 13C and 15N isotopes from fungal sporocarps to probe how R. vinacea accesses nutrients in high nitrogen environments, and to test whether R. vinacea is, in fact, an EMF, or if it behaves as a saprotroph in high N contexts. I am collecting R. vinacea sporocarps from multiple N addition treatments at the Harvard Forest, including 0 N added, 50 and 150 kg N/ha/yr added, and extracting tissue for isotopic analysis in elemental analyzer machines. If the isotopic signature of R. vinacea is enriched in ∂13C and ∂15N, it will infer that the fungus is still accessing nutrients from a live plant symbiont. However, if the signature is depleted in ∂13C and ∂15N, a contrast to what is known about the isotopic signatures of EMF in general, data will suggest this fungus has found a new way to access nutrients in high nitrogen environments. The method I am developing is likely to pin point how surviving EMF are functioning in N enriched environments.
The early growth of conidia in an extraordinarily stressing environment is influenced by water activity during conidial production.
- P. Ruijten
- H. Huinink
- O. Adan
Fungal growth has negative aesthetical, structural and even medical effects. Growth prevention strategies are therefore desired. The influence of water on the growth of fungi is crucial when considering these strategies. Growth often prevails in a surrounding where both water and nutrients are scarce. The impact of this highly stressing environment during sporogenesis on subsequent growth is often neglected. The goal of this study is to investigate the effect of varying water stresses, in combination with depleted nutrient availability, on the early growth of spores cultivated under various circumstances. A nutrient-depleted substrate was therefore constructed. This was done by treating glass with ozone, thereby deleting carbon as a nutrient source. The water conditions during sporogenesis, as well as during subsequent growth, were varied. Spores of Penicillium rubens were harvested from colonies grown on Malt Extract Agar, MEA, plates with a varying water activity, aw. These spores were placed on the carbon depleted glass plates and placed in incubation chambers. The parameter describing the state of the water in the incubation chamber is the relative humidity, RH. Glycerol solutions were used to generate various RH values. The system under consideration is thus an extremely stressing environment: no carbon source is present, and water is provided solely via the vapour phase. Due to this harsh environment, only few spores could germinate, roughly 1 %. For the germinating spores the germination time, tg and initial growth rate, μ, were monitored. Despite this stressing environment, the expected proportionality between growth behavior and RH was found: tg decreases and μ increases with increasing the RH, for any value of aw during sporogenesis. Varying the spore history, via the aw, has an effect which depends on the RH of the environment. At low RH, spores produced at low aw have a lower tg and higher μ compared to those grown at high aw; i.e. the spores germinate and grow faster when grown at low aw. This is not found in a high RH environment, where the growth history had no effect. This last result was remarkably pronounced when the substrate was not only depleted of nutrients, but also made hydrophobic: growth only occurred when spores were developed at low aw and placed in a high RH. It has recently been found that pores grown on lowered aw can attract more water. This was linked to a varying amount of compatible solutes in these spores. This is hypothesized to justify the reported growth behavior. By deleting carbon as a nutrient source, the substrate used allows one to investigate the effect of a single parameter: water. Water availability during sporogenesis and during germination was independently varied. The influence of sporulation conditions on germination kinetics, which becomes even more pronounced in a more stressing environment, is clearly demonstrated. The fundamental role of compatible solutes in the survival and growth of fungi under stressed conditions can thus not be overlooked.
Population genomics of the deadly human fungal pathogen, Coccidoides (causing Valley Fever) in the southwestern United States
- J. Uehling
- M. Teixeira
- H. Mead
- B. Barker
- R. Brem
- J. Taylor
- A. Sil
The disease Valley Fever is caused by the fungal pathogen Coccidioides, which can kill otherwise healthy humans (i.e., those who are not immunocompromised, <10% of infections) when they inhale fungal spores from soils. Valley Fever (VF) is common in deserts of the Southern United States including California. In the last 10 years, VF infection rates have increased by over 8-fold. Although research is ongoing there are currently no vaccines, and for the deadliest infections treatment with currently available antifungal drugs (azoles) control infections while the strongest drugs (amphotericin) are not tolerated well by all patients. Elegant observational studies of Coccidioides have established its complex life cycle, disease etiology, and its interaction with the mammalian immune system. However, the molecular basis of VF infection biology remains very poorly understood. To date, studies have implicated a handful of Coccidioides genes in growth and virulence; but the pace of molecular genetics research has been limited by the requirement for BSL3 containment and the paucity of genomic tools available. Thus, the urgent need in the field is to accelerate the discovery of genes and proteins that underlie virulence-relevant traits in Coccidioides, which can ultimately serve as the targets for novel therapies and/or vaccines. Here we use population, evolutionary, and phylo- genetics to ascertain the fungal genetic diversity in Coccidioides populations from the Southeastern United States and to characterize target virulence genes with potential to guide the development of novel therapeutics. To evaluate population structure among the strains of this population, we extracted genotypes at microsatellite loci from a previously published study (Teixeira et al. 2015) and re-analyzed them with population-genetics and phylogenetic approaches. We found that clinical and environmental isolates are not genetically isolated and that there has been recent genetic interchange between them. Future data analyses will be centered on the generation of full genomes for individuals in this population and by genome wide association studies (GWAS) to elucidate genetics of virulence measured by growth and sporulation rates, production of melanin, antifungal resistance, and the ability to evade human immune system. Results of the Valley Fever population diversity analyses will be presented and discussed.
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.
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.
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.
Biological control of Fusarium wilt of tomato by application of Penicillium spp. and Chenopodium murale
- A. Javaid
- S. Naqvi
- A. Shoaib
Fusarium wilt of tomato, caused by Fusarium oxysporum f. sp. lycopersici, is an economically important soil-borne disease of tomato especially in warmer regions of the world. Fungicides used to control this disease also pollute the environment and cause health hazards. In the present study, this disease was managed by application of two antagonistic species of Penicillium namely P. digitatum and P. expensum, and dry biomass of a weed Chenopodium murale as soil amendments. The antagonistic fungi and different doses of dry biomass of the weed (1%, 2% and 3%) were applied in pathogen inoculated pot soil either separately or in combinations. The highest disease incidence (100%) was recorded in positive control where only fungal pathogen was applied. Different treatments of soil amendments reduced disease incidence to 3 – 23%. The lowest disease incidence (3%) was recorded in 2% C. murale biomass + P. expensum treatment. All the soil amendment treatments significantly enhanced shoot and root growth as well as fruit yield as compared to positive control. The highest fruit biomass was recorded in 2% C. murale biomass + P. digitatum treatment. The highest activities of peroxidase (POX), catalase (CAT) and polyphenol oxidase (PPO) were recorded in positive control. These enzymatic activities were significantly lowered when soil was amended with antagonistic fungi or C. murale biomass. Effect was more pronounced where C. murale biomass was applied either alone or combined with Penicillium spp. In laboratory bioassays, 100 mg mL-1 concentration of n-hexane and n-butanol sub-fractions of methanolic leaf extract completely controlled the growth of F. oxysporum f. sp, lycopersici. GC-MS analysis of these two fractions revealed that hexadecanoic acid; methyl linolenate and β-sitosterol were the most abundant compounds in n-hexane sub-fraction, and 1-heptanol; 3-hydroxyhexanoic acid; 1,2-decanediol and etiracetam were predominant constituents of n-butanol sub-fraction which may be responsible for antifungal activity of C. murale against the pathogen. This study concludes that application of 2% C. murale biomass + P. digitatum has the potential to significantly reduce Fusarium wilt of tomato and enhance tomato growth and yield.
Hyphomycetes in Pernambuco: a study in the tourism destination Ilha de Itamaracá, Brazil
- E. Malosso
- G. Da Silva
- W. Tavares
- R. Castañeda-Ruiz
The tropical Atlantic rain forest in Brazil consists of one of the largest reservoirs of biodiversity in the world. The region occupied by this forest has numerous rivers, streams and lakes surrounded by riparian vegetation in different status of conservation. The water systems of the Atlantic Forest is responsible for the maintenance of almost half of the Brazilian population, however, due to the beautiful landscapes, these regions are also very popular as tourism destinations and the intense flow of visitors in some areas have proven to impact the environment and endanger freshwater supply to some villages, like those in Itamaracá Island. To keep the riparian forest balanced it is important an effective cycling of nutrients from plants to soil/microbial biomass and back to plants. Therefore, a number of fungal species are found decomposing organic matter both in the soil and inside the water bodies. Among the taxa that are found in the aquatic environment and the riparian zone are the hyphomycetes, that are mostly asexual Ascomycota. This study aimed to investigate richness of hyphomycetes colonizing the leaf litter in the riparian zone and submerged in the Lagoa da Mata, Itamaracá Island, Brazil. This lake is approximately 43 Km² and is located inside the Environmental Protection Area (APA) of Santa Cruz, although it is used for tourism and swimming and open fire is not prohibited. Decomposing leaf litter was collected in the riparian zone and inside the lake in 6 sampling stations. The leaf litter was taken to the laboratory and processed according to the recommendations for each type of sample. After 3 days of incubation, microscope slides were mounted with fungal structures for identification of specimens. The analysis of the soil leaf litter resulted in 34 taxa of asexual Ascomycota with Circinotrichum maculiforme Nees, Beltrania rhombica Piroz. and Beltraniella portoricensis (F. Stevens) Piroz. & S.D. Patil as the most abundant species. From the submerged leaf litter, 13 taxa were detected with Xylomyces acerosisporus M.S. Oliveira, Malosso e RF Castañeda, Triscelophorus acuminatus Nawawi and Triscelophorus monosporus Ingold as the most abundant. The lowest number of taxa was found in the dry season. This is the first study reporting asexual hyphomycetes for Itamaracá Island. Financial support: CAPES and CNPq.