Welcome to IMC 2018 International Mycological Congress
Searching for microorganisms with a potential of plastic degradation (PET, LDPE).
- P. Goyes
The low cost and relatively short amount of time of fabrication of plastics, added to the disposable character of plastic-made products and low rate of recycling, has turned plastic pollution into an environmental concern. Plastic is one of the most remarkable causes of death of marine animals. This is a strong reason for us to do research on how to degrade plastic-made materials and remove them from the ecosystems. This study aims to evaluate the potential for plastic degradation among microorganisms like fungi and bacteria, found in soil and degrading biomass from trees in the Amazon, Rain forest and Andean region in Ecuador.
Differential regulation of Pleurotus ostreatus dye peroxidase genes in response to carbon source and dyes
- J. Cuamatzi-Flores
- E. Esquivel-Naranjo
- S. Nava-Galicia
- M. Bibbins-Martinez
The fungus Pleurotus ostreatus is a basidiomycete with worldwide distribution and is able to metabolize a wide variety of lignocellulosic and xenobiotic substrates. It has been the subject of many studies focused on bioremediation processes due to its multi-enzymatic machinery, which essentially comprise laccase (Lac), manganese peroxidase (MnP), versatile peroxidase (VP) and accessory enzymes such as dye peroxidase (DyP) and hydrogen peroxide generating peroxidase (H2O2). DyPs (E.C. 184.108.40.206) are hemeperoxidases that catalyze oxygen transfer reactions similar to the oxygenases, using H2O2 as a co-substrate electron acceptor and also an electron donor compound, which is oxidized to its respective radical. Its name reflects its ability to oxidize several types of dyes.
This research has determined the effect of different carbon sources and dyes on both the expression of Pleos-DyP genes via real time PCR (RT-qPCR) and dye peroxidase activity during the time-course of the submerged fermentation of Pleurotus ostreatus supplemented with either yellow azo (AA), remazol brilliant blue R (ARBR) and acid blue 129 (AA129) dyes, as well as glucose or glycerol as a carbon source.
With regard to the effect of the carbon source on enzyme activity, it was found that glycerol induced the activity levels up to three times compared to basal fermentation in which glucose was the carbon source. All fermentations supplemented with dyes also presented maximum activity values that were higher than those for the basal fermentation. The maximal activity in the basal fermentation was 1550 IU/L, compared to 4900, 3298, 2828 and 1744 IU/L for glycerol, AA, ARBR and AA129 fermentations, respectively.
On the other hand DyP gene expression profiles displayed up/down regulation over the fermentation time in the three DyP genes evaluated (Pleos-DyP1, Pleos-DyP2 and Pleos-DyP4). Glycerol induced Pleos-DyP1 and Pleos-DyP2 genes presented a relative expression (log2) of 10.01 and 11.96-fold increase, respectively, with both findings obtained at 504 h. AA addition caused the highest induction level for the gene Pleos-DyP4 with a relative expression (log2) comprising a 14.1-fold increase at the end of the fermentation process (552 h). Furthermore, the addition of ARBR caused the lowest induction level for the gene Pleos-Dyp2 with a relative expression (log2) of 5.05-fold at 360 h. On the other hand, the addition of AA129 caused the highest repression level for Pleos-DyP1 at 168 h with a negative relative expression (log2) comprising a -14.6-fold change.
In conclusion, the enzyme activity and gene expression profiles obtained in the different conditions evaluated show the influence of the carbon source, the nature of dyes and the growth phase on the differential regulation of the DyP gene expression in P. ostreatus.
Anaerobic fungi as a source of novel enzymes and antimicrobials
- R. Evans
- G. Griffith
- T. Callaghan
- S. Huws
Anaerobic rumen fungi (phylum Neocallimastigomycota) occupy the gastrointestinal tract of many herbivorous animals, and play an essential role in degrading food with a range of powerful hydrolytic enzymes. Whilst these enzymes are important to rumen efficiency, they can also be useful to biotechnological and biomedical industries. The rumen microbiome presents an underexplored source for novel microbial enzymes and metabolites, the former with biotechnological potential and the latter as potential drug candidates to target ever-increasing antimicrobial resistance. Rumen and faecal samples were collected from various large herbivores, and fungal cultures were grown and maintained under anaerobic conditions. After roll tube culture to isolate single-zoospore cultures, sequencing of LSU was undertaken to identify the fungi to species level. Analysis of genomic data from these cultures alongside data from several published studies has been undertaken to explore the diversity of lipases and antimicrobial peptides (AMPs) within the genomes of these fungi.
Projections for the production of enzymes of industrial interest by Ceriporia lacerata
- L. Carmona Saldarriaga
- L. Marín Palacio
Mushrooms have been widely studied for their production of extracellular enzymes with lignin-degrading ability. Enzymes are biological catalysts of great importance at cellular level, but also, they are of great interest at industrial level since they are key for certain reactions to occur, as they increase the rate of reactions without changing the equilibrium. Typically, enzymes are produced during fermentation processes of microorganism. However, low efficiencies and high costs are usually associated with their production. Therefore, bioprospecting for new microorganism for the production of enzymes is an important topic of research. In particular, Ceriporia lacerata, a native white-rot fungus found in Colombia, has not been largely explored for its ability to produce biologically active metabolites. Given the natural growth conditions of this fungus, it is likely that lignin-degradative enzymes are produced, such as: pectinases, amylases, laccases and cellulases. With this project, we aim at exploring the ability of C. lacerata to produce these four lignin-degradative enzymes under submerged fermentation conditions. First, the effect on fungal biomass and enzymatic production were assessed using two different flasks geometries and four different growth media, according to literature reports. Fungal biomass production was measured by dry weight, while enzymatic activity was determined using specific protocols depending on the kind of enzyme that wanted to be evaluated. In general, the enzymatic extract of each medium was added to a substrate solution, depending on the evaluated enzyme and either change in viscosity or absorbance values were recorded. The results of these evaluations showed that the geometry of the flask had a significant effect on biomass production but it did not affect enzymatic production. On the other hand, greater enzymatic activities were found for pectinases and cellulases than for amylases and laccases. In fact, for the latter enzyme, we have not been able to determine the enzymatic activity under submerged fermentation; even after evaluating the addition of different waste/by-products of food industry to the culture media. However, we found that when adding ABTS-like inductor under solid fermentation conditions, some degradation occurred, suggesting that laccases were produced. In conclusion, we found that C. lacerata was able to grow under different flask geometry conditions, while baffled-shake flasks increased the production of biomass. Also, we found that C. lacerata produced pectinases, celullases, amylases and laccases, although the greatest potential was observed for the production of cellulases and pectinases under the evaluated conditions.
Designer yeast strains for C1/3/5/6 biorefinery
- S. Han
Heterogeneous structure of lignin imparts plants with structural rigidity and also serves to protect cellulose and hemicellulose from degradation. Thus, prior to fermentative production of ethanol from the cellulose by yeast strains such as Saccharomyces cerevisiae and Pichia pastoris, the materials are degraded and hydrolyzed to release monomeric sugars. In this study, designer cellulosome was assembled in yeast Saccharomyces cerevisiae for utilizing of cellulose as the substrate. For utilizing of cellulose part in lignocellulosic biomass by simultaneous saccharification and fermentation, a recombinant scaffolding protein from Clostridium cellulovorans and a chimeric endoglucanase from Clostridium thermocellum were assembled as complex system. Compared to the results for single subunit, assembly of cellulosome-based enzyme complexes caused a noticeable increase in the level of enzyme activity. The resulting strain was able to ferment cellulose part in pretreated barley straw into ethanol with the aid of beta-glucosidase from C. thermocellum. The use of complexed enzyme systems is one of the strategies for effective lignocellulosic biomass hydrolysis. Enzyme complexes were formed via the interaction of a dockerin domain with cohesin modules in the scaffolding protein. Accelerating the biological degradation of lignocellulosic materials will benefit from the development of useful recombinant enzymes with hydrolysis ability. In future research, construction of designer enzyme complexes containing other lignin degrading enzymes could be used to develop biocatalysts that can completely degrade lignocellulose into single sugars.
Aspergillus terreus itaconic acid fermentation technology reflects the physiological requirements of overflow metabolism
- L. Karaffa
- Á. Molnár
- I. Kolláth
- B. Kovács
- Á. Soós
- C. Kubicek
- E. Fekete
Itaconic acid (2-methylenesuccinic acid; IA) is a five-carbon dicarboxylic acid, with one of the carboxyl groups being conjugated to a methylene group – a feature that makes IA easy to (co)polymerize. It is thus used as a building block chemical for the synthesis of a variety of compounds with a broad range of applications, such as manufacturing plastics, coatings and resins. IA is commercially produced by large-scale submerged fermentations employing the filamentous Ascomycete fungus Aspergillus terreus. Although production of IA is the result of overflow metabolism similar to that leading to citric acid (CA) production in A. niger, available data for IA yields are significantly lower than for CA. CA is known to accumulate to high levels only at high concentrations of rapidly assimilating carbon sources, strong aeration and severe Mn(II) ion limitation. Here we show that these three parameters similarly influence IA production and yield by A. terreus. Mn(II) ion concentrations lower than 3 micrograms per liter were shown necessary to obtain highest IA yields. However, Mn(II) ion concentrations higher than that could be antagonized by 50 milligrams per liter Cu(II) ions. Yields are also dependent on the concentration of the carbon source (D-glucose) – highest yields (0.9) only obtained at concentrations of 12 – 20 % (w/v). As for high aeration requirements, we demonstrated that A. terreus is capable of growth-supporting respiration in the presence of KCN. Responsible for this activity is the cyanide-resistant alternative oxidase (AOX) that is located on the matrix side of the inner mitochondrial membrane and branches off the canonical cytochrome-dependent pathway at the level of ubiquinon, leaving complexes III and IV of the electron transport system bypassed. The alternative pathway thus moves much fewer protons across the inner mitochondrial membrane to generate a proton motive force, and provides less ATP for energy conservation. In effect, AOX uncouples the re-oxidization of NADH from ATP synthesis, thereby allowing intense carbon assimilation to continue – a prerequisite for high IA yields. The genome of A. terreus specifies two paralog genes (aodA and aodB), each putatively encoding an AOX. During IA fermentations mimicking industrial conditions, aodB displayed constitutive but weak expression at each of the time-points tested. In contrast, the aodA expression levels strictly and positively correlated with increasing dissolved oxygen (DO) levels as well as with the final IA yield. Hence, high DO levels required for high-yield IA fermentations on D-glucose may be related to AodA, putatively encoding a cyanide-resistant alternative oxidase.
Production and characterization of Penicillium purpurogenum MM1 cellulases and its application in biobolishing of cotton textile
- M. El-Metwally
The modern textile industry has become familiar with the use of cellulases in a number of mechanical and chemical operations to improve the comfort and quality of textiles. For this purpose deteriorated cotton textiles samples were used to isolate cellulolytic fungi that produce cellulases in quantity and quality suitable for the bioboloshing process. Among many isolates, Penicillium purpureogenium MM1 recorded the highest enzyme activity reaching to 45.7 , 8.0 and 117.8 u/ml for CMCase, Avicelase and b-glucosidase respectively and liberated the largest amount of reducing sugar (144.1 mg/ml) in cotton textile treatment. The partially purified cellulases have optimum temperature of 40°C and optimum pH 4 with good half life time reach to four hours at 60°C for CMCase and Avicelase, three hours at 70°C for b-glucosidase. In biobolishing trials carried out at the Masr textile company, P. purpureogenium MM1 cellulases have the same efficiency as the imported ones being used (Denimax 992).
A novel laccase from white rot fungus Trametes orientalis: Purification, characterization and application
- J. Si
- F. Zheng
- B. Cui
- Y. Dai
A novel laccase (Tolacc-T) from white rot fungus Trametes orientalis was enriched to apparent homogeneity with a specific activity of 20.667 U/mg protein and recovery yield of 47.33%. The SDS-PAGE gave a single band indicating that Tolacc-T appears a monomeric protein with a molecular mass of 44.0 kDa. Domain structure analysis revealed that Tolacc-T contained a typical copper II binding domain and shared three potential N-glycosylation sites, but had no copper I binding domain, demonstrating that the enzyme is really a laccase, but a novel laccase. Optimal pH and temperature of Tolacc-T was 4.0 and 80 °C, respectively, and it retained more than 80% of its original activity after 2 h incubation at 10 °C to 50 °C. The enzyme exhibited strict substrate specificity towards ABTS but showed no or trace activities towards other substrates. Among the metals tested, Mn2+ was proved to be the best activator for enhancing the laccase activity. A strongly inhibiting effect was found when NaN3, L-cysteine, and DTT were added to the enzyme. However, Tolacc-T activity was little bit inhibited in the presence of chelator EDTA. Furthermore, the enzyme was capable of degrading structurally different synthetic dyes in the absence of a redox mediator.
DNA-free genome editing in Beauveria bassiana (Hypocreales, Ascomyota) with CRISPR-Cas9 ribonucleoproteins
- M. Hyun
- G. Sung
- J. Kim
- J. Cho
- S. Lee
- K. Lee
- W. Kong
- J. Han
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–Cas9 is an adaptive immune system of prokaryotes. Recently it became the most powerful molecular biology tool, which can induce double strand breaks (DSBs) via non-homologous end joining (NHEJ) or homology-directed repair (HDR). The CRISPR-Cas9 system facilitated the precise genome editing in microorganisms, plants and human, with relatively simple protocol. Recently RNA-guided engineered nucleases (RGEN) delivery method that consists of the preassembled Cas9 protein and gRNA, have been successfully applied in plant and human cells. In this convenient straightforward delivery system, the codon optimization and other genetic manipulation for specific organisms are not necessary. Despite these advantages, the CRISPR-Cas9 system has not been widely adapted for fungi since the routine plasmid-based transformation has the complicated process to generate mutant in laboratory. In the present study, we purposed to improve the fungal transformation efficiency with DNA-free CRISPR Cas9 method, with the entomopathogenic fungus Beauveria bassiana. Obtained putative transformants were sub-cultured 3 times to confirm their mitotic stability. The RGEN induced mutations and the genome editing efficiencies for target genes were verified using Sanger and high-throughput deep sequencing. Our results revealed that DNA-free CRISPR-Cas9 is an efficient and useful gene editing system for B. bassiana.
Beauveria bassiana: Potential Biological Control against Coffee Berry Disease
- L. Serrato-Diaz
- Y. Marino-Cardenas
- A. Soto-Bauzo
- P. Bayman-Gupta
Beauveria bassiana (Bb) has been used successfully as a biological control against the coffee berry borer (CBB). Although interactions of Bb, CBB and Coffee Berry Disease (CBD) are frequent, information about the use of Bb as a biological control for plant pathogens is limited. During 2016 - 2017, a survey was conducted to evaluate the potential of Bb in controlling CBD in commercial and experimental coffee farms in Puerto Rico. A total of 8162 fruits were collected and evaluated. Coffee fruits with and without Bb were dissected to evaluate internal and external fruit rot. Coffee fruits with Bb had significantly less internal and external rot than coffee berry fruits without Bb (12.4% vs. 18.8%), suggesting Bb as a potential biological control against CBD caused by Colletotrichum spp. To test effectiveness of Bb against CBD, three isolates of Colletotrichum spp. and one isolate of Bb were inoculated on coffee fruits. Experiments were conducted in laboratory and field conditions and were repeated twice. A total of 300 coffee fruits were drilled and inoculated. All rotten coffee fruits were plated on PDA and fungi were identified, fulfilling Koch postulates. In laboratory and field conditions, coffee fruits inoculated with both Bb and Colletotrichum spp. had significantly lower percentage rot than fruits with Colletotrichum spp. alone (as did control fruits). The protective effect of Bb against CBD caused by Colletotrichum spp. is an important discovery and suggests an additional role for Bb in programs of disease management in Puerto Rico and other coffee-producing countries.
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.
Exploiting lichen-associated bacteria for biocontrol of soil-borne pathogens and stress protection of plants
- M. Grube
- T. Cernava
- G. Berg
AbstractSymbioses are a hotspot for cross-kingdom communication and control. Bacterial communities were identified as stable, specific and structurally integrated partners in the classical lichen symbiosis. Their roles in the symbiosis of lichens are supported by metagenomic, -proteomic and metabolomic data. Functions include (i) nutrient supply, especially nitrogen, phosphorous and sulfur, (ii) resistance against biotic stress factors (that is, pathogen defense), (iii) resistance against abiotic factors, (iv) support of photosynthesis by provision of vitamin B12, (v) fungal and algal growth support by provision of hormones, (vi) detoxification of metabolites, and (vii) degradation of older parts of the lichen thallus. The results were confirmed by bridging multi-omics with culture studies. Our findings showed the potential of lichen-associated bacteria to interact with the fungal as well as algal partner to support health, growth and fitness of their hosts. As the microbiome of lichens comprise a high number of bacterial species (>800 in Lobaria pulmonaria) we started to exploit this diversity for biotechnological applications. We found that bioactive volatiles produced by lichen-associated bacteria are able to suppress soil-borne pathogens. Metatranscriptomic datasets from bacterial communities of the lung lichen also showed that host-associated bacterial communities are well-adapted to dehydration by stress protective mechanisms and changes of metabolic processes. The results indicate an intense interplay in holobiont function under drought stress. These capacities can be exploited for plant protection strategies.
A first look at culture-dependent endophytic fungal diversity of wild Rubiaceae in Costa Rica.
- E. Escudero
- H. Castillo
- P. Juárez
- M. Granados
- E. Alvarado
- P. Chaverri
Global warming effects, like longer rainy seasons and higher temperatures are increasing diseases on coffee plantations around the world and thus affecting the producers' economies. Endophytic fungi with plant-beneficial properties have been proposed as an alternative to the use of fungicides. Because the coffee family, Rubiaceae, is highly diverse in Costa Rica, we recently started to characterize their endophytes with the idea of finding beneficial fungi that could be applied in improving coffee-plant health. The objectives of this preliminary study were to identify the culturable endophytic fungi from several wild Rubiaceae species and do in planta antagonism tests against the pathogen Mycena citricolor. Culturable endophytes were isolated from 49 Rubiaceae species: Allenanthus erythrocarpus, Bertiera bractiosa, Guettarda macrosperma, Palycourea eurycarpa, Pentagonia costaricencis, and Psychotria pubescens, among others, collected from two regions in Costa Rica, i.e., Guanacaste Conservation Area and the Osa Peninsula. Cultures were identified using the nrDNA ITS region. The culturable endophyte fungi mainly obtained from leaves were e.g., Clonostachys spp., Endomelanconiopsis endophytica, Lasiodiplodia theobromae, Simplicillium aff. lamellicola, Diaporthe spp., Phyllosticta spp.; and others like Trichoderma deliquescens, T. virens and T. rifaii were extracted from the stems. Psychotria solitodinum and P. panamensis were the most diverse in the Osa Peninsula, and Bertiera bracteosa, Coutarea hexandra, Psychotria pubescens, were the most diverse in the Guanacaste Conservation. At the moment, 419 taxa are identified, 397 from Ascomycota and 21 from Basidiomycota. The most frequent families in the Ascomycota are Botryosphaeriaceae, Glomerellaceae and Pestalotiopsidaceae; and in the Basidiomycota Ceratobasidiaceae and Meripilaceae. Colletotrichum was the prevalent genus in both areas and within Rubiaceae species. A preliminary antagonism was performed applying T. rifaii on two varieties of coffee plants, “Catuaí” and “Caturra” in order to try to reduce the infection caused by Mycena citricolor. Results show a significant reduced infection on the plants treated previously with T. rifaii specially on the “Caturra” variety. Additional molecular markers will be used to refine the taxonomy of cryptic species and more in vivo essays will be done. We think these preliminary results are capable to predict a noticeable improvement of the coffee plantations health.
The discovery and characterization of Ustilaginomycetes yeasts reveal enzymatic activity, resistance to antifungal drugs and species complexes.
- T. Rush
- A. Pringle
- E. Dominguez
- W. Bankeeree
- A. McTaggart
- P. Chong
- S. Prasongsuk
- H. Punnapayak
- T. Seelanan
- P. Lotrakul
The plant biodiversity of Thailand and Malaysia is estimated at over 10,000 species, each growing in an unique geography and climate. These plants and their surrounding habitats offer opportunities for the discovery of yeasts with novel physiological and enzymatic properties, and in recent years there has been a steady increase of yeast discoveries from these and other developing countries. From November 2012 – January 2013, three unique strains (TAR 509, TAR 520, and TAR 523) of anamorphic, basidiomycete fungi were cultured from a leaf of Pistia stratiotes (water lettuce) growing in Bangkok, Thailand and from a Hibiscus sp. in Kuala Lumpur, Malaysia. Despite having only one strain for each of the novel fungi reported here, we reveal novel physiological and chemical characteristics as well as phylogenetically distinct fungal lineages. Using concatenated nuclear ribosomal gene sequences of the large subunit and internal transcribed spacer regions, we determine that TAR 509, isolated from P. stratiotes, belongs to the recently discovered family Fereydouniaceae, in the Urocystidales. TAR 520, isolated from Hibiscus, is a novel species of Dirkmeia, and TAR 523 is the first characterization of the anamorphic stage of Ustilago sparsa, both in the Ustilaginales. We present evidence that these three strains have strong production of amylase, cutinase, lipase, and xylanase. Furthermore, scanning electron microscopy reveals that Dirkmeia sp. strain TAR 520 has resistance to antifungal drugs in the echinocandin class, measured by minimum inhibitory concentration analysis.
Moreover, data identify possible species complexes within the genera Fereydounia and Dirkmeia, based on both maximum likelihood and Bayesian analyses. We believe that Fereydounia sp. strain TAR 509 is a new species based on differences in its growth in osmotic pressure and temperature ranges, as well as morphological and ecological differences when compared to the type species of F. khargensis. Finally, a recent study showed that foliar applications of Dirkmeia churashimaensis strain RGJ1 on pepper generated antiviral and antibacterial resistance against pathogens. However, our phylogenetic analyses suggest that this strain is not D. churashimaensis, instead, it clusters with our novel species of Dirkmeia. Our newly described species is the second know species of the genus. Our discoveries are evidence that many novel species of Ustilaginomycetes may occur in understudied tropical areas.
Where do you fit in the mycological genealogy?
- C. Mayers
- D. Haelewaters
- J. Stajich
- M. Blackwell
AbstractDo you know where you belong in the great family tree of mycologists? Academic Family Tree (https://academictree.org/) is a collaboratively-edited, continuously updated online database of scientists connected in a family tree with advisors in place of parents and their students in place of children. Trees are being built in many fields, from law and crystallography to biomechanics and Drosophila genetics. An offshoot of this project is Mycotree, a database of mycologists, which is currently highly underpopulated. While we did not initiate Mycotree, we want to raise awareness for this unique resource and encourage participants of IMC11 to contribute their “family” in this unique opportunity to preserve mycological history in one place. The accompanying poster includes a representative segment of Mycotree beginning at the authors’ leaves and spanning back to such luminaries as Heinrich Anton de Bary and Ernst Haeckel, as well as an example biographical profile. Scientist profiles are more than just nodes in a tree; they allow additional biographical details to be added, such as university positions, dates of supervision of students, links to published biographical sketches, etc. The poster also covers instructions for helping contribute your lineage and knowledge. Adding or editing biographical entries and connections in Mycotree simply requires a free account and making connections between mentors and students is easy. By leveraging the diverse attendees at this year’s meeting to combine their knowledge for the greater mycological good, we hope that Mycotree will grow to accommodate a truly global perspective of the long and storied history of mycology!
Oral history for mycology
- M. Blackwell
- R. Samson
Relatively few videos of mycological reminiscences exist. Imagine if Margaret Barr, Lilian Hawker, Richard Korf, Erast Parmasto, Emory Simmons, and C.V. Subramanian had left their stories for us on video in addition to their still images and memories printed in memorials. The Congress offers an opportunity to record interviews with mycologists, including their assessments of changes that occurred over their careers and the experiences of other mycologists who are just beginning their careers. Three participants represent the variety of mycologists who will participate in a scheduled session at IMC 11. General questions will be given to participants in advance to provide direction for thoughtful, smooth-flowing interviews. Videos will be housed at the Hunt Institute for Botanical Documentation, Carnegie Mellon University, which makes botanical and mycological materials of historical interest widely available. Interviews would be available for teaching. Additional interviews will be recorded throughout the Congress, so please sign up here.
Impact of Aspergillus nidulans signaling mutants on growth and secretion on non-preferred carbon sources
- S. Reese
- L. Ries
- T. Reis
- G. Goldman
- S. Harris
Filamentous fungi are the leading producers of fermentation enzymes. The use for these industrial enzymes are vast ranging from; food fermentation, textile production, leather modification, and chemical detergent processing. The biotechnological supply for enzyme production is in growing demand, and unlocking the knowledge of enzyme secretion pathways is vital to industrial profits. Secretion pathways are coherently linked to fungal morphology, and studying fungal growth patterns could provide insight into these elusive pathways. In filamentous fungi, the Cdc42 and Rac1 GTPases are required for normal hyphal morphogenesis, but their roles in enzyme secretion and morphological adaptation to carbon source shifts have not been investigated. We characterized the effects of Aspergillus nidulans Cdc42 and Rac1 deletion mutations on the activities of secreted cellulase and xylanase. In addition, we determined how these mutations impact morphological responses to a shift from glucose to non-preferred carbon sources. These small GTPases mutants were grown in rich media then switched to a non-preferred carbon source for further growth. The carbon source shifts were marked with a Wheat Germ Agglutinin (WGA) fluorescein labeling, and pictures where taken with a GFP filter. Using ImageJ, the hyphal growth patterns were analyzed for differences in hyphal dimensions. Our results suggest that Cdc42 regulates the morphological transition to non-preferred carbon sources. Cdc42 may also selectively regulate the expression of specific hydrolytic enzymes (cellulases vs. xylanases). Conversely, Rac1 appears to play a more global role in the expression or trafficking of hydrolytic enzymes. Further study of these GTPases and their effectors should provide additional insights into the relationship between fungal morphology and secretion pathways.
Neurospora crassa has AP180, a clathrin adaptor in the endocytic collar but not clathrin
- M. Garduño-Rosales
- F. Lara-Rojas
- O. Callejas-Negrete
- S. Bartnicki-Garcia
- R. Mouriño-Pérez
Endocytosis is essential for polarized growth and morphogenesis in filamentous fungi. It is highly concentrated in a specialized collar at the hyphal subapex, very close to the hyphal tip. Whether endocytosis in the subapical collar is clathrin dependent is still not clear. In this work, we examined the dynamics and localization of AP180, an adaptor of the clathrin coat, by fluorescent-protein tagging in Neurospora crassa, using laser scanning confocal microscopy, spinning disk confocal microscopy and total internal reflection fluorescence microscopy. Additionally, we analyzed the deletion mutant, and a mutant where the ENTH endocytic domain is absent. We observed that AP180 was located forming patches in the endocytic collar and in septation sites. Fluorescence signal was also detected as highly dynamic scattered patches and tubular structures along the hypha, which moved both in retrograde and anterograde direction. Some patches seemed to be aligned, and some even fused with each other. The deletion mutation of AP180 was lethal for N. crassa, while the deletion of the ENTH domain is not lethal but it affects the protein localization at the subapical collar. It is curious that AP180 localizes at the endocytic collar even if clathrin does not. In conclusion, AP180 is an essential protein, part of the endocytic machinery, the absence of clathrin in the endocytic collar suggest that AP180 might be working as part of a clathrin independent pathway.
Identification and functional testing of Wsc1-3, Mid2 and Mtl1 protein-protein interactions forming novel stress signaling complexes in yeast
- E. Santiago-Cartagena
- J. Rodríguez-Medina
- V. Vélez-Segarra
- N. Martínez-Matías
- M. Babu
- B. Rymond
- I. Stagljar
AbstractIntroduction & Objectives: Signaling proteins required for activating the yeast PKC1-Cell Wall Integrity (CWI) pathway are attractive targets for antifungal drugs because they contribute to cell viability under stress. The objective of this research is to identify proteins that interact with stress sensor proteins Wsc1p, Wsc2p, Wsc3p, Mid2p and Mtl1p of the PKC1-CWI pathway and determine if they contribute to cell survival under stress conditions. Our hypothesis is that these interacting partners are required for resistance to antifungal drugs and environmental stress.
Methods: To identify novel interacting partners, the integrated Membrane Yeast Two-Hybrid (iMYTH) technique was applied. Confirmatory tests were Immunoprecipitation coupled to Mass Spectrometry (IP-MS) and Affinity Purification with Western blot (AP-WB). To test the functional importance of specific interactions, viability assays were performed under stress conditions induced by Caspofungin (CS, 75ng/ml), temperature, and an oxidizing agent (1mM H2O2). PKC1-CWI pathway activation was assayed by Western blot.
Results: At 30°C, 14 novel interactors were confirmed for Wsc1p, 31 for Mid2p, 14 for Wsc2p, 5 for Wsc3p, and 15 for Mtl1p. Interacting proteins were associated with biological processes of signal transduction, stress response, cell wall organization, cytoskeleton organization, and unknown functions. Secondary tests confirmed 14 interactors for Wsc1p, 13 for Mid2p, 2 for Wsc2p and 3 for Wsc3p. Confirmatory tests for Mtl1p interactors are underway. Null mutant strains of stress sensors or their interacting partners acquired sensitivity to H2O2 (3) and CS (6). Double mutant strains atx1Δmid2Δ, grx1Δmid2Δ, ras2Δmid2Δ and ras2Δwsc1Δ acquired sensitivity to both CS and H2O2. Ras2p expression was required for PKC1-CWI pathway activation by oxidative stress.
Conclusions: Novel stress signaling proteins involved in PKC1 regulation were identified. These were shown to be associated with yeast cell survival under stress conditions.
Acknowledgements: This research was supported by grants from NIMHD-G12MD007600, 8U54MD007587, NIGMS-RISE-R25GM061838, NIGMS/NIAID-SC1AI081658, NIGMS-INBRE 5P20GM103475, Canadian Institutes of Health Research (MOP: 125952), Ontario Genomics Institute (RP005766), Canadian Cystic Fibrosis Foundation (2847), Canadian Cancer Society (700406, 702109), and University Health Network (ORF GL2-01-018).
Cleaning a multinuclear single cell during fruiting body formation of myxomycetes
- Y. Yajima
Myxomycetes (true slime molds) have a multinuclear single cell stage, plasmodium, which makes fruiting bodies containing numerous myxomycete spores. It has been fragmentary reported that degradation of cellular comportments such as organelle degeneration and deterioration of a layer of peripheral cytoplasm during fruiting body formation. The possible roles of such degradations in the life cycle of myxomycetes also have been barely discussed, however, the risk of its insufficiency has not been emphasized. For cellular integrity, it is critical to clean the cell of damaged organelles, aggregated proteins and invaders. The objective of this study is to determine how a single cell itself cleans up own used/aged cell components to generate pre-gamete cells, i.e., myxomycete spores. Sequential steps of cell cleaning behavior in myxomycete fruiting body formation analysed by two- and three-dimensional imaging. Organelle clearances especially on nucleus were observed with broad visual field and electron microscopic 2D/3D. DNA fragmentation was examined with cryo-fixed cell. Cleaning process during fruiting body formation were different among species, and the relation with construction of fruiting body structures was observed. DNA fragmentation was detected in multiple nuclei, and its distribution in a single cell differed in the stages of fruiting body formation. The selection of reduction processes in the number of nuclei was also depended on the stages. The results suggested that a myxomycete cell behave sensibly during the fruiting body formation, and cleans up own single cell without rupture or complete cell death.
Aureoboletus projectellus - new invasive bolete, rapidly spreading in Europe as an opportunity to study invasions of macromycetes.
- Ł. Istel
- J. Pawłowska
- A. Okrasińska
- Ł. Banasiak
- M. Gorczak
- M. Wrzosek
American bolete Aureoboletus projectellus was reported in Europe in the beginning of the century. At first it was only observed on the Baltic Sea shore. Since 2014 the occurrence range of the species has increased significantly. It now reaches more than 150 km into land and covers eight countries. Main aim of the study was to establish a framework for studying invasion of macrofungus in statu nascendi based on the spread of A.projectellus. A model prepared in MaxEnt was used to estimate distribution of potential niches of A. projectellus in Europe. Comparing model together with data about species distribution in invasive range were combined to predict way of future dispersion and to help to prepare a list of location that should be monitored. Baltic Sea coast line seems to be one of the most suitable area for invasive range. The continuous ring of favorable conditions all around Baltic sea shows that the invasion is unstoppable but can be suitable for studying invasion process. Other disjunctive areas of favorable habitats include Alpine foothills and small remote mountains located on the south of Europe (for example Apennines and Pyrenees). Invasion in remote islands can be probably slowed down or even stopped by preventing the transport of edible fruit bodies or planting pines which is mycorrhizal partner of A. projectellus. Every attempt to stop the spread of invasive fungus is important because A. projectellus can be a threat to domestic/native species of mushrooms (eg, Tricholoma focale or T. apium).
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.
Long-term nitrogen fertilization alters nitrogen isotopes and concentrations in ectomycorrhizal fungi, hosts, and soil in pine forests
- E. Hobbie
- N. Hasselquist
To assess how nitrogen (N) availability affected functioning of ectomycorrhizal fungi and interactions among ectomycorrhizal fungi and plants, we measured %N and δ15N in soils, foliage, and nine ectomycorrhizal fungal taxa in six-year (Rosinedalsheden) and 40-year (Norrliden) N addition experiments in Pinus sylvestris stands in northern Sweden. The %N of the F horizon correlated strongly with sporocarp %N, reflected nitrogen addition history, and served as a useful proxy for N availability. Both sporocarp %N and soil %N increased with fertilization, implying that nitrogen uptake per unit fungal growth and protein content of sporocarps increased with N fertilization. In addition, foliar %N correlated strongly and positively with foliar δ15N, with both increasing with N additions. Our prior growth chamber studies indicated that this positive relationship is related to declines in carbon allocation to ectomycorrhizal fungi at high N availabilities. Thus, we suggest that δ15N in ectomycorrhizal Pinus can be used as a proxy for carbon allocation patterns to ectomycorrhizal fungi and a useful indicator of the extent of alteration of plant-fungal interactions across N availability gradients. Patterns in fungal and soil δ15N across the two sites suggested that N acquisition was primarily from the H horizon for Cortinarius traganus and Russula aeruginea, from the S horizon for Lactarius rufus and Paxillus involutus, and from the F horizon for the other five taxa. Higher 15N enrichments in Cortinarius semisanguineus, Suillus variegatus, and Paxillus involutus relative to source N with increased N availability suggested enhanced transfer of N to plants (higher transfer ratios), whereas other taxa did not change transfer patterns. Our results indicated that sporocarp nitrogen concentrations and δ15N patterns were useful integrators of fungal responses to N addition, and should be useful in gauging fungal responses to other environmental perturbations.
Ergosterol and nitrogen levels provided by ophiostomatoid fungi associated with the mountain pine beetle (Dendroctonus ponderosae) host tree colonization
- S. Guevara-Rozo �
- A. Hussain
- J. Cale
- N. Erbilgin
Mountain pine beetle (Dendroctonus ponderosae, Coleoptera: Curculionidae) has killed pine trees on millions of hectares of forest over the past decade in western North America. In Canada, beetle outbreaks had been historically restricted to central British Columbia, but has been recently expected its host range into jack pine (Pinus banksiana) forests in Alberta. The beetle inoculates fungal spores of symbiotic, ophiostomatoid (Ascomycota: Ophiostomataceae) fungi as it colonizes host pines. Three most common fungi species associated with the mountain pine beetle in Alberta are: Grosmannia clavigera, Ophiostoma montium, and Leptographium longiclavatum. These fungi play important roles in beetle host range expansion and survival as they help to overwhelm tree defenses and are an important source of dietary nitrogen and ergosterol for developing beetles. Nitrogen is an important constituent of proteins, nucleic acids, and hormones and can increase in the phloem when the fungi associated with the mountain pine beetle are present. Sterols, on the other hand, are needed for beetle metamorphosis and reproduction and in the case of the ergosterol, its availability changes depending on the fungus species. Our objective was to determine the relative benefit of each of the fungi to mountain pine beetle in terms of their ability to concentrate nitrogen in pine phloem and produce ergosterol along tree stems. Eighty healthy jack pines occurring in two forest stands in Lac La Biche (Alberta, Canada) were inoculated with live plugs from cultures of the three fungi or non-colonized media (control) at three heights along the tree bole: 1m, 3m, and 5m (twenty trees per treatment). Phloem samples were taken during inoculation process to determine constitutive nitrogen levels prior to fungal growth at each height. Six weeks after inoculation, phloem samples were taken from inside and outside of phloem lesions resulting from fungal infections. These samples were analyzed for total nitrogen and ergosterol levels through dry combustion and ultra-performance liquid chromatography, respectively. Nitrogen and ergosterol levels by fungi species and heights will be presented and discussed.
Mycorrhizas, masting, and monodominance in the Neotropical leguminous canopy tree Dicymbe corymbosa
- T. Henkel
- J. Mayor
Pulse-like bursts of heavy seeding followed by several low-to-no reproductive years (i.e. “masting”) are well known from ectomycorrhizal (ECM) trees which dominate high latitude forests, e.g. Pinaceae, Fagaceae. In tropical rainforests masting has been documented in ECM tree species of Fabaceae subfam. Detarioideae and Dipterocarpaceae, which are often locally dominant or co-dominant. Masting may be an evolved trait based on an “economy of scale” mechanism for enhancing seedling establishment. In remote tropical rainforests of Guyana, from 1998–2017, we investigated annual seeding, seedling survivorship and growth, and ECM colonization of the monodominant canopy tree Dicymbe corymbosa (Fabaceae subfam. Detarioideae) – a species with highly synchronous supra-annual seed production. Seed output, predation, and nutrient investment were quantified for each seeding year in primary D. corymbosa forests following the 2003 mast. Establishment, survival and mycorrhization of seedling cohorts were monitored, and climatic conditions associated with seeding events were assessed. Mast seeding occurred in 1998, 2003 and 2016, with three intervening low seed years, indicating a strongly bimodal seeding pattern for D. corymbosa. El Niño-intensified dry season conditions preceded each masting event and regionally synchronized the flowering. The attendant increased solar irradiance appears to magnify flowering and fruiting through increased carbohydrate production. Similarly, ECM-mediated phosphorus (P) cycling appears paramount to facilitating these masting events by (1) refueling stored foliar P to threshold levels and (2) returning subsequent P losses in reproductive parts. Seedlings require rapid ectomycorrhization to persist in high densities following masting events. While > 300 species of ECM fungi occur in these D. corymbosa stands, the ECM fungi dominating seedlings were mainly a handful of Tomentella species which also dominate adult trees. Seedlings persist with little growth for decades in the heavily shaded understory, but are able to respond quickly to canopy openings. Masting contributes the greater part of standing seedling crops, and along with efficient nutrient recycling by ECM fungi, is critical for persistent monodominance in D. corymbosa.
Ectomycorrhizal fungal communities associated with Abies koreana in relation to abiotic environmental factors
- L. Jieun
Ectomycorrhizal (ECM) fungi have a symbiotic relationship with woody plants and they play an essential role in nutrient cycling and maintaining the ecosystem. Abies koreana is an endemic species in Korea and found mainly in the sub-alpine areas of Mt. Halla in Korea. The A. koreana population has declined and has been designated as critically endangered, which might be due to climate changes. The climate changes also affect the communities of host plants and ECM fungi. This study was conducted to investigate the effects of abiotic factors on ECM fungal community associated with A. koreana. We sampled the roots and rhizosphere soils of two host plant species, Abies koreana, Taxus cuspidate, in two different climate zones, subalpine and temperate, of Mt. Halla. We identified ECM using morphological characteristics of ECM root tips and sequence analysis of internal transcribed spacer regions of rDNA. ECM fungi in the soil were analysed using Illumina Miseq. We detected a high correspondence between fungal diversity by a molecular analysis of the root tips and soil samples. The ECM fungus communities in the roots and soils were significantly different in terms of biotic factors. The results suggest that ECM fungal communities differed in different climate zones and were affected by abiotic environmental factors.
Biogeographic pattern of Collembola fauna associated with the insecticidal mushroom Strobilurus ohshimae in Japan
- T. Nakamori
- K. Kamata
Organisms interact with various other species, and the differences in the associated species drive diversification. The variability in the interactions between macrofungi and fungivores are poorly understood. The basidiomycete Strobilurus ohshimae, which fruits exclusively on fallen twigs of Cryptomeria japonica, has sporocarps covered with secretory projecting cystidia that can kill Collembola on contact. Collembola are wingless soil microarthropods, some of which feed on macrofungal sporocarps in huge numbers. Some Collembola species avoid S. ohshimae sporocarps, while others feed on them. We investigated the geographic distribution of Collembola species feeding on S. ohshimae sporocarps in 12 native C. japonica forests in Japan. The presence of potential sporocarp feeders was surveyed using bait-traps containing Shiitake mushrooms. The preference for S. ohshimae was evaluated by determining the density of S. ohshimae per sporocarp mass relative to that for Shiitake mushrooms. The presence, species, and mode of feeding of Collembola preferring S. ohshimae varied with the site. In Akita, Ceratophysella tergilobata fed mainly on the gills of S. ohshimae, while Ceratophysella sp. 1 fed mainly on the interior parts of the stipes. In Yamanashi, Kyoto, Wakayama, Oki Is., Shimane, and Kochi, Ceratophysella pilosa or closely related taxa preferred S. ohshimae and fed mainly on the interior parts of caps. In Miyagi and Yakushima Is., no Collembola showed preference for S. ohshimae. Ceratophysella denisana, which is distributed widely, and Ceratophysella aff. horrida never showed preferences for S. ohshimae. Ceratophysella sp. 2 preferred S. ohshimae in Shizuoka, but not in Kochi. Morulina alata fed on S. ohshimae in Akita, Sado Is., Toyama, and Oki Is., but did not show strong preferences. These results suggest that the strength and mode of impact of Collembola grazing on S. ohshimae varied geographically.
Fungi of Mortierella section lignicola as potential partners of ants
- M. Wrzosek
- I. Siedlecki
- M. Gorczak
- M. Kopczyński
- J. Pawłowska
Mortierella is a genus containing nearly 100 species. It belongs to one of the basal phylogenetic lines of terrestrial fungi (Mucoromycota). Mortierella species are generally known as prototrophic organisms living in oligotrophic habitats. Wagner et al. (2013) provided a comprehensive molecular phylogeny of the order Mortierellales based on nuclear ribosomal DNA and divided all members of the order into seven clades. The aim of the study was to trace the presence of fungi belonging to Mortierella on ant’s bodies and check the insect’s response to presented mycelia in nature. We used the method of isolation of fungal strains straight from cadavers to artificial media and analyzed films documenting ant’s behavior in presence of fungal mycelia. Results of our studies revealed that most Mortierella strains isolated from healthy ants belonged to Wagner’s clade "3" also called lignicola section. Furthermore some strains from CBS collection used by Wagner also were sourced from infrabuccal pellets of Campanotus and Formica ants - both from America and Europe. We described new species - M.formicae located in that clade. The strain, characterized by compact clusters of gemmae was isolated from Formica pratensis workers. We also obtained M.beljakovae from Formica rufa, F. cinerea and F. pratensis ants, as well M.gemmifera from Lasius flavus. All of above mentioned species characterize by abundant formation of chlamydospores. The studies of Myrmica scabrinodis also revealed unexpectedly high occurrence of Mortierella on this species. The behavioral observations with test of choice suggest that ants prefer Mortierella mycelium more than mycelia of other species. The tendency for transporting the mycelium particles to the nest was observed. We propose a hypothesis that Mortierella is used by different ant species of temperate zone as food or source of enzymes. Insects nests could be used by fungi as a suitable habitat. The workers and queens participate in fungal dispersion. The clusters of chlamydospores produced by fungi of lignicola clade could be an adaptation for such symbiosis. The study was supported by the National Science Centre, Poland under grant No. 2016/23/8/N28/00897
A twenty-year morphological and molecular study of macrofungi in the driftless (unglaciated) region of southwestern Wisconsin, USA.
- S. Aspenson
- T. Osmundson
- T. Volk
Southwestern Wisconsin was unaffected by the glacial drifts that ended some 10,000 years ago, and is now referred to as the driftless area. This area is known for the forested hillsides of limestone-based soils with deep valleys called coulees. These ridges and coulees centered on the Mississippi River offer relicts of forests, prairies, wetlands and grasslands that house a diverse array of organisms. A 20-year survey of fruiting bodies of three older growth forests found approximately 1200 species of macrofungi that include some species with disjunct geographical distributions. Previous graduate students as well as students from the UW-La Crosse Mycology class have been making these collections every year on a three-week rotation. At least 500 students and visitors from three mycological forays have been involved in these collecting efforts. Despite their physical proximity, it is surprising that the total overlap within the three sites is just 303/1200 species (~25%). Some Appalachian relict species have been collected (e.g. Boletus frostii and Ciboria americana), as well as unusually high population densities of some fungi considered to be rare elsewhere (eg. Phlebia coccineofulva). Preliminary data from groundwork DNA sequencing suggest that, because of its isolation, the driftless area may also be home to cryptic species of fungi. In our study, all 1800 specimens of nearly 1200 morphological species from the three study sites are being sequenced, using the ITS region as the primary region of interest. Primers with reference tags are added to the specimens DNA, with different tags on both the forward and reverse strands. These tags allow the delineation of species after the samples are sequenced through Illumina sequencing. Having both morphological and sequence identification will allow for the better understanding of fungi associated with this area, along with confirmation of the species already identified by morphology. These data will also allow for comparisons of geographical regions and revelations of cryptic species. This study is novel because of the large number of specimens and species involved, as well as the length of time (20 years) over which the collections were made.
Towards a Colorado Mycoflora: Molecular diversity of mushrooms from the Southern Rockies.
- S. Wright
- A. Wilson
- V. Evenson
The diversity of macrofungi across North America is staggering and, traditionally, morphology has been the primary means of identification. While morphology is still considered useful, the advent of cheap and effective DNA sequencing has enhanced the exploration of taxonomic relationships among mushroom forming fungi. Currently, the identities of many North American macrofungi are based on morphological concepts that borrow from Eurasian species. The North American Mycoflora Project was created to test species concepts of the continent’s mushroom forming fungi using DNA sequence data. The North American state of Colorado boasts a wide range of fungal diversity. This project aims to study the specimens vouchered in Vera Evenson’s book “Mushrooms of Colorado and the Southern Rocky Mountains,” and housed in the Sam Mitchel Herbarium of Fungi at Denver Botanic Gardens, in order to produce a regional mycoflora of Colorado and the Southern Rockies. Using ITS sequence data this project intends to expand upon our understanding of the region’s diversity of macrofungi. The results of this project represent the projects first steps, using ITS sequence data of over 100 species Colorado mushrooms and allies, and exploring whether the names traditionally used for the region’s fungi hold up to direct scrutiny.
Creating a regional mycoflora – an Indiana case study
- S. Russell
- M. Aime
Attaining a reasonable understanding of the biodiversity of macrofungi for any given region has mostly eluded researchers working in North America. Despite over 200 years of mycological progress, few regions have up-to-date surveys of the macrofungi that occur in their area; this is especially true for surveys that are broadly supported by genetic data. As a part of the North American Mycoflora Project, a full-scale, state-wide survey of macrofungi is currently underway in Indiana. This includes three primary components – extensive field collecting, broad environmental sequencing, and high-level engagement of citizen scientists. Here we report preliminary progress towards a mycoflora of Indiana involving over 4,000 new vouchered and sequenced specimens deposited in the Purdue Kriebel Fungarium since 2015. These sequenced specimens will provide the “faces” for large-scale environmental sampling efforts being conducted across the state. Methodologies for successful citizen scientist engagements are also discussed. Together, these three components combined will significantly enhance our understanding of regional diversity and phenology. It will also form a core dataset to be used in future ecological research.
More than 80% of fungi in northern Thailand are new to science and have amazing biotechnological potential
- K. Hyde
Fungi are an understudied, but essential, fascinating and biotechnologically useful group of organisms. We have been studying the fungi of northern Thailand at Mae Fah Luang University since January 2008. During this time, we have grown from a single researcher, to a large team that has published more than 500 SCI papers. Our studies have been diverse, from phylogeny and taxonomy of microfungi, to growing novel mushrooms, and correctly identifying plant pathogens. In this presentation, I will discuss the importance of fungi and the advances we have made in the Center of Excellence in understanding the biodiversity of fungi in the region. We have made huge advances in the understanding of the fungi at the higher levels. We have inventoried a large number of new fungi for the region, but a huge amount is still to be done. For example, in the edible genus Agaricus, we have introduced more than 20 new species, and many more are waiting description. In these relatively well-known genera we are finding about 80% of species we collected are new to science. In the microfungi, which are relatively poorly studied, the percentage even higher. At the same time, we have been finding ways to exploit these fungi. Our work has resulted in the discovery of at least ten new species which are being developed as novel industrial mushrooms. We have isolated at least ten novel medicinal compounds from Thai fungi and are also looking at ways to exploit them in biocontrol. Selected examples from this study will be given. Our work is just a beginning. Fungi have been generally neglected over time, despite the fact that they provided Penicillin, Lovastatin and various important medicines. Fungi have been poorly exploited and yet have a huge potential in biocontrol, bioremediation, novel compound discovery as well as basic industrial organisms (mushroom, fertilizers and cosmetics).
Diversity and novel species of Entomopathogenic fungi in a conservation area: Banphao Thai community forest, Thailand
- S. Mongkolsamrit
- S. Lamlertthon
- W. Noisripoom
- K. Tasanathai
- D. Thanakitpipattana
- J. Luangsa-Ard
In Thailand, entomopathogenic fungi are one of the species diverse groups. They play an important role in the control of population of insects. Banphao Thai forest in Phitsanulok province is a community forest located in the upper central part of Thailand, and covers an area of 0.64 square kilometers. The ecosystem of Banphao Thai community forest is unique by having cassava plantations surrounded by wilderness. The diversity and molecular phylogenetic studies of entomopathogenic fungi were the objectives that have been established in a collaboration between the Centre of Excellence in Fungal Research (CEFR), Naresuan University and Microbe Interaction and Ecology laboratory, BIOTEC, Thailand. During a field study carried out from 2016 to 2017, specimens were recorded, collected, then preliminarily identified according to their morphological characteristics and then subjected to molecular phylogenetic study. The diversity and abundance of entomopathogenic fungi is high during the rainy season from May to November. A total of 393 entomopathogenic fungi belonging to six genera include Beauveria, Cordyceps, Conoideocrella, Ophiocordyceps, Metarhizium and Polycephalomyces. Their insect hosts were classified to Coleoptera (75.3%), Hemiptera (6.9%), Hymenoptera (0.3%), Isoptera (8.7%), Orthoptera (0.5%), Lepidoptera (8.4%). Shimizuomyces sp. growing on fruit plants were also found in the ecosystem. Interestingly, the interactions between the hyperparasitic fungus Polycephalomyces and their diverse hosts has been observed in this ecosystem. The novel fungal pathogen Polycephalomyces phaothaiensis is a parasite of coleopteran larvae which occurs in rainy season. Meanwhile P. phaothaiensis also plays a role as hyperparasitic fungus which infects numerous fungal hosts such as Ophiocordyceps sp.1 and Ophiocordyceps cf. brunneipunctata, found predominantly during the rainy season. From our investigations, we found 21 species of entomopathogenic fungi and a first record of the genus Shimizuomyces. Polycephalomyces phaothaiensis has multiple hosts and little is known about its life cycle and ecology.
Decontamination of aflatoxin B1 and mycocinogenic activity evaluation by using yeasts belonging to the complex Meyerozyma guilliermondii
- M. Etcheverry
- A. Montemarani
- S. Robledo
- A. Nesci
This study pursued understanding the modes of action by which yeast isolates belonging to the complex Meyerozyma guilliermondii affect AFB1 production by Aspergillus flavus. Isolates Y16 and Y25 were not able to produce killer toxins. However, whole cells and some cellular constituents were able to reduce AFB1 concentrations in different evaluated conditions. Cell-free supernatant of isolates decreased AFB1 levels in liquid medium. For this reason, the effect on ground maize grains was studied. The characterization of the cell-free supernatants and the optimization of the biological decontamination activity of the toxin were tested. The experimental design showed that the best conditions for the cell-free supernatant of yeast Y16 to produce a significant reduction of AFB1 were 39° C, pH 8 and the presence of Mg+. The cell-free supernatant of yeast Y25 was effective at 28° C, pH 5 and presence of Mg+. These results made us suppose a possible enzymatic effect in the reduction of AFB1. To determine whether the effect of the cell-free supernatant of the yeasts on the accumulation of AFB1 was attributable to a protein activity, they were subjected to heat inactivation and proteinase k treatment. No changes on the reduction of the toxin levels were detected. The toxin reduction doesn’t correspond to an enzymatic effect. The cell-free supernatant from the two yeast isolates added to ground maize grains were able to reduce the levels of AFB1 when the toxin was already present in the substrate. Characterization of cell-free supernatants and optimization of the biological decontamination activity of the toxin are necessary steps in the process of selecting a treatment applicable to stored maize.
Phenotypic and phylogenetic studies of selected philippine invertebrate pathogenic fungi
- L. Maghari-Capio
- E. Tadiosa
Invertebrate pathogenic fungi (IPF), are fungi that grow superficially on invertebrate exoskeleton and progress all throughout the body. They encompass an extensive set of phenotypically, phylogenetically, and ecologically distinct fungal species on invertebrates. The present study involves host specificity, phenotypic and phylogenetic studies of IPF. Isolation was done using Potato Dextrose Agar (PDA) and using Lactophenol Cotton Blue to stain the structures for phenotypic analyses. Cetyltrimethyl-Ammonium- Bromide (CTAB) method was used for DNA extraction. PCR amplification was performed in a total of 25 ul volume using DNA markers of EF1 and ITS. PCR products were checked using Agarose Gel Electrophoresis and Ethidium Bromide used to stain the gel. Purified PCR products were sent to Macrogen South Korea for sequencing and Alignment was performed using BioEdit (Hall, 1999). Phylogenetic Analyses was executed using a maximum parsimony employed in MEGA 6 (Tamura et al., 2013). Phenotypically and Sequence Phylogeny from ITS gene loci confirmed the identification of IPF to Simplicillium sp, Cordyceps sp. and Isaria sp. same with the EF1 gene marker. Species specificity on spider in the family of Sparassidae was the highest (15, 50.00%), followed by Araneidae (5, 16.66%), Tetragnathidae (4, 13.33%), Salticidae (2, 6.66%) and Oxyopidae (1, 3.33%). The present study discussed the evolutionary relationship of IPF in the country and further study is needed to support the results.
The Rio Claro as species refuge of arthropodopathogenic fungi Cordyceps in the Magdalena river valley, Colombia
- T. Sanjuan
- A. Osorio
- A. Franco-Molano
The Rio Claro canyon is a private reserve located at 350 m.a.s.l. in the middle of the Magdalena River basin which lies between the eastern and central Andean cordillera of Colombia. It lies within the Neotropical region and belongs to the Magdalena biogeographic zone, characterized by having biogeographical units which are considered Pleistocene refugia and are used to delimit areas of endemism, that is, they are the result of a process of fragmentation of the area and the temporal isolation of the species contained in these units. The Rio Claro canyon is part of one of these units and according to research carried out during the last three decades, the area has special geological and geomorphological phenomena, such as tropical karstifications in marble, which are the result of the erosion of the mountain range during the last six million years. The forests therefore grow on very particular soil conditions, with a humidity above 75% and temperatures of 28°C on average, and are distinctive. This special vegetation leads to a wide diversity of species of amphibians, mammals, reptiles, and birds, many of them endemic. With these geomorphological characteristics it would be expected that the fungi would also exhibit a high diversity of species and endemism. To investigate this, the species of the arthropodopathogenic fungus Cordyceps, which is linked to the arthropod populations, was sampled in the years 2014 to 2017 during the rainy season, They were sampled along a 7 km portion of the canyon. In total, 70 specimens were collected, comprising 29 different species, two of which were new records for Colombia. The most common species were Cordyceps tenuipes and Ophiocordyceps amazonica, while Polycephalomyces ramosus and P. nipponica were the new records for the country. Interestingly, the epizootic species O. unilateralis on carpenter ants and O. araracuarensis on Cicadae nymphs were always found in an area of the forest containing wild cacao trees. The results show that Cordyceps has a high diversity corresponding with the previous findings that of Rio Claro canyon as a center of endemism. The conservation of the Rio Claro basin becomes a priority for the government given the current proposals for mining, with the goal to conserve the germplasm that constitutes this Pleistocene refuge.
Interactions between Inonotus obliquus and a successor fungus Hericium coralloides
- P. Veteli
- R. Linnakoski
- M. Cortina Escribano
- V. Marjomäki
- H. Vanhanen
Species of wood-inhabiting fungi succeed each other during the process of tree-killing and wood decomposition. The interactions in deadwood are diverse, including some specific relationships where the successor prefers a resource previously occupied by a certain other fungus. The coral tooth fungus, Hericium coralloides (Scop.) Pers., is an edible species widely found in Finland, but restricted to sites with abundant deadwood. This saprotrophic species is considered a successor fungus, occurring often on hardwood trees killed by the pathogenic Inonotus obliquus (Fr.) Pilát. However, detailed understanding of the association between these two fungal species is lacking. In this study, the interspecific interactions between the H. coralloides and I. obliquus were studied in two approaches: 1) in co-cultures on three different agar media, and 2) micromorphological study of the structural features of the interaction, under light and helium ion scanning (HIM) microscopy. The isolates used included three strains of dikaryotic H. coralloides obtained from fresh basidioma collected from Southern and Eastern Finland in 2016 and 2017 (H1-3), and one dikaryotic I. obliquus strain from Eastern Finland. The agar media used were 2 % Malt Extract Agar (MEA), 2 % MEA enriched with 3% birch sawdust (MEA+SD), and water agar with 3 % birch sawdust (WA+SD). Each pairing was replicated three times on each media. The co-cultures were incubated for 100 days, during which the radial growth was measured, and the interactions were reported as: 1. no contact, 2. deadlock, 3. partial replacement or 4. complete replacement. With the H. coralloides strains H1 and H2, the radial growth of I. obliquus stopped at a consistent distance from H. coralloides, whereas loose mycelium of H. coralloides grows through I. obliquus mycelium. After contact, I. obliquus mycelium in the through growth zone thickens, with abundant aerial hyphae and often increased guttation. This was followed by gradual dying of I. obliquus hyphae, with the H. coralloides eventually replacing I. obliquus. Partial replacement of I. obliquus was the result in 56% of cases , whereas in 28 % of cases, alive I. obliquus hyphae was not found among now very dense H. coralloides. With the H. coralloides strain H3, the outcome varied considerably. The strain of I.obliquus used replaced H3 partially on MEA, deadlocking on WA+SD, and was partially replaced on MEA+SD. The samples studied by microscopy revealed that H. coralloides hyphal tips coming into contact with I. obliquus branch and coil around latter’s hyphae, which subsequently lose turgor and structure. Lysis and vacuolation of I. obliquus hyphae appear to follow. In addition, when in contact with I. obliquus, terminal chlamydospores emerge in H. coralloides hyphae. Our results support the assumed predecessor-successor relationship between the two species, and suggest it may involve a phase of direct physical antagonism, with features typically associated with mycoparasitism. H. coralloides occurs also on hardwoods that are not known to host I.obliquus, such as Populus, so further study in the specificity of this strategy is needed.
Funnelimoris mosseae alters soil fungal community dynamics and composition during litter decomposition
- H. Gui
- W. Purahong
- K. Hyde
- P. Mortimer
Although arbuscular mycorrhizal fungi (AMF) are believed to be non–saprophytic, recent studies have indicated that AMF are able to influence litter decomposition through interacting with the soil fungal community. However, it remains unclear exactly which constituent groups of the soil fungal community respond to AMF during litter decomposition, and in what ways. In order to fill this knowledge gap, we investigated the effect of AMF on soil fungal communities in a subtropical forest in southwestern China. Our experimental set–up included a dual microcosm unit with two treatments: inoculated with AMF (AM) and uninoculated (NM). Destructive soil sampling was carried out at different times (T0, T90, T120, T150 and T180) and Illumina sequencing was used to detect changes in soil fungal community composition. We found that the composition and operational taxonomic unit richness of the fungal community, at higher taxonomical levels (e.g. phyla, order), remained stable across treatments. However, the relative abundance of some key genera including Mycena, Glomerella, Pholiotina, and Sistotrema were significantly affected by AMF inoculation. Soil fungal community structure was also significantly altered by AMF inoculation during the later stages of litter decomposition, but the diversity of the soil fungal community was unaffected. Our study provides new insight into understanding the interaction between AMF and soil fungal communities during litter decomposition.
Assembling the phylogenetic tree of life of the parasite Escovopsis
- Q. Montoya
- R. Bizarria Júnior
- M. Martiarena
- A. Rodrigues
Symbiotic interactions play an important role in the evolution of life and allow structuring complex systems in nature. Multipartite symbiotic associations with microbes enabled the evolutionary success of fungus-farming ‘attine’ ants (Formicidae: Myrmicinae: Attini: Attina). These insects collect various substrates for feeding a mutualistic fungus, which is farmed as food source for their colonies. Nevertheless, fungi in the genus Escovopsis can affect this mutualism eventually leading attine ant colonies to death. These fungi are thought to be specialized parasites of the attine ants’ fungal cultivar. Until now Escovopsis is the only known parasite that exploits the ant-fungus mutualism. However, the lack of a broad phylogenetic study hampered our knowledge of the parasite diversity as well as the phylogenetic position of some strains. Here, we shed light on the Escovopsis tree of life. Amassing a large number of isolates (365) spanning from several regions in Brazil and other Latin American countries we carried out a robust phylogenetic analysis using five molecular markers (ITS, LSU, tef1, rpb1 and rpb2). Combining phylogenetic with morphological analysis, our results support that Escovopsis (sensu lato) comprise multiple fungal genera. It includes E. kreiselii and E. trichodermoides, which form two well-defined phylogenetic clades distinct from Escovopsis (composed by 28 and 1 subclades, respectively). In addition, two other genera were found (with four and five subclades). Escovopsis (sensu stricto) comprehends at least 32 sub-clades including the five formally described species with vesiculate coniophores. Lack of studies in Escovopsis systematics made impossible the recognition of these genera. Our results open several questions whether more than one fungus coevolved as parasites like Escovopsis and how multiparasitism relationships modelled the life of attine ants.
Parasites of rust fungi from South America
- P. Gomez Zapata
- M. Aime
The genera of fungi and dipterans, Sphaerellopsis and Mycodiplosis, respectively, are known to be cosmopolitan parasites of a wide range of rust fungi. These have the potential to control rust diseases as one alternative to fungicides. Although geographic distribution and genetic variation have been studied in North America and part of Europe for some species, records and data analysis from South America are currently very scarce. Given the high biodiversity in the tropics, the number of genera of fungi and insects feeding on rusts may be underestimated. We are currently studying rust specimens collected from South America deposited in the Arthur Fungarium (PUR) at Purdue University in terms of geographic distribution, genetic diversity and species richness. Every rust sorus from each specimen will be scanned on a stereomicroscope, and any sign of parasites, such as pycnidia, mycelium, and/or larvae will be extracted from the rust sori for DNA isolation. Internal transcribed spacer (ITS) region and mitochondrial cytochrome c oxidase subunit 1 (COI) for fungi and insects, respectively, will be used to study the genetic variation of these parasites. From the first 207 collections examined so far out of approximately 10,000 collections from South America, 48 specimens were infected by mycoparasites and 28 specimens by fly larvae, finding even—in some collections—two different parasites per specimen. Fly larvae such as Mycodiplosis spp., and mycoparasites such as Eudarluca caricis, Ramularia uredinicola, Setophoma sp., and Sphaerellopsis paraphysata, have been so far found on Uredinopsis and Melampsora species. These parasites were discovered in specimens from Argentina, Bolivia, Brazil, Colombia, and Uruguay. We also found the first report of R. uredinicola in Argentina associated with Melampsora epitea on Salix babylonica, suggesting that scanning more specimens may lead to the discovery of more first reports and maybe new unknown species from South America.
Endophyte communities in limber pine in relation to resistance and susceptibility to white pine blister rust
- R. Sitz
- A. Schoettle
- J. Stewart
The non-native pathogen Cronartium ribicola causes the lethal disease white pine blister rust (WPBR) on five-needled pines throughout much of North America. A susceptible species, limber pine (Pinus flexilis), is a high elevation pine valued for its ability to colonize environmentally harsh sites, its role in early forest succession, and as a food source for wildlife. Limber pine is declining from the disease in the Northern and Central Rocky Mountains and C. ribicola continues to spread to the Southern Rockies populations. A single R gene, conferring qualitative complete resistance (aka major gene resistance) to WPBR, is present at low frequencies in limber pine. To better understand mechanisms of limber pine resistance to WPBR, endophyte communities in limber pine trees previously determined to be susceptible or resistant were examined. Endophytes are known to buffer biotic and abiotic stresses in their plant hosts, and may play a role in resistance to WPBR or provide an indication of physiological differences between resistance and susceptible trees that affect the resident endophyte community. The objectives of our study are to (1) document the endophyte communities present in limber pine stands in the Southern Rocky Mountains, and (2) determine whether differences in endophyte communities exists upon geographic gradients, or (3) in the colonization of WPBR resistance and susceptible limber pine. To determine the fungal endophyte diversity in limber pine needles, Illumina Sequencing to amplify the ITS-1 region of the internal transcribed spacer region. Mothur was used to classify sequences into operational taxonomic units, and then we determined overall fungal species diversity, and differences between geographical gradients as well as resistant and susceptible trees.
Mycoparasitic ability and host range of Buchwaldoboletus hemichrysus
- M. Caiafa Sepulveda
- M. Smith
The majority of the taxa belonging to the family Boletaceae form ectomycorrhizal associations with plants. However, two sister groups (the Chalciporus lineage and the Pseudoboletus lineage) appear to be non-ectomycorrhizal and putatively mycoparasitic. The genus Buchwaldoboletus (Chalciporus lineage) fruits directly on wood and is considered a non-mycorrhizal saprobe. Buchwaldoboletus lignicola was recently confirmed as a mycoparasite of Phaeolus schweinitzii in laboratory confrontation tests, suggesting that all taxa in this group may be parasites. In the southeastern United States, Buchwaldoboletus hemichrysus regularly fruits on pine stumps in association with a wide variety of wood decay saprobes, including Ganoderma curtisii and Gymnopilus species. These observations suggest that B. hemichrysus is also a mycoparasite, but the natural host fungi remain unknown. The objective of this study was to test the mycoparasitic ability of B. hemichrysus against 11 species of pine decay fungi and two species of fungi that decay hardwood trees (Pleurotus sp. and Lentinus crinitus) to determine the potential host range of B. hemichrysus. We carried out confrontation experiments in Petri dishes on Modified Melin Norkrans medium (MMN) where we challenged B. hemichrysus against each of the 13 different wood-decay fungi species from four different orders (Agaricales, Gloeophyllales, Polyporales and Russulales). We also carried out confrontation experiments with B. hemichrysus against itself as a control. Confrotation experiments showed that B. hemichrysus was able to parasitize five species (Ganoderma curtisii, G. meredithiae, Gymnopilus sp., Leiotrametes lactinea and P. schweinitzii) but was not able to parasitize the other 8 species. Two of the confrontations led to a deadlock where B. hemichrysus was able to persist but not grow further (Gloeophyllum separium and Ceriporiopsis sp.). Six fungi (Dichomitus sp., Lentinus crinitus, Pleurotus sp., Pluteus sp., Schizophyllum commune and Stereum hirsitum) were able to inhibit the growth of B. hemichrysus and to partially overgrow it. In all the confrontations, B. hemichrysus grew slower than all tested wood decay fungi. Despite the rapid growth of all wood decay fungi, an inhibition halo was observed at the contact zone in the cases of Gloeophyllum separium and Ceriporiopsis sp.. All wood decay fungi produced notable pigments that permeated the media in the presence of B. hemichrysus but these pigments were significantly reduced in control plates. At the contact zone of the 5 species that were parasitized, we observed hyphae of B. hemichrysus wrapping around the hyphae of the wood-decay fungi. Buchwaldoboletus hemichrysus also produced abundant conidia during confrontations with those fungi it parasitized and in confrontations that led to a deadlock. Conidia were abundant on confrontation plates but production was limited on control plates. These results show that B. hemichrysus is a mycoparasite like others species in the Chalciporus clade and suggests that all fungi in this group are likely mycoparasitic. Our work also confirmed that B. hemichrysus was able to parasitize a wide phylogenetic range of pine decay fungi (Agaricales and Polyporales) but was not able to parasitize taxa that primarily decay hardwoods.
Unearthing ephemeral mycoparasites – molecular studies of Pyxidiophorales (Laboulbeniomycetes, Ascomycota)
- M. Gorczak
- D. Haelewaters
- Ł. Istel
- A. Okrasińska
- M. Wrzosek
Laboulbeniomycetes is a class of ascomycetous fungi consisting of three orders with strikingly different biology and morphology. Two of them – Laboulbeniales and the recently described Herpomycetales – are obligate biotrophic ectoparasites of arthropods. They share a number of characters that makes them unique among ascomycetes: determinate growth, lack of hypha and absence of asexual stage. Less is known about the free-living, mycelial order Pyxidiophorales. Those mycoparasites are rarely recorded but their intricate arthropod-dependant dispersal strategies have been well documented. Being a difficult and mostly unculturable group, Laboulbeniomycetes have been poorly represented or omitted in major molecular phylogenetic studies resolving evolutionary relationships in fungi. Similarly, due to lack of reference sequences no Laboulbeniomycetes have been identified in metabarcoding studies. The main objectives of this study were to resolve phylogenetic relationships among lineages of Laboulbeniomycetes and to analyse existing DNA sequence databases (such as NCBI GenBank) to assess the diversity of uncultured and undescribed members of Laboulbeniomycetes. Newly generated marker sequences were obtained from cultures and unculturable specimens of Pyxidiophorales. We used these sequences to distinguish Laboulbeniomycetes OTUs in publicly available databases. Phylogenetic trees were generated using maximum likelihood and Bayesian approaches based on three ribosomal DNA markers (SSU, ITS, and LSU) and two protein-coding genes (RPB2 and MCM7). Review of sequence databases revealed many environmental OTUs belonging to Pyxidiophorales. This suggests that the group is much more diverse and widespread than currently accepted. Pyxidiophorales may be often overlooked because of their minute, ephemeral fruitbodies and non-characteristic anamorphs. The addition of sequences from new Pyxidiophora isolates significantly increased support for major branches in Laboulbeniomycetes phylogenetic reconstructions. Our analyses provide strong support for the class Laboulbeniomycetes and the orders Herpomycetales and Pyxidiophorales. The basal-most node of the order Laboulbeniales generally has weak support although major clades within are well supported. In our reconstructions, Herpomycetales is basally positioned, sister to both Laboulbeniales and Pyxidiophorales. This evolutionary scenario would imply the reversion from determinate thallus growth to typical ascomycete mycelial lifestyle. More likely would be the independent development of insect ectoparasitism by two lineages.
Low molecular weight organic acids as key molecules in bacterial-fungal interaction
- F. Palmieri
- A. Lohberger
- A. Estoppey
- L. Pétremand
- J. Challacombe
- G. House
- D. Rodrigues
- J. Young
- S. Bindschedler
- P. Chain
- P. Junier
Low molecular weight organic acid (LMWOA) production is widespread in the fungal kingdom and can have important roles in processes as diverse as pathogenesis, competition, mineral weathering or lignocellulose degradation. More recently, LMWOA have been identified as being involved in the interaction between fungi and bacteria. This is particularly true in the case of oxalic acid, especially in soils. Oxalogenic fungi are known to interact with oxalotrophic bacteria in soils within the oxalate-carbonate pathway. Moreover, oxalic acid can also serve as a signaling molecule for mycophagous bacteria to localize their fungal target. Due to this multitude of functions, LMWOA appear crucial in bacterial-fungal interactions and might be part of a general mechanism in the mediation of reciprocal bacterial-fungal behavior. Therefore, the aim of this study is to assess the role of LMWOA, and in particular oxalic acid, as key molecules in bacterial-fungal interactions. We tested the role of LMWOA consumption by oxalotrophic bacteria on fungal growth inhibition. Fungal strains of the phyla Ascomycota, Basidiomycota and Zygomycota were selected and LMWOA production was confirmed by acid detection on a pH-indicator-containing medium followed by HPLC identification. Fungal-bacterial co-cultures confronting the selected fungi to three soil bacteria (two oxalotrophic and one non-oxalotrophic) were performed in media with differing nutrient composition (to trigger differential LMWOA production). Co-cultures were then observed with fluorescence stereoscopy and confocal microscopy. LMWOA consumption by bacteria was assessed by MALDI-Imaging Mass Spectrometry. Half of the fungal strains screened produced large amounts of various LMWOA. For those strains, oxalotrophic bacteria appear to control LMWOA production, but this depends on the media composition, with nutrient-poor media (closer to soil solution) favoring bacterial control of the fungal growth. Likewise, bacterial survival varied depending on the fungal species and nutrient conditions of the medium, once again with nutrient-poor media favoring bacterial survival. With this study, new insights into the functional role of LMWOA in bacterial-fungal interactions are highlighted.
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.
Description of aquatic yeasts and their interactions with plants and Aedes mosquitoes
- T. Billingsley Tobias
- J. Hunt
- R. Viadero
- C. Miller-Hunt
- A. Porras-Alfaro
Yeasts play important roles in food webs, degradation of organic matter, and nutrient cycling in aquatic ecosystems. The diversity and abundance of yeasts in aquatic systems remains poorly characterized. In addition, their functional roles and interactions with other aquatic species are becoming increasingly important. The purpose of this project is to describe fungal communities in urban streams and examine potential endophytic links between aquatic yeasts and riparian trees and the potential roles of mosquitos in yeast dispersal. Fungal traps containing apples, pears, and cherries were placed in an urban stream in west central Illinois from April through November. Fungal communities were characterized using cultured-based methods and DNA sequencing. Three hundred and seventy-three fungal isolates were cultured. ITS rDNA indicated 71 OTUs and further multigene phylogenies will be conducted to determine species level identification. Seventy-eight percent of the fungi were isolated from traps containing pears and 53% of all fungi cultured were yeasts. The greatest yeast diversity was cultured from traps set in November with 19 OTUs (50%). Yeast identified as the same species using ITS rDNA showed wide variation in morphology and color. Tremellomycetes (39%) and Saccharomycetes (29%) were the most frequent fungal classes. Both classes were represented by seven genera. Preliminary phylogenetic analysis indicated that 40% of the OTUs were closely related to plant endophytes or associated with insects. The most abundant species cultured were in the genera Meyerozyma, Candida, Pichia, and Cryptococcus. These yeasts are commonly associated with mosquito microbiota. Yeast bioassays will also be conducted to determine potential mosquito-fungal interactions including the role of yeasts in oviposition, egg hatching, and larval growth. Mosquito eggs will also be examined using microscopy to determine potential fungal interactions. To test potential endophytic life stage, leaves from the most abundant trees were collected at the same sites where fungi were trapped and will be isolated and characterized using molecular methods. The nature of the interactions between mosquitoes and yeasts and their effects on the abundance of populations remains mostly unknown.
Profiling fungal community diversity at the Martin Peña Channel
- L. Serrano-Torres
- J. Pérez-Jiménez
The Martín Peña Channel is a tidal creek that connects the San Juan Bay with the San José and Los Corozos Lagoons (San Juan, Puerto Rico, USA). The channel has a long history of pollution as unauthorized communities settled in its banks. However, microbial metabolic activities have been reported in the area, including biodegradation of alkanes. The mix of freshwater and seawater found in estuaries creates an environment where unique microorganism can thrive. Estuaries are suited for fungi distribution studies because of the gradient of environmental conditions, air currents, and water transit. Fungi decomposition of complex substrates, lignin or cellulose, contribute to sustain the ecosystem. The objective of this study is to describe the richness and distribution of fungal communities in soil samples in the Martín Peña Channel. The hypothesis is that diverse fungal communities will be present along the channel. Soil samples were gathered along the Martín Peña Channel. Total genomic DNA was extracted from each sample for the amplification of the ITS region. Terminal restriction fragments length polymorphism (TRFLP) analysis was used to describe fungal communities. TRFLP profiles of the fungal ITS region showed that there is little abundance and diversity of fungal phylotypes among the sites. Most samples displayed one phylotype, while one sample had two. Furthermore, many samples showed phylotypes which fragment length were beyond the detection range. The low abundance and diversity of fungal communities found suggest limited taxa richness and the need of more informative approach, as simple of alternative restriction enzyme. Analysis of soil quality suggests that environmental factors (pH ranges from 5.43-8.33) and nutrient content (organic matter percentage ranges from 3.48%-38.27%) should not restrict fungal proliferation. Apparently, high pollution levels may affect the fungal communities in the sites since similar approaches have been applied to forest soil and greater diversity was reported. Other restriction enzymes may have the potential of better describing fungal communities in this area. Cultivation efforts and the application of next generation sequencing are in progress to better disclose fungal diversity on tidal banks heavily polluted.
Fungal community assembly on the roots of Costa Rican coffee (Coffea arabica) and native Rubiaceae species.
- E. Sternhagen
- L. Aldrich-Wolfe
- S. Vink
Myriad plants are grown as crops far beyond their native ranges, and these plant species have novel interactions with their new surroundings. Understanding the interactions of non-native crops with belowground fungal communities is important for maintaining crop and soil health and for conserving fungal diversity. Soil and root fungal community assembly may be influenced by abiotic factors like light availability and soil nutrients, or by biotic filters manifested as phylogenetically-determined plant traits. In this study, we used coffee (Coffea arabica), a plant of African origin which is now cultivated throughout the tropics, to examine how belowground fungal biodiversity of forest and agricultural systems might interact. Root and rhizosphere fungal communities of coffee and of native Rubiaceae species were sampled in the Monteverde region of Costa Rica. Environmental variables included soil nutrients, moisture and temperature, and photosynthetically active radiation. Root samples were taken to assess fungal colonization by arbuscular mycorrhizal (AM) fungi and non-AM fungi. In both habitats (forest or coffee field) at each of three sites, samples were taken of roots and rhizosphere soil, as well as of background soil not associated with any specific plant. DNA was then extracted from all samples. Illumina sequencing of the ITS2 region of the fungal DNA and the PIPITS bioinformatics pipeline gave a dataset of fungal communities based on closest known species matches in the Warcup database. Both AM and non-AM fungi differed between coffee and forest, and between species in the forest. AM and non-AM fungi in coffee were positively correlated with soil K and negatively correlated with Cu and Zn. These differences may be effects of agricultural chemicals since K is a common ingredient in fertilizer and Cu is found in fungicides. In forest Rubiaceae, AM fungal colonization was negatively correlated with Ca and P, while non-AM showed a negative correlation with Fe and pH. Since AM fungi are essential to plants for P uptake, it is logical that under low P conditions, plants benefit from higher AM colonization. Additionally, the relationship between pH and non-AM fungi may exist because acidic soils tend to favor fungal growth. However, these abiotic soil factors did not differ by tree species and thus do not explain the observed fungal differences between species of Rubiaceae. These results suggest that, while abiotic factors influence root fungal communities, a biotic effect of plant host is also important consider.
Thermotolerant mycobiota of Israeli soils
- I. Grishkan
Israel is a country with more than 60% of the territory covered by deserts. Its climate is characterized by hot and dry summers even in the Mediterranean part, thus making temperature one of the most important microclimatic factors influencing development and distribution of fungal communities in the soils. The objective of the study was to reveal the composition, structure, and spatiotemporal dynamics of thermotolerant mycobiota in the soils of Israeli deserts and northern territories. Microfungi from the upper soil layers of 0-2 cm from sun-exposed open localities (bare or crusted) and under the nearby shrub or herb canopies were isolated at 37oC using the soil dilution plate method. A comparatively rich thermotolerant microfungal biota contained 165 species from 82 genera, with Aspergillus and Chaetomium being the most numerous genera – 25 and 22 species, respectively. Aspergilli (Aspergillus fumigatus and A. niger), teleomorphic ascomycetes (Canariomyces notabilis, Chaetomium nigricolor, and Ch. strumarium), and zygomycetous Rhizopus arrhyzus comprised the basic part of the thermotolerant communities. The desert areas remarkably differed from the northern areas by a much higher abundance of A. fumigatus known as one of the most frequent and abundant thermotolerant and thermophilic species in a variety of desert regions, and teleomorphic species, as well as by a lower abundance of A. niger and R. arrhyzus. The cluster analysis based on species’ relative abundances showed that the thermotolerant microfungal communities from the geographically distinct Israeli and Spanish deserts were much more similar to each other than the communities from the Israeli desert and northern regions. Seasonal dynamics revealed for a canyon in the southern Negev desert was expressed mainly in the variations of species richness (substantially lower in the winter), and abundances of A. fumigatus (dominant in the summer) and A. niger (dominant in the winter). Importantly, the composition of thermotolerant mycobiota was almost entirely different from the composition of mesophilic mycobiota isolated at 25oC. It, therefore, can indicate that the melanized species with protective multicellular spore morphology, which overwhelmingly prevailed in the desert soils at 25oC and were almost entirely absent at 37oC, possibly occur in the summer in a dormant spore state. At the same time, aspergilli dominant at 37oC, accompanied by teleomorphic species with perithecial fruit bodies producing comparatively large (10 µ and more) dark-colored ascospores, were apparently able not only to survive but also to germinate at high temperatures and be active during a long hot period in the desert.
Biological soil crusts microbial diversity in mojave desert, USA
- N. Pombubpa
- P. De Ley
- N. Pietrasiak
- J. Stajich
Up to 40% of global terrestrial land surface consists of desert environments. Naturally water availability is limited in these areas, which increase the vulnerabilities of desert organisms especially plants. Desert ecosystems are not usually covered with dense vegetation and any occurring vascular plants are normally found associated with important microbial communities in the plant interspaces called “biological soil crusts (biocrust).” Biological soil crusts (biocrusts) also known as biological, cryptogamic, cryptobiotic or microbiotic soil crusts contain diverse variety of microbial communities that are essential to desert environments. Bryophytes, lichens, eukaryotic algae, cyanobacteria, bacteria, and fungi combine with soil particles to form different types of biocrusts. Biocrusts are now widely recognized as a living skin of dryland environment, and biocrusts research is critical for desert ecosystem protection. Although scientists have recognized crusts as an important part of desert ecosystems, biocrusts are often ignored and disturbed by the general public with substantial effects on ecosystem health. Disturbances on biocrust microbial communities are often not visible and rarely quantified. Likewise, the functions of biocrusts microbiome (especially functions and interactions related to fungi) are poorly characterized. The stability and temporal variations in microbial community composition are also poorly understood. Work to classify biocrusts has been largely focused on external morphology of biocrusts, which may underestimate the functions and diversity of microbial communities. As a result, this project aims to explore the composition of microbial communities, to capture the biocrust microbiome, and to investigate spatial and temporal patterns of the community as well as the impact of disturbance on community composition. Understanding how microbes interact to form crust layers requires detailed accounting of their microbial diversity, community structure, and measurable functions. Therefore, deeper and higher resolution in biocrusts microbial community is needed. We have used amplicon sequencing of environmental DNA to assess the composition of bacterial and fungal communities. Biocrusts were collected from Joshua Tree National Park, Granite Mountain, and Kelso Dunes within Mojave Desert in California, USA. Preliminary results showed that major bacterial phyla were mainly Cyanobacteria, Proteobacteria, Actinobacteria, and Acidobacteria. Major fungal phyla were mainly Ascomycota (Dothideomycetes, Eurotiomycetes, and Pezizomycetes) and Basidiomycota (Agaricomycetes and Tremellomycete). Although alpha diversity analysis showed no different species richness between sites, beta diversity analysis reviewed geographical pattern among three sites. We also found correlation between increasing species richness and raining event, which was an indication of temporal/seasonal effects on biocrusts microbial community. Core microbiome analysis and also abundant taxa were used to determine and prioritize culturing efforts while further analysis on functional diversity and biotic interactions will be examined on fungi, which will be isolated from biocrusts.
Checklist of fungi from a semi-arid region of South Africa established through environmental and Sanger sequencing approaches
- M. Gryzenhout
South Africa has a rich fungal biodiversity, which is unfortunately poorly studied due to a lack of human capacity and expertise. Although some species have been identiﬁed, the majority are new or dubiously named and fungi are rarely included in conservation and biodiversity initiatives. It also frustrates a large and growing group of citizen scientists eager to identify fungi they encounter. The taxonomic dilemma is too great to wait for species to be adequately named and described before fungi can be rightfully included in national and international initiatives. Molecular tools can be predictively used to determine locations and indicate hotspots, which is useful to plan future surveys and strategies for more complete assessments. This is especially useful in drier environments where fungi will not usually fruit and be visible. In one such location, namely Bloemfontein, South Africa, environmental samples (humus, soil, dead plant material) were sequenced directly using Illumina sequencing. The mini-barcodes were used in phylogenetic diversity analyses to ascertain the diversity for the area and establish, for the first time, a checklist of genera present. In addition, fruiting bodies that could be found over the past two years were sequenced using Sanger sequencing to provide a higher level of resolution for identification. These results were also linked to the data obtained with environmental sequencing. Despite the area being semi-arid, a high level of diversity was detected and more than 60 genera, of which some were never before observed in the area, were detected. The approaches developed in this study can thus be useful in the future to generate much-needed biodiversity data for South African macrofungal species that will stimulate further study.
The thread connection between Hotaea weneckii and Dunaliella atacamensis: triggering symbiosis from spiderwebs to in vitro culture
- L. Muggia
- P. Zalar
- A. Azua-Bustos
- N. Gunde Cimerman
Microorganisms have evolved multiple and novel strategies to survive in exreme environments. Iconical examples are lichens, in which the association between fungi and algae enable the symbionts to outcompete in habitats where otherwise the single organisms could not survive. The green algal genus Dunaliella and the black yeast Hortaea are exemplar in being adapted to extremely saline, aquatic environments. Only one single species of the genus Dunaliella, D. atacamensis, is known from subaerial environments. It thrives on spiderwebs on walls at the entrance-twilight transition zones of caves in the Atacama desert (Chile). A closer inspection of the colonies has shown the presence of black yeast cells of Hortaea werneckii between the algal cells. As symbiotic interactions between Dunaliella species and the Hortaea fungi have not been known so far, we performed a series of co-cultivation experiments to test whethere the two symbionts can co-grow in vitro and develop lichen-like symbiotic structures. We set up co-cultures with the axenic strains of Hortaea werneckii isolated from the salterns and Hortaea werneckii isolated from the Chilean spiderwebs together with the salt tolerant Dunaliella salina and the aerial D. atacamensis. We used different growth media and culture approches to trigger the association between the fungi and the algae and documented our observations by light microscopy.
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.
Diversity and composition of fungal communities associated to biological soil crusts in a fog oasis of the Peruvian desert
- X. Pinchi
- P. Castellanos
- C. Arana
Biological soil crusts (BSCs) are describe as a thin layer of upper soil where soil particles are aggregated by a community of highly specialized organisms such as cyanobacteria, bacteria, microfungi, algae, lichens, and mosses. BSCs are essential components of arid and semiarid ecosystems due to their importance in soil stability, water retention and soil fertility; however, they remain poorly studied with respect to their fungal diversity. The objective of this study was to compare the composition, structure and diversity of fungal communities associated to three different BSCs (site 1 to 3; which are at different stages of biocrust formation, being 1 the earliest stage) and superficial soil with vegetation (SSV;site 4) at Lomas de Lachay Natural Reserve, Lima. Five SSV and BSCs samples were taken from a 100 m transect. Soil moisture was determined using the gravimetric method and pH was analyzed using a digital pH meter. Fungi were isolated using the dilution plate technique (10-1; 10-6). An aliquot of 0.1 ml was taken from each dilution and plated in Petri dishes containing Malt Agar (AM) and Czapeck Agar (ACZ). Pure cultures were identified using the microculture technique. Soil moisture was slower in SSV but pH was higher than in BSCs. A total of 142 strains from 46 species belonging to Zygomycetes (4 species) and Ascomycetes (42 species) were isolated. The most abundant genera isolated from SSV were Penicillium, Aspergillus and Fusarium while for BSCs were Aspergillus, Penicillium, Fusarium, Paecilomyces, Cladosporium and dark-colored species. Likewise, melanin-containing microfungi species were higher in BSCs (33%) compared to SSV (15%). Shannon-Wiener diversity index did not show significant differences among sites where biocrust was found but it does between BSCs and SSV. Moreover, diversity is higher in later stages of biocrust development. A NMDS analysis showed clearly three different fungal communities (site 1, 2 and 4) while site 3 seems to be heterogeneous. These results suggest that diversity of fungal communities increases as biocrusts are in later stages of development and more vascular and non-vascular plants are present. The significantly lower percentage of thermotolerant and UV-resistant species and the higher abundance of cellulose degrading species in SSV were caused by the presence of vegetation and debris. In the same way, annual plants that regrow due to the increase of humidity during seasonal fog allows the dominance of species with high growth rates such as Penicillium, Aspergillus and Fusarium in BSCs.
Molecular identification of filamentous fungi diversity in north coast beaches of Puerto Rico
- L. Echevarría
- M. Arroyo
Puerto Rican beaches are a significant tourist attraction. The Northern region has a great variety of beaches with diverse microbial characteristics. Beach sands receive direct contamination from the garbage generated by people, which serves as nutrient for fungi growth. The objectives of this investigation were to assess the filamentous fungi diversity of four popular beaches; identify the genus and species; and identify the taxonomic relationship between the most abundant fungi. The beaches studied are located in the towns of Vega Baja, Manatí, Barceloneta and Arecibo. One sample of dry sand per month from three equidistant points were acquired every month for a year in each beach. The samples were homogenized according to dry (December-April) and humid (May-November) seasons, for a total of four composite samples per season. The DNA of each sample was isolated and quantified; and, upon sequencing, evaluated by metagenomics analysis with MG-RAST. There were 104 fungi species identified by DNA sequencing analysis. The most abundant were: Aspergillus penicillioides, Aspergillus terreus, Microascus sp., Arthrographis kalrae, Paramicrosporidium sp., Dokmaia sp., Gliomastix polychroma and Aspergillus sp. The taxonomic analysis demonstrated that there is no relationship in the genus of the most abundant species. As significant finding, 66 species of new registries were identified, including Malassezia restricta, Arthrographics eremomycyces, and Cephaliophora tropica. Not only were many of the species pathogenic, several genus of filamentous fungi have been previously isolated from patients in nasal culture, and can cause eye-, respiratory- and skin disease. The majority of these fungi use direct contact and air transport as transmission vehicle to the host.
Evaluating Helminthosporium solani, causal agent of potato silver scurf blemish disease, for sensitivity to the fungicide azoxystrobin
- S. Macchiavelli Giron
- Y. Chen
- J. Spychalla
- A. Gevens
Globally, potato is the third most important food staple crop, which is critical in this time of population growth and increasing world hunger. Silver scurf of potato, caused by the ascomycetous fungus Helminthosporium solani, is a tuber blemish disease causing negative quality and storability impacts of great and increasing concern in the U.S. Potato producers in Wisconsin (WI) and other major potato-producing states have had to rely heavily on fungicides in addition to variably-effective cultural controls for silver scurf management. After H. solani developed field resistance to the once effective fungicide thiabendazole, QoI fungicides (such as azoxystrobin) were adopted to improve control. However, recently there has been an increase in disease affecting quality and storability. Genetic mutations associated with QoI fungicide resistance have been identified in other potato pathogen populations, such as Alternaria solani. For this reason, we hypothesized that H. solani might currently be poorly controlled due to QoI resistance after over 2 decades of use. In our preliminary screening of the pathogen population in Wisconsin, we selected five H. solani isolates representing different production regions and management programs. Five of these isolates were exposed to azoxystrobin at varying dose rates to determine fungicide sensitivity in vitro. Isolates were grown on clarified V8 agar amended with azoxystrobin in various concentrations (0, 0.001, 0.01, 0.1, 1, 10, 50, 100 μg/mL). Relative growth was measured after 20 and 40 days of incubation at 23ºC under dark conditions. There were significant differences in growth between isolates, as well as between treatments. One of the isolates grew on all azoxystrobin concentrations, including the highest concentration, suggesting some level of fungicide insensitivity in vitro. Further studies will focus on evaluating a larger isolate collection for sensitivity and determining presence of mutations associated with QoI sensitivity in other potato pathogens.
Thermal sensitivity of Calonectria henricotiae and Calonectria pseudonaviculata conidia and microsclerotia: developing a tool for managing boxwood blight in nurseries
- M. Miller
- N. Shishkoff
- M. Cubeta
An understanding of asexual fungal propagule thermal sensitivity is useful for developing disease management approaches that deploy heat to inactivate conidia and microsclerotia of plant pathogens. Heat treatment of cuttings, plant debris and soil that harbor conidia and microsclerotia would provide a useful management tool for suppressing the pathogenic activity of Calonectria pseudonaviculata (Cps; present in the US) and C. henricotiae (Che; a quarantine pathogen not present in the US) and boxwood blight disease. The objective of this study was to determine the thermal sensitivity of conidia and microsclerotia of Che and Cps treated in water at 45 C, 47.5 C, 50 C, 52.5 C, and 55 C. For conidia, as time of exposure increased at each temperature, the proportion of germinated conidia decreased. The predicted time required to kill 90% of Cps conidia (LD90) decreased as water temperature increased from 45 C to 55 C and ranged from 35.4 to 5.6 min, respectively. Conidia inactivation was dependent on isolate, species of Calonectria, and length of exposure at each temperature tested. Microsclerotia of Che and Cps displayed reduced germination with increasing exposure and higher water temperatures. Microsclerotia of Che were significantly more resistant to heat treatment than Cps at 47.5 C and 50 C, while microsclerotia of both species were rapidly killed at 55 C. In conclusion, these studies provide baseline knowledge on the thermal sensitivity of Che and Cps conidia and microsclerotia. This information is currently being used to complement thermal sensitivity data for commonly grown boxwood species and plant-based infection assays with the goal of developing a method for managing blight disease on cuttings during the boxwood propagation process.
ZnO-Nanoparticles as antifungal agent limiting growth and mycotoxins production by Aspergillus flavus and Fusarium proliferatum on a maize based-medium
- G. Pena
- M. Cárdenas
- G. Planes
- C. Barbero
- L. Cavaglieri
- S. Chulze
In Argentina, around 60% of maize is exported and the remaining is used as feedstuff. Aspergillus flavus and Fusarium proliferatum are two mycotoxigenic species frequently isolated from maize that produce aflatoxins and fumonisins, respectively. ZnO-Nanoparticles (ZnO-NPs) have been used as efficient antimicrobial agents. ZnO is a non toxic compound and a strategy of low cost and low environmental impact to reduce mycotoxin accumulation in stored maize. The aims of this study were: (i) to synthesize and characterize ZnO-NPs; (ii) to evaluate their effect under presence/absence of white light during fungal incubation on growth rates and aflatoxin B1 (AFB1) and fumonisin B1 (FB1) accumulation by A. flavus and F. proliferatum, respectively, and (iii) to determine fungal morphological alterations by SEM. ZnO-NPs were synthesized according to the drop by drop mixing method and characterized by SEM. Aspergillus flavus RCAF016 and F. proliferatum ITEM 15699 strains were grown on a 2% maize based medium (0.995 aW) containing 0, 10, 50 and 100 mM ZnO-NPs. The inoculated plates (by triplicate) were incubated at 25°C, 21 days in darkness or under 12/12 h photoperiod cold white and black fluorescent lamps. SEM analysis of ZnO-NPs showed thin flakes of 200 × 200 nm and thickness of ~30 nm. Aspergillus flavus and F. proliferatum strains were able to grow in presence of 0, 10, 50 and 100 mM ZnO-NPs. However, growth rates were reduced (in relation to the control) at the three concentrations evaluated under photoperiod or darkness incubation conditions. Hypha and conidia morphological alterations were observed in both A. flavus and F. proliferatum treated with ZnO-NPs by SEM analysis (2000-5000 X). The alterations were: hyphal deformations, less conidiation and unusual bulges. Mycotoxin accumulation was also affected by the ZnO-NP treatments. After 3 days of incubation in darkness and 10 mM ZnO-NPs, Aspergillus flavus RCAF016 showed AFB1 production higher than the control, but at 50 and 100 mM the toxin production was inhibited at not detectable (ND) levels. After 7 and 14 days, AFB1 was reduced at ND levels at 10, 50 and 100 mM ZnO-NPs, whereas, at 21 days the toxin was reduced by 85% and at ND levels at 50 mM and 100 mM, respectively. Under the photoperiod incubation condition AFB1 was not detected in both controls and treatments. Fumonisin B1 accumulation by F. proliferatum ITEM 15699 was also reduced by ZnO-NP treatments. Both incubations conditions showed reduction in FB1, in general, at the three ZnO-NPs concentrations evaluated. The reduction was between 73 and 99% after 14 and 21 days of incubation. This study showed that ZnO-NPs could be used for limiting growth and AFB1 and FB1 accumulation by A. flavus and F. proliferatum, respectively in a maize based medium. This environmental friendly strategy of low cost could be applied during maize storage.
Using essential oils as a chemical control for fungal plant pathogens
- K. El Mounadi
- A. Digiugno
- E. Coberly
Fusarium head blight is a devastating disease of wheat and barely caused by the pathogen Fusarium graminearum. F. graminearum also produces the mycotoxin deoxynevalenol which causes a wide range of toxicological effects in both humans and animal fed contaminated feed. Despite the continued release of cultivars resistant to fungal pathogens and chemical fungicides, F. graminearum remains difficult to control. Thus there is an urgent need to find safe and durable strategies to limit the infection of plants by this pathogen. We tested the ability of ten essential oils; castor oil, fenugreek oil, clove oil, peppermint oil, cinnamon oil, eucalyptus oil, fennel oil, thyme oil, clary sage oil and marjoram oil, to inhibit the growth of F. graminearum in vitro and in planta. All tested oils inhibited the growth of F. graminearum in vitro with MICs ranging from 0.037 to 0.3 µM. Clover oil, eucalyptus oil, Thyme oil and cinnamon oil displayed higher antifungal activity with an MIC<0.0375µM. These results suggest that essential oils have the potential to contribute to the development of new class of antifungal agents to protect crops from fungal diseases.
The influence of fungicides on the grapevine wood mycobiome: a case study on tracheomycotic ascomycete Phaeomoniella chlamydospora
- G. Del Frari
- A. Gobbi
- P. Talhinhas
- H. Oliveira
- L. Hestbjerg Hansen
- R. Boavida Ferreira
Grapevine trunk diseases are the major threat to viticulture of our days. They affect the perennial organs of grapevines (Vitis spp.) causing decline, loss in quantity and quality of yield, and early death of the plants, resulting in enormous costs for the industry. Phaeomoniella chlamydospora is a tracheomycotic ascomycete which colonizes the xylem of grapevines causing wood discoloration and necrosis, plant decline, and it is believed to be indirectly responsible for other symptoms as well. Phaeomoniella chlamydospora is associated mainly with four syndromes of grapevines: brown wood streaking of rooted cuttings, Petri disease, grapevine leaf stripe disease and esca proper. The incidence of these syndromes in vineyards has greatly increased over the last 20-30 years and the most puzzling question remains – why has P. chlamydospora become so successful? This work aimed to answer this question by formulating the hypothesis that fungicides commonly used in vineyards to control downy mildew (Plasmopara viticola) and powdery mildew (Erysiphe necator) interact with the wood mycobiome causing a change in its composition, which may favor the wood colonization by P. chlamydospora. To test this hypothesis, we worked on one-year old rooted cuttings of 'Cabernet Sauvignon', under greenhouse conditions. Grapevines were inoculated with either a spore suspension of P. chlamydorpora (strain CBS 161.90), with an artificial mycobiome or with a combination of both, and treated with three combinations of fungicides and a control. We used metabarcoding (Illumina sequencing) with two sets of universal primers (ITS1F2F/ITS2R and ITS86F/ITS4R) in order to understand the changes that the wood mycobiome incurs due to the application of fungicides and Real-Time PCR to quantify the abundance of P. chlamydospora. Our results show that the application of different fungicides changes the relative abundance of several fungal species in the grapevine wood, including that of P. chlamydospora. The colonization of the wood by this ascomycete is also greatly reduced when co-inoculated with the artificial mycobiome. Sequencing results are partially supported by quantitative analyses. This study demonstrates that fungicides applied as foliar sprays can indirectly interfere with the fungal communities in the grapevine wood giving an advantage to some communities over others, allowing speculations on the role that this changes play in triggering disease mechanisms.
Response of potato to the black scurf disease pathogen Rhizoctonia solani Kühn AG-3
- R. Grosch
- F. Genzel
- P. Franken
Black scurf disease on potato caused by Rhizoctonia solani AG3 occurs worldwide and is difficult to control. It has been observed that potato cultivars show differences in susceptibility to R. solani AG3. The cultivation of potato cultivars with high resistance level to Rhizoctonia diseases represents an ecological and economic sustainable control strategy. Presently, the degree of resistance is based on symptom assessment in the field, but molecular methods could offer a more effective screening procedure for cultivars with high degree in resistance. We hypothesized that field resistance to black scurf disease in potato cultivars is associated with defense-related gene expression levels and salicylic acid (SA) concentration. In a comparative analysis, the expression levels of common defense-related genes of two cultivars with moderate and high degree in resistance to black scurf disease were studied. Besides RNA accumulation, salicylic acid (SA) concentrations in potato tissues were measured. A higher constitutive expression level of defense-related genes was found in the highly resistant cultivar. A significant increased expression level of these genes upon pathogen infection was only observed in the moderately resistant cultivar. In addition, the constitutively higher expression level correlated with increased amounts of SA compared to the moderately resistant cultivar. R. solani AG3 DNA density reflected differences in resistance. The results indicate that expression levels of defense-related genes and the amount of SA in potato tissues can potentially be used as indicators of potato field resistance to black scurf disease.
Mycovirus isolated from three species of the genus Pseudopestalotiopsis
- K. Watanabe
- K. Kusano
- K. Komatsu
- H. Moriyama
- K. Ando
- N. Phay
- D. Hop
- S. Nozawa
Mycoviruses are viruses that infect fungi and have been detected as dsRNA in several fungal species since 1999. More than 200 cases have been reported, but the number of reported mycoviruses is extremely small compared with the number of mycoviruses infecting animals and plants. We also know very little about how mycoviruses infect and affect fungi. Our current understanding states that most mycoviruses are transmitted by hyphal anastomosis from carrier fungi to healthy fungi, and have no pathogenicity to fungi. In recent year, the cases were reported the transmission of mycovirus via an insect and carrier fungi reducing pathogenicity to plant. Interactions between fungi and mycoviruses are therefore not yet fully understood. In this study, we aimed to characterize interactions among fungi, mycoviruses and plants. We obtained mycoviruses from the fungal species of the genus Pestalotiopsis sensu lato, composed of the Pseudopestalotiopsis, Pesatlotiopsis, and Neopestalotiopsis genera including pathogenic, endophytic and saprophytic fungi in plants.
Of the 130 strains belonging to the genus Pseudopestalotiopsis, three strains (F0083, F0015, and F0033) were found to contain dsRNA. DsRNA segments were analyzed using agarose gel electrophoresis and were estimated to be approximately 2.9 kbp in length. In a previous study, we reported used Next-generation sequencing and RT-PCR and reported that dsRNA obtained from Pseudopestalotiopsis sp. F0083 belonged to a novel mycovirus within unirnavirus (Kusano et al, 2017). This virus encoded two ORFs (unknown protein and RNA-dependent RNA polymerase) and these were detected using an ORF finder.
In the present study, we used specific primer pairs designed on the unirnavirus genome of F0083 to confirm that dsRNA detected in F0015 and MM0033 originated from the same unirnavirus. Both dsRNA sequence data in F0015 and MM0033 were mostly shared with dsRNA sequence data in F0083. A protein BLAST search provided the estimates to encode RdRps similar to ORF2 of Penicillium janczewskii Beauveria bassiana-like virus 1 and Beauveria bassiana RNA virus 1 belonging to unirnavirus. Phylogenetic analysis based on the putative RdRp amino-acid sequence revealed that the dsRNAs found in F0015 and F0033 belonged to the same member of unirnavirus. These results indicate that dsRNAs found in different species of Pseudopestalotiopsis are unirnavirus and these mycoviruses are the closest strains to each other.
Intercepted fungi: New and interesting species from the Caribbean and Central and South America
- J. McKemy
- M. Romberg
Global commerce and trade continues to increase. With this comes the potential for the unintentional introduction of non-indigenous species of fungi into new environs. During the last decade imports of various host genera have risen greatly. Among these are: Eucalyptus (Myrtaceae), numerous genera in the Proteaceae (Leucadendron, Leucospermum, Protea), Coccinia (Cucurbitaceaea) and others. In an effort to protect American agriculture, inspection of imported host material is performed as shipments arrive at various ports of entry into the US. Inspected host material suspected of possibly harboring diseases is forwarded to the National Mycology Laboratory in Beltsville, MD. In order to provide more information on the diversity of fungi encountered on the above hosts we have studied more closely the fungi occurring on these hosts intercepted over the past 3 years. Morphological and molecular characterization of several intercepted fungi have provided numerous specimens representing undescribed species. Many others represent species with new distributional records. Genera intercepted on these hosts include Phyllosticta, Cercospora, Colletotrichum, Diaporthe (Phomopsis), Neofusicoccum, Pyrenophora and Pestalotiopsis. New records, species, and distributional information can affect quarantine decisions and have trade implications. Many widely traded host genera represent taxa for which there is a lack of information on associated fungi. Information on the fungi associated with these hosts supports informed regulatory decision-making.
Powdery mildews (Erysiphales) in Victorian horticulture: DNA isolation to rediscover an old foe hidden in herbaria
- R. Smith
- T. Sawbridge
- R. Mann
- J. Kaur
- T. May
- J. Edwards
Powdery mildews pose a significant threat to the global food trade and can cause massive crop losses throughout agricultural and horticultural industries. In Victoria, the horticultural industry is focused around the production of almonds, pome, stone fruit, grapes, citrus, and berries, with powdery mildews responsible for reducing crop yields across all of these major crops. Possessing accurate and current plant pathogen records is vital for Australian import and export of horticultural crops to prevent incursions of new and exotic pathogens. Maintaining up to date plant pathogen data is vital for Australian Biosecurity. The Victorian Plant Pathology Herbarium (VPRI) houses over 42,000 plant pathogen specimens. Of these the powdery mildews (Erysiphales) represent ~2700 specimens, collected from agricultural, horticultural and ornamental host plants. The VPRI plant pathogen database feeds directly into the Australian Plant Pathogen Database (APPD) providing specimen based records of current plant pathogens. Traditionally, morphology and host plant associations were used for the identification of powdery mildews but this has been proven unreliable. The modern approach to fungal taxonomy is to use morphological characters together with genetic analyses. The purpose of this study is to update the VPRI Erysiphale specimen database by traditional fungal morphological character description and genetic analyses.I will present research on the molecular characterisation of VPRI powdery mildew specimens, focusing specifically on horticultural crops of Victoria, Australia. An integral part of this research is the establishment of reliable DNA extraction methods and identification of appropriate gene regions for species identification of preserved powdery mildews specimens. The aim of this project was to test a range of DNA extraction protocols which used varying source material of different ages (fresh, recent -200 years old and ancient) to compare the DNA quantity and quality yielded from the VPRI powdery mildew specimens. We also evaluated fungal gene markers for use in herbarium fungal species identification as it was detected that current markers are too large for powdery mildew herbarium DNA.Once DNA extraction protocols and suitable gene markers have been identified we will first focus on powdery mildews of the Rosaceae family, notably pome and stone fruit, for this study. Once VPRI powdery mildew DNA is obtained we will perform PCR to produce gene marker amplicons for species identification. Confirmation of powdery mildew species will be achieved through amplicon sequencing on the Illumina MiSeq platform. Simultaneously, whole genome sequencing of suitable Rosaceae powdery mildew herbarium DNA will be run on the Illumina HiSeq platform to produce reference genomes for powdery mildews. These reference genomes will fill the current knowledge gap in horticultural powdery mildew genomics as currently there are only three reference genomes available for Blumaria graminis (barley), Erysiphe pisi (pea) and Golovinomyces orontii (cucurbits).
Microsatellite and SNP discovery for population genetic studies of Gemmamyces piceae
- G. Adams
- L. Winton
- S. Everhart
- Z. Kamvar
- K. Černý
- K. Černý
Gemmamyces piceae, a psychrophilic fungus, causes bud blight disease of Picea species in Northwestern Europe and Central European mountains. The pathogen was first identified in Alaska in 2016, although symptoms were first noticed in 2013. Ninety percent of the forest land in Alaska is boreal forest dominated by Picea species. By 2017, blighted spruce buds were recorded at over 200 locations in Southeast, South Central and Interior Alaska. However, examination of numerous samples using microscopy and sequencing of the ITS barcode revealed that there are actually three different fungi causing bud blight in Alaska: G. piceae, Dichomera gemmicola, and Camarosporium strobilinum. These fungi are indistinguishable in the field. 182 permanent plots were established to evaluate the statewide distribution and presence/absence of bud blight. The pathogenic fungi causing bud blight occurred in 65 of the 182 plots; 117 plots lacked symptoms of bud blight. The occurrence of G. piceae is confirmed from Anchorage to Fairbanks, Alaska. However, we did not find G. piceae during surveys of Southeast Alaska. To investigate the origins of G. piceae in Alaska and elsewhere, population genetic studies have been initiated utilizing microsatellite and single nucleotide polymorphism (SNP) genetic markers. Whole genomes of isolates of Alaska and Czech Republic were used for microsatellite library development. Genomic DNA of two Alaska and one Czech isolates were purified and subjected to Illumina HiSeq sequencing, following which, assembly of paired reads was performed and bioinformatically mined using msatcommander software, which aids in identifying repeat type, repeat length, and candidate primer pairs.
Population structure of Teratosphaeria pseudoeucalypti, causal agent of Teratosphaeria leaf blight on Eucalyptus in Uruguay
- C. Perez
- N. Ramírez
- S. Simeto
- G. Balmelli
- B. Wingfield
- D. Tuan
- M. Wingfield
Teratosphaeria pseudoeucalypti, the causal agent of Teratosphaeria leaf blight, was recently reported in South America where it causes severe damage on Euclalyptus camaldulensis, E. tereticornis and related species in plantations. Genetic resistance is the most effective management option for the disease, but success depends greatly on thepopulation diversity of the pathogen. The aim of this study was to characterize the population structure of T. pseudoeucalypti in Uruguay. Isolations were made from leaves on E. camaldulensis, E, tereticornis and hybrids trees widely distributed throughout the country resulting in a collection of 217 strains.. Strains were characterized based on morphology and using molecular markers. The complete genomes of two strains were sequenced, and mating type regions were analyzed. Only the MAT1-1 was found in all analyzed strains, suggesting that the fungus is heterothallic and thus likely asexual in Uruguay. The population was also characterized based on SNP´s of four genomic regions. A single haplotype of T. pseudoeucalypti was found to be present in Uruguay, corresponding to haplotype KE8 reported from Queensland (Australia) in 2010. The results of this study suggest that Eucalyptus breeding and planting stock developed for resistance to T. pseudoeucalypti will have effectiveness against the entire population, and its durability will depends on the effectiveness avoiding new introduction. Thus, highlights the importance of reinforcing quarantine regulations such that new genotypes, possibly including a MAT 1-2 mating type are not introduced into Uruguay in the future.
Developing the tools to monitor populations of Cercospora species associated with Cercospora Leaf Blight on soybean
- H. Tonry
- Z. Carver
- S. Albu
- H. Hutchins
- V. Doyle
The purpose of this research is to develop diagnostic tools that differentiate species of Cercospora associated with Cercospora Leaf Blight (CLB) and Purple Seed Stain (PSS) on soybean. Cercospora cf. flagellaris, C. kikuchii, and C. cf. sigesbeckiae are three closely related species that have been associated with CLB and PSS but are difficult to distinguish based on phenotypic characters. It is imperative that we are able to correctly and rapidly identify these pathogens in order to monitor the pathogen population and better understand the etiology of the disease. We used a bioinformatics approach to identify species-specific primers with sequence data from twenty-three Cercospora species across sixteen loci to develop specific primers sets for C. cf. flagellaris, C. kikuchii, and C. cf. sigesbeckiae. We have successfully developed primers that will discriminate among C. cf. flagellaris, the dominant species in Louisiana, as well as C. cf. sigesbeckiae and C. kikuchii. These primers will allow us to monitor populations of Cercospora associated with CLB and PSS and potentially quantify fungal biomass. These diagnostic tools will be used in ongoing efforts to identify sources of inoculum and understand patterns of dispersal in order to improve disease management in soybean.
Epitipification and phylogenetic relationship of cercosporoid fungi associated with plants of the brazilian Cerrado
- A. Silva
- D. Pinho
Cercosporoids are commonly associated with leaf lesions in several plant species. Currently, there are over 2000 species, most of which are known in a single host. This group of fungi is the most representative in the brazilian Cerrado, but to date the majority of species have been described exclusively by morphological characteristics. The inclusion of molecular data in the taxonomy of cercosporoid fungi revealed that the group is polyphyletic and demonstrated the need for sequence comparison for the accurate identification of the species. Therefore, this work has the objective of epitypify the specimens of cercosporoid fungi associated to the Cerrado plants and to understand their phylogenetic relationship. For precise identification, isolates were obtained from leaf lesions on Piper aduncum (Piperaceae), Solanum sp. (Solanceae), Palicourea rigida (Rubiaceae), Smilax japecanga (Smilacaceae), Cybistax antisyphilitica, Handroanthus ochraeus, Handroanthus heptaphyllus, Handroanthus serratifolius, Tabebuia aurea, (Bignoniaceae), Passiflora setacea (Passifloraceae), Acrocomia aculeata, Mauritia flexuosa (Arecaceae) and Vellozia squamata (Velloziaceae). After confirmation of identity by morphological comparisons, the genomic DNA was extracted using the Wizard® Genomic DNA Purification kit. Nucleotide sequences from the ITS region were obtained and compared to sequences of type species and specimens available from GenBank. In total, 31 isolates were obtained from 13 host plants belonging to eight botanical families. The isolates obtained belong to the Mycosphaerellaceae and are grouped into ten distinct clades. This study confirms the report of Pseudocercospora piperis associated with Piper aduncum, with high phylogenetic support. The epitypes of Cercospora tabebuiae-impetiginosae, Pseudocercospora passiflorae-setaceae, P. cybistacis, P. tabebuiae-caraibae, and P. tabebuiae-ochraeae associated with Handroanthus heptaphyllus, Passiflora setacea, Cybistax antisyphilitica, Tabebuia aurea and Handroanthus ochraceus, respectively, were designated for the first time and a probable new species of Pseudocercospora associated with Vellozia squamata will be proposed following the norms of the International Code of Nomenclature for algae, fungi, and plants. The specimens examined in this study represent a small fraction of cercosporoid fungi reported in the Cerrado and demonstrate the need to re-collect these organisms to understand their phylogenetic relationship. Financial support: FAP-DF, Capes, CNPq and UnB.
Multiple lines of evidence suggest alternative hosts are involved in the development of Cercospora Leaf Blight of soybean in Louisiana
- V. Doyle
- Z. Carver
- P. Price
Recent phylogenetic studies have changed our understanding of the diversity of Cercospora species associated with Cercospora leaf blight (CLB) and Purple seed stain (PSS) of soybean. Cercospora kikuchii has long been considered the causal agent of CLB and PSS, but we now recognize a composite of species are associated with the disease. These studies suggest most of the species associated with CLB and PSS are not host specific, in contrast to our current understanding of C. kikuchii. Given the limited host range of C. kikuchii, the frequently observed uniformity of disease symptoms in the field, and the fact that the pathogen is seed borne, it has been assumed that the primary source of inoculum for the disease is soybean seed. However, this assumption needs to be revisited in light of the diverse host preferences of C. cf. flagellaris and C. cf. sigesbeckiae, two of the predominant species associated with CLB and PSS. The aim of our current work is to determine the principal sources of inoculum of CLB and PSS through field trials and molecular ecology/epidemiology. We examined the role of seed borne inoculum by eliminating all microbes from the seed prior to planting and monitoring disease incidence and severity over the course of two field seasons. We also characterized the diversity of Cercospora species on soybean seed prior to planting, from blighted leaves, and from harvested seed to examine changes in the composition of the Cercospora community over the course of a season. Finally, we looked for direct evidence of gene flow between alternative hosts both proximate to and distant from agricultural fields. Despite eliminating all microbes from the soybean seed prior to planting, we did not observe a reduction in disease incidence or severity at two different sites in Louisiana over two field seasons, consistent with planted seed not being the primary source of inoculum. We also found that the composition of Cercospora species present in the community at the time of planting (seed) significantly differs from that responsible for leaf blight and seed stain at the time of harvest. In the first season, regardless of the frequency of each of C. kikuchii, C. cf. sigesbeckiae, and C. cf. flagellaris on seed at the time of planting, C. cf. flagellaris dominates the community on blighted leaves and harvested seed. This shift in the community over the course of the field season also indicates seed is not the primary source of inoculum. We are currently working on replicating this study with collections from a second field season. Finally, genotype data of C. cf. flagellaris (n=72) and C. cf. sigesbeckiae (n=14) at 19,670 SNP loci indicate ongoing gene flow between alternative hosts proximate to agricultural areas and those on soybean. However, the lack of differentiation among isolates from seed in distant geographical areas coupled with significant differentiation among agricultural and non-agricultural populations (alternative hosts), suggests the movement of seed has historically been a significant source of inoculum in soybean fields.
Exploring the diversity of Colletotrichum species in Louisiana and the Gulf South.
- D. Cooke
- H. Hutchins
- V. Doyle
The genus Colletotrichum is globally distributed with diverse ecological niches, including endophytes, pathogens, and saprobes, and ranks among the most important genera responsible for both pre- and post-harvest losses. According to the Louisiana Plant Disease Management Guide (LA-PDMG), Colletotrichum is a pathogen of more than 64 ornamental plant species, 12 major crops, and 9 vegetable groups in Louisiana. The development of agricultural practices that minimize the impact of fungal pathogens to Louisiana’s $1.78 billion agricultural industry is reliant on knowing the species responsible for plant diseases. However, many of the species in the LA-PDMG remain unidentified despite significant advances in Colletotrichum systematics and taxonomy over the last decade. We aim to identify the Colletotrichum species associated with plants in Louisiana and the Gulf South by placing isolates into a broader phylogenetic context. We collected endophytes and pathogens from host species listed in the LA-PDMG as well as other introduced and native plant species in the Gulf South. We isolated Colletotrichum from healthy and diseased plant tissue, extracted genomic DNA, and sequenced the APN2-MAT IGS or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) regions. These specific loci have been previously shown to be useful for species identification in Colletotrichum. Among the 56 plants collected, we isolated Colletotrichum from 21, including Liriope spp. (monkey grass), Magnolia (Magnolia grandiflora), Paw Paw (Asimina triloba), Ligustrum (Ligustrum spp.), and ornamental Olive (Olea europaea cv. 'Arbequina'). Our results will have implications for fungal systematics, fungal ecology, and plant pathology by providing a more thorough understanding of Colletotrichum diversity in Louisiana and the Gulf South and the role of individual hosts in the movement of these endophytes and plant pathogens.
Role of motility and phytohormone biosynthesis of an endohyphal bacterium from Rhizoctonia solani.
- P. Zhang
- K. Obasa
- J. Huguet-Tapia
- P. Zhao
- F. White
Fungal-bacterial interactions have profound impact on mycorrhizosphere environment of crops, while the nature of these symbiotic relationships remain understudied. An endohyphal bacterium Enterobacter sp., named En-Cren, was isolated from the pathogenic soil fungus Rhizoctonia solani AG2-2IIIB that causes brown patch disease on cool-season turf grass (Agrostis stolonifera L.). The virulence of the fungal pathogen is associated with the presence of En-cren. En-cren can be released as a free-living bacterium when fungal hyphae are damaged and migrate rapidly along the outside of the fungal mycelia (including the aerial hyphae). The major objectives of this study were to identify the genetic nature of hyphal motility of the bacterium and whether the bacterium contributes to fungal virulence through the production of phenylacetic acid (PAA), a phytohormone that had been hypothesized to play a role in the interaction between R. solani and host plants. Single gene mutations for flagella hook protein flgE and type IV pilus secretin pilQ in En-cren did not prevent hyphal movement. Double mutants of both motility apparati will be tested. En-cren produces PAA and indole acetic acid (IAA) in minimal media supplemented with precursor compounds, while mutation at the gene for indole-3-pyruvate decarboxylase suppressed PAA and IAA production in cultures of the free-living bacterium. The study will provide more information for our understanding of fungal-bacterial interaction in the mycorrhizosphere and of the virulence mechanism of the broad range soil pathogen Rhizoctonia solani.
Importance of basement topography as a distribution factor of Pythium in rotational paddy fields with barley, wheat and/or soybean in hilly and mountainous areas in Japan
- Y. Shimizu
- S. Mori
- H. Mochizuki
- K. Matsumori
- A. Kawaguchi
- K. Nomiyama
- K. Tomioka
We aimed development of techniques for low cost and stable production of rice, barley, wheat and soybean in hilly and mountainous areas in western Japan, based on their rotational cultivation. Poor growth of barley and wheat was found in experimental fields located in Hiroshima Prefecture, Japan, in December 2016. Severe waterlogging was observed in those areas caused by excess soil water due to poor drainage. Pythium spp. were frequently isolated from the plants that showed poor emergence in the waterlogging areas. Aerial photos taken by an unmanned aerial vehicle revealed a clear relationship on the spatial distribution between waterlogging areas and poor growth of barley and wheat. It was also clarified that the waterlogging areas located above former river channels which had been buried in the fields, through spatial analysis with overlapping the aerial photos and former topographical maps using geographical information system. From these results, it was revealed that basement topography should affect to growth of barley and wheat. Pythium known as an aquatic microorganism prefers to wet condition such as in-water or water-soaked areas. Understanding of basement topography will be important to know distribution of Pythium in fields in hilly and mountainous areas in Japan. The following species of Pythium have been recorded as pathogens of browning root rot of barley or wheat in Japan; Pythium iwayamai, P. paddicum, P. horinouchiense, P. graminicola, P. okanoganense, P. spinosum, P. sylvaticum, P. ultimum var. ultimum, P. vanterpoolii, and P. volutum. The present Pythium isolates from barley and wheat were tentatively identified as species similar to P. aphanidermatum or P. myriotylum based on their morphological and cultural characteristics. In future, we will investigate their pathogenicity to barley and wheat.
A deep sequencing dual RNAseq approach investigating the interaction between Rhizoctonia solani AG1-IB and a commercial Lactuca sativa cultivar (cv. Tizian)
- B. Verwaaijen
- D. Wibberg
- A. Winkler
- R. Zrenner
- H. Bednarz
- K. Niehaus
- R. Grosch
- A. Pühler
- A. Schlüter
The phytopathogenic species Rhizoctonia solani is capable of infecting a wide range of economically important crop plants like; soybean, rice, sugar beet, potato and lettuce. Isolates of R. solani AG1-IB are responsible for bottom rot on lettuce. The genome of R. solani AG1-IB (isolate 7/3/14) was recently established. At present, little information regarding the molecular responses in R. solani during the interaction with its host plants is available though. Therefore, the transcriptome of R. solani AG1-IB (isolate 7/3/14) was studied during the course of its pathogenic interaction with the host plant lettuce using a leaf infection model. Samples were taken from three distinct pathogen-host interaction zones which covered different phases of disease progression on leaf tissue inoculated with the AG1-IB isolate 7/3/14. The zones can be defined by, symptomless leaf tissue in zone 1, zone 2 with light brown discoloration and zone 3 with dark brown necrotic lessions. These zones were also verified by means of microscopy. We found decreased auto fluorescence of chlorophyll in zone 2 and 3 accompanied by fungal infection structures, whereas zone 1 only contained symptomless runner hyphae. For transcriptome analysis, we sampled these zones for high throughput Illumina RNA sequencing. This approach improved sensitivity and robustness of the dual RNAseq transcriptome analysis, enabling the detection of over 3000 significantly differential expressed genes, by cross comparison of the three sampling zones. A group of 21 previously undescribed protein coding transcripts was detected that were almost exclusively transcribed in zone 1. This group could contain potential novel R. solani effectors. Furthermore, we were able to give the first account of R. solani RSA lectin transcription levels during infection. Within zone 2 and 3 R. solani RSA encoding transcripts were among the overall most abundant. Overall we found that in zone 1 transcripts related to proliferation, metabolism and energy turnover are low in abundance, in zone 2 transcription of the corresponding genes is highly upregulated whereas in zone 3 proliferation is decreased again and signs indicative of programmed cell death can be found. To date, in a follow up study, we are investigating the transcriptional response of lettuce towards infection by R. solani.
Redefining genera in the Ophiostomatales
- M. Proctor
- Z. De Beer
- T. Duong
- M. Wingfield
The Ophiostomatales is an order of Ascomycete fungi that contains many economically important tree pathogens, sap-staining fungi and even a few human pathogens. Most species are associated with tree- or plant infesting arthropods such as mites and bark- and ambrosia beetles, while some occur in soil and other unique niches, for example Protea infructescences. The taxonomy of the Ophiostomatales was confused for more than a century due to the dependence on morphological characters for species and genus definitions. The availability of DNA sequences helped to align the taxonomy of these fungi with phylogenetic relatedness, and resolved the confusion between Ceratocystis (Microascales) and Ophiostoma (Ophiostomatales). However, the sexual and asexual states of many species in the Ophiostomatales were still treated in different genera, with genera typified by sexual states having priority. The abandonement of dual nomenclature necessitated a re-evaluation of nomenclature in the Ophiostomatales. The aim of this study was to reconsider the generic boundaries and unresolved nomenclatural issues in the Ophiostomatales based on phylogenetic analyses. Sequences of 155 gene regions were extracted from whole genomes available for 40 taxa representing all the major lineages in the order, and a maximum likelihood tree was generated based on the concatenated data. In addition, sequences of four gene regions were determined for more than 240 species, and single gene trees as well concatenated trees were generated based on these data. The respective trees provided a much clearer understanding of where generic boundaries, supported by morphology and ecology, should be drawn. Based on the results we accept current definitions for Sporothrix, Ceratocystiopsis, Aureovirgo, Graphilbum, Fragosphaeria, Hawksworthiomyces, Esteya and Afroraffaelea. The generic boundaries for Leptographium, Raffaelea and Ophiostoma had to be redefined, and the names Grosmannia and Dryadomyces were re-instated. Six new genera are being described. Based on these results, most genera in the Ophiostomatales can be recognized based on morphology and ecology. However, DNA sequences remain essential to confirm generic placements and species delineation.
Assessing the clinical relevance of fungal genotype in Blastomycosis infections
- K. Laux
- S. Bentivenga
- J. Anderson
- G. Gauthier
- J. Meece
Blastomycosis is a fungal disease caused by infection with the fungus Blastomyces which grows as a mold at ambient temperatures and produces infectious spores. When inhaled, the spores undergo a temperature induced transition into yeast which cause infections such as pneumonia, skin lesions, meningitis, bone infections, and sepsis. In endemic regions in the United States, the incidence of infection of blastomycosis ranges from 1-2 per 100,000 annually. However, some counties in Wisconsin have reached up to 41.9 per 100,000 in humans and up to 1,420 per 100,000 in dogs which makes Wisconsin hyper-endemic for this disease. In 2013, multi-locus sequence typing revealed two distinct species of Blastomyces were responsible for blastomycosis—Blastomyces dermatitidis and Blastomyces gilchristii. Despite differences in genotype and demonstrated differences in clinical presentation between these two species, the clinical relevance of this distinction had yet to be thoroughly explored. As physicians are currently blind to species type, the goal of this study was to assess whether the recent distinction had clinical relevance for care providers. Clinical isolates (N=112) were obtained from patients throughout the state of Wisconsin who were diagnosed with blastomycosis from 2008-2016. Fungal DNA was extracted and the ITS region of the genome was amplified and sequenced. A single nucleotide polymorphism in the ITS sequence was used to distinguish Blastomyces dermatitidis from Blastomyces gilchristii. Fungal genotype was compared to patient demographics, sensitivity and specificity of the urine antigen test, and patient treatment collected from clinical records. In keeping with previous studies, patients infected with B. dermatitidis infections were 15 years older, more likely to have a pre-existing condition, and more likely to be male than those with B. gilchristii infections (p<0.05). Patients infected with B. gilchristii were diagnosed earlier than those with B. dermatitidis (p=0.05) which may indicate differences in disease severity. The urine antigen test displayed false negatives 38% of the time in B. dermatitidis infections as opposed to 18% false negatives in B. gilchristii infections. A false negative was defined as failure to display positive result throughout the course of a known blastomycosis case. Quantities of detected antigen were also significantly lower in B. dermatitidis infections as opposed to B. gilchristii infections (p<0.05). While 97% of physicians made use of itraconazole to treat blastomycosis, only in 50% of cases physicians used the itraconazole serum concentration test. We found evidence to support use of therapeutic drug monitoring regardless of fungal genotype as 61% of patients displayed at least one value that merited dose modification or patient education. While in 97% of blastomycosis cases physicians made use of radiographic imaging in diagnosis and post-diagnostic monitoring with a range of 1-66 X-rays per patient, there are currently no guidelines for how these should be performed. Fungal genotype has clinical relevance in terms of urine antigen test performance, patient demographics, and clinical presentation. Knowledge provided by this study will improve clinical decision making and patient care in cases of blastomycosis.
Dimorphic pathogen, Emergomyces africanus, identified in Cape Town air samples
- E. Levetin
- J. McLoud
- I. Schwartz
- D. Berman
- A. Botha
- B. Lerm
- R. Colebunders
- C. Kenyon
Emergomyces africanus is a recently described dimorphic species in the Ajellomycetaceae that causes an AIDS-related systemic mycosis in South Africa. Infections are believed to result from inhalation of conidia or other infectious propagules, although no attempts had previously been made to isolate E. africanus from the air. The present study was undertaken to develop molecular techniques for the identification and quantification of E. africanus propagules from air samples. The atmosphere was monitored with a Hirst-type 7-day spore trap (Burkard Manufacturing, Co. Ltd., Rickmansworth, Hertfordshire, England) equipped with an alternate orifice to increase sampling efficiency for small spores. The sampler was stationed on the roof a building 4 meters above ground in Bellville, an urban area of Cape Town, Western Cape, South Africa and sampling occurred from 15 Sep 2015 to 29 Aug 2016. Melinex tape was attached to the sampler drum and greased with petroleum jelly; sampler drums were changed weekly. One-day (24 hour) segments of the exposed Melinex tape were cut into smaller pieces and DNA extracted. For each week, fractions of the DNA extract from the 7 daily air samples were pooled to produce a weekly air sample; this allowed testing of both daily and weekly periods. Quantitative PCR was performed using E. africanus species-specific primers and TaqMan probe that hybridized to a region of the ITS I gene, and propagules were calculated using a standard curve which had a lower limit of detection of 5 propagules. Emergomyces africanus DNA was detected in 11 of the 50 weekly samples. Ten of these 11 weeks produced ITS target numbers that were within the range of the standard curve and could be quantified. The week of 26 Jul to 1 Aug 2016 had 63 propagules in the pooled sample, which was the greatest number detected. Aliquots of the DNA from the daily air samples from the 11 positive weeks were also analyzed for E. africanus. From these 77 daily samples, 34 were positive; however, only 11 were within the range of the standard curve and quantified. The highest numbers of E. africanus propagules were found on 30 July and 1 Aug with 26 and 24 propagules, respectively. This study showed that E. africanus propagules were detected in the atmosphere of an urban industrial setting on approximately 10% of the days during the period analyzed and suggests that airborne exposure to the pathogen is common in Cape Town. More research is needed to determine the extent of exposure in other areas where infection has been diagnosed.
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.
MALDI-TOF mass spectrometry, a rapid identification tool for the newly revised Cryptococcus neoformans/C. gattii species complex
- K. Grosso
- N. Krueger
- S. Suh
Cryptococcus neoformans and C. gattii are clinically important yeasts that can cause infections in humans, including meningoencephalitis and Cryptococcosis. Although the two species are phylogenetically closely related to each other, the former primarily infects immunocompromised patients, while the latter primarily is diagnosed in otherwise healthy persons. Recently, Hagen et al. refined the species complex into seven distinct species and four hybrid genotypes based on phenotypic and genotypic diversity, such from the sequence comparison of eleven genetic loci, serotyping, electrophoretic karyotyping, etc. The ATCC Mycology portfolio contains a significant number of yeasts in this species complex isolated from various sources and regions, and a rapid and accurate identification tool is strongly desired to appropriately classify these yeasts within the revised species complex. In this study, we evaluated MALDI-TOF mass spectrometry as a cost-efficient rapid identification method for the revised C. neoformans/gattii species complex. Type strains of the seven newly identified species and an additional five reference strains were used to develop reference spectra. The protein profiles were acquired from cultures on at least two different media (Sabouraud dextrose agar and YM agar) at two different incubation time (24 hrs and 48 hrs). A dendrogram generated from the protein profiles using a VITEK® MS plus system (bioMérieux) showed that each species was clearly distinct from other species in the complex, although a few profiles from 48 hr cultures were clustered with other closely related sister taxa. Reference spectra of each species were evaluated by testing with those strains fully characterized by the multilocus DNA sequencing of ITS, D1D2, IGS, and GPD gene. The results indicate that MALDI-TOF mass spectrometry shows promise as a rapid and efficient tool for identifying these closely related yeasts by their protein fingerprints.
Inflammatory responses in mice after intratracheal instillation of spores of some Aspergillus and Penicillium spp monitored in Southwest Nigeria.
- A. Odebode
- A. Adekunle
- F. Ebenezer
Fungi are an increasing public health problem worldwide because they have a great impact on human health. In this study, comparative analysis of airborne fungal spores for two year (May 2014 – April 2016) in ten different locations in Lagos and Oyo States, Nigeria. Identification and characterization of fungal species was carried out by amplification of internal transcribed spacer 1 and 4 gene followed by quantification of allergenic gene by reverse transcriptase quantitative polymerase chain reaction in the most abundant fungal isolates. A mouse model was devised to elucidate and compare the adverse effects provoked by the four most abundant fungi isolated from various locations in all locations. Sixty Balb/b albino mice were grouped into nine treatments of six per cage with a control group. The animals were exposed intranasally to the spores of A.flavus, A. penicilloides, Penicillium citrinum and Penicillium chrysogenum at 2.3 x 10 7 and 3.2 x 10 5 m/L. Both dose-response and time-course inflammatory and toxic responses were investigated after a single dose of the microbes. Biochemical parameters and histopathology revealed that all the microbes studied provoked inflammation after a single dose but the magnitude and its characteristic features were different. The spores of A.flavus, and A. penicilloides provoked a very intense acute inflammation indicated by production of increased malondialdehyde, myeloperoxidase, total protein production in the lungs, recruitment of inflammatory cells into the airways and expression of neutrophils, eosinophils and basophils in the blood. The inflammatory cell response in the lungs was more severe and varies with each organism at different concentrations. Histopathology result for all inoculated organisms on mice lung showed that there was mild thickening of the alveolar interstitium and moderate haemorrhages. There was also accumulation of inflammatory cells around blood vessels suggestive of vasculitis. The results showed that A. penicilloides in addition to A. flavus had highly significant lethality on lungs of mice than those of P. citrinum and P. chrysogenum. This study has also confirmed that production of fungal spores is indicative of weather parameters. Environmental conditions such as relative humidity (RH), temperature and wind velocity exert a significant effect on the amount of microorganisms in the air therefore airborne microbial quantity and quality can vary with time, year and location.
Identification of dermatophytosis and onychomycosis etiologic agents by classic and by MALDI-TOF MS phenotypic methods
- J. Gómez-Velásquez
- N. Loaiza
- J. Romero
- N. Lima
- S. Florez-Muñoz
- A. Mesa-Arango
Skin, nail and hair infections are a frequent cause of dermatological consultation in tropical countries. The fungi involved in these infections are usually dermatophytes, Candida spp. and some non-dermatophyte filamentous fungi (NDFF). Within the NDFF group, Neoscytalidium dimidiatum has been increase as common agent of onychomycosis. The identification of these etiologic agents, in routine mycological diagnosis, is mainly based on macro- and micro-morphology characters. However, this approach is subjective and the identification could be not accurate. This is an obstacle for the implementation of appropriate therapy, since that antifungal susceptibility profiles may vary among species. Currently, more sensitive, specific, rapid and cost-effective techniques are being introduced for the identification of microorganisms, such as Matrix Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI-TOF) mass spectrometry (MS) with the protein profile of each microorganism. The aim of our study was to compare the identification by classic and MALDI-TOF MS phenotypic methods fungal isolates collected from patients with a clinical diagnosis of onychomycosis or dermatophytosis. In total, 134 clinical isolates were included in the study and identified by classic macro- and micro-morphology characters from de cultures. In addition, the isolates were analysed by MALDI-TOF MS. An in house spectra library was establish in order to identify N. dimidiatum, because this species is not included in commercial libraries. Identification by classic approach resulted in 76 dermatophytes with T. rubrum (41), T. mentagrophytes complex (15), T. tonsurans (6), E. flocossum (2), M. gypseum (1) and M. canis (11); 26 Candida with C. albicans and C. tropicalis representing 5 and 4 isolates, respectively; for NDFF the prevalent species was N. dimidiatum with 25 out of 31 isolates, follow Fusarium spp (4), Aspergilllus flavus (1) and Penicillium sp (1). One Trichosporon sp was also identified. For MALDI-TOF MS the dermatophytes isolates identified as T. rubrum and M. canis reduced to 16 and 4, respectively and the prevalent group became Trichophyton spp (37). For NDFF, MALDI-TOF MS confirmed the identification at species level of 19 N. dimidiatum, the isolate A. flavus and at genus level the 4 Fusarium spp. Within the NDFF isolates, the Penicillium sp isolate was correctly identified by MALDI-TOF MS as A. versicolor. The Trichosporon sp isolate was also confirmed. Finally, the identification of Candida isolates by MALDI-TOF MS were resolved at species level with C. parapsilosis (6), C. haemulonii (1) and C. guilliermondii (1). The inconsistencies observed in the results from the two approaches used with more evidence for filamentous fungi than for yeasts identifications can be due to: 1) the established score to define the identification up to genus or species level; 2), sample preparation and the quality of the extraction of proteins; and, 3) the lack of spectra or low number of species included in the commercial libraries could interfere in the final outputs. The results of this work clearly show that the classic approach is prone of errors, much more work is needed to make MALDI-TOF MS identification a routine in clinical laboratories and to clarify these results identification by molecular biology is advised.
Establish in Medellin (Colombia) an in-house library to identify clinically important filamentous fungi by MALDI-TOF MS
- J. Gómez-Velásquez
- N. Loaiza
- J. Romero
- N. Lima
- A. Mesa-Arango
Epidemiology of fungal infections has changed in an important manner in the last decades, however timely diagnosis and treatment are still a current challenge.Accurate and swift identification of the agents that cause fungal infections are paramount, since sources of infection, differences between therapeutic regimes, and in-vitro susceptibility profiles may vary amongst species and strains. The application of Matrix Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI-TOF) mass spectrometry (MS) for the identification of fungal samples is currently well-established based on the remarkable reproducibility for the measurement of constantly expressed and highly abundant proteins, such as ribosomal proteins, that are used to generate a fingerprint profile. However, the use of this tool for filamentous fungi identification in routine clinical laboratories is still limited due to several reasons, such as, 1) the biological variations among fungal clinical isolates or 2) the lack of a spectrum of reference in commercial libraries for the identification of emerging, endemic and prevalent fungi in tropical regions. Consequently, the establishment of an in-house library that will contain spectra for prevalent fungi found in routine mycological diagnosis at a local level, and of those not included in commercial libraries, is a demand.The aim of this research was to establish a library of spectra for the identification of clinically important filamentous fungi through MALDI-TOF MS in a mycological diagnosis laboratory. In order to establish the in-house spectra library, 21 strains were used, including dermatophytes (Trichophyton (3), Microsporum gypseum (1) and Microsporum canis (1)), Aspergillus (7), Fusarium (4), Neoscytalidium dimidiatum (1) and Sporothrix (4). Afterwards, the spectra were validated with 17 strains and 35 clinical isolates identified by classic and molecular methods. In addition, 16 of 17 strains and 29 of 35 clinical isolates were subjected to identification in commercial filamentous fungi libraries and in BDAL (Bruker Daltonics, Germany). The results obtained from the three libraries were compared. For the acquisition of the reference spectra for each genus and for some species, it was necessary to standardize growth and assay conditions.The in-house generated library identified clinical isolates/strains down to species in the proportion of 82.5%/93.0% of the cases, and down to genus in 88.6%/100.0%. In addition, 11.4%/0.0% of the fungi not being identified. With the filamentous fungi library, identification down to genus and species were of 62.0%/64.3% and 31.0%/42.8%, respectively for clinical isolates and strains, while 37.9%/35.0% proportion remained unidentified. With the BDAL library identification the proportion for clinical isolates/strains was of 42.3%/7.1% for genus and 3.4%/0.0% for species; identification was not achieved in 51.7%/92.8% of the cases. Optimal growth time required to obtain proteins varied among genera and among some species. With the in-house built library, it was possible to identify strains and clinical isolates, in some cases, more accurately than with commercial libraries. In addition, fungal spectra that are not included in commercial libraries were included; this foster the standardization of the growth conditions for the different strains, the protein extraction technique, as well as the definition of criteria for the acceptance of a spectrum of reference.
Improving tree potassium content effectively controls the occurrence and development of apple Valsa canker
- G. Sun
AbstractValsa canker disease, caused by the fungus Valsa mali, seriously impedes apple production in China and other eastern Asian countries. Infection can take place throughout the year, and pathogen colonization produces localized cankers that kill twigs, limbs, and, finally, the entire tree. Because V. mali penetrates into the host phloem and xylem, resulting in a perennial canker, fungicide application is ineffective. All commercially important apple varieties are susceptible to the pathogen, and the average disease incidence is over 50% across all tree ages in China. To better understand the reason for the prevalence of Valsa canker and provide a theoretical basis for its control, field disease investigation and fertilization experiments were performed. Field investigation of the occurrence of Valsa canker in 24 apple orchards in concert with foliar nutrient analysis found a significant negative correlation of leaf potassium (K) content with incidence and severity of Valsa canker. Greenhouse fertilization experiments showed that increasing tree K content enhanced resistance to pathogen colonization and establishment. Apple trees with leaf K content greater than 1.30% exhibited almost complete resistance to V. mali. Field trials demonstrated that increasing K fertilization could significantly reduce disease incidence. Efforts are ongoing to dissect the underlying mechanisms for the observed K effects on Valsa canker. Preliminary result showed that K deficiency reduced the accumulation of cinnamic acid and 4-coumaric acid upon challenge inoculation, these phenolic compounds inhibited V. mali growth on PDA. Our study demonstrates the feasibility of managing Valsa canker on apple with adjustments to K mineral nutrition, and implies that K affects Valsa canker resistance via regulating phenolic compound metabolism.
Will the real pecan truffle please stand up: use of molecular tools reveals cryptic diversity within the Tuber lyonii complex and an updated phylogeny of the Tuber rufum clade
- A. Grupe
- R. Healy
- A. Mujic
- M. Jusino
- G. Bonito
- G. Guevara
- T. Brenneman
- A. Kinoshita
- J. Trappe
- M. Smith
Tuber species are among the most iconic mushrooms of the culinary world. They share a rich, cultural history throughout the world. While the European black truffle (T. melanosporum) and the European white truffle (T. magnatum) are well known, there is a growing market demand for the native North American pecan truffle (Tuber cf. lyonii). For 110+ years, the true identity of the pecan truffle has been ambiguous because spore morphology and size are not useful metrics to determine species identity within this lineage. Tuber lyonii, originally described by Butters in 1904 from Minnesota, and T. texense described by Heimsch in 1958 from Texas, are the names that have been applied to multiple cryptic species of the T. rufum clade. The objectives of this study are to elucidate the true identity of the pecan truffle, obtain sequence data from the holotypes of T. lyonii and T. texense, and build an updated phylogeny for the Tuber rufum clade. Through use of herbaria in Asia, Europe, and North America, we obtained specimens of target taxa and holotypes of T. lyonii and T. texense before sequencing the Internal Transcribed Spacer (ITS) region. Sanger sequencing was successful for generating a preliminary tree as well as obtaining sequence data from the holotype of T. lyonii. An Illumina MiSeq metagenomic barcode for the ITS1 region was performed on the holotype of T. texense and yielded an ITS1 sequence. Although traditional molecular markers work for species level delineation, they lack the ability to resolve the cryptic species within the “T. lyonii” complex. However, an ITS sequence from the holotypes of T. lyonii and T. texense have shown that these holotypes are distantly related to the commonly recovered species that have been previously called by these names. Here we use Sanger sequencing and sequence capture techniques to resolve the species within this cryptic, yet commonly recovered complex. We present the most robust phylogenetic analysis of the T. rufum clade to date. The results and future implications of this research on fungal systematics, utility of herbarium specimens, and the developing market for pecan truffles are also discussed.
Boletes of Bangladesh: taxonomy and phylogeny
- M. Hosen
- Z. Yang
- T. Li
Boletes are fascinating mushrooms in the family Boletaceae comprising ca. 70 genera. A good number of them are frequently collected for human consumption. Bangladesh is a part of tropical South Asia, has the widest and most scattered distribution of Shorea robusta, a tree of Dipterocarpaceae which plays symbiosis association with many groups of fungi. However, macrofungi in Bangladesh are poorly understood. The aim of the study is to investigate the species diversity of boletes and their evolutionary relationships with other boletes of the word. Boletes samples were collected from eight districts of Bangladesh under the monodominant stands of Shorea robusta. Species delimitation was documented using morphological data and molecular approaches of the four genes (28S+tef1-α+rpb1+rpb2). This is the first survey and taxonomic report of boletes from Bangladesh. An extensive field survey was conducted during the monsoons of 2011–2016, resulted over 250 boletes samples, representing eight genera and 19 species. From amongst the collection of boletes, herein are reported two new genera, 13 new taxa, and three new lineages at generic level (uncertain phylogenetic position) based on morphology along with molecular evidence. In conclusion, this is the first attempt to discovery of boletes, and their diversity, distribution and evolutionary relationships will be addressed during the congress.
Studies in Floridian boletes
- A. Farid
- A. Franck
- J. Garey
DNA sequencing of macrofungi continually shows that many species are yet to be described and many species belong in different genera than originally thought based on morphology. The boletes are remarkably diverse in Florida, yet very few have been sequenced. Numerous boletes were described my William Alphonso Murrill (1869–1957) and Rolf Singer (1906–1994); the protologues and type specimens of many of these species require critical re-examination to understand their application. We have approached the study of boletes in Florida with field work, herbarium studies, microscopy, and targeting the main phylogenetic loci for fungi (ITS, LSU, TEF1, and RPB), sometimes requiring the design of novel primers for successful amplification. Studies of phylloporoid fungi in Florida revealed one species which requires a new genus, adding another example of the independent gain of gills in boletes. A Tylopilus-like species collected on the University of South Florida campus (Tampa, Florida) likely represents another novel bolete genus. Additional sequencing suggests several species are undescribed, some belong in recently described genera, and other species are newly confirmed for Florida.
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.
Cortinarius clavatus sp.nov. a new species in section Hinnulei genus Cortinarius from Pakistan
- J. Khan
- H. Sher
- A. Khalid
This study is a part of larger project dealing with exploration of macrofungal species concentrated in forest and woodland ecosystems of northern areas of Khyber Pakhtunkhwa, Pakistan. The study area is predominantly mountainous encompassing well-known ranges of Himalaya and Hindu-kush. In this paper a new species of Cortinarius (C. clavatus sp.nov.) is reported, described and illustrated. Specimen of this new species were collected from coniferous forest of Miandam valley, district Swat, Pakistan during summer 2015–2017. Morphological characters along with molecular data (nrITS) was used to confirm species identification. Basidiospore ornamentation was checked by using electron microscopy. Cortinarius clavatus is characterized by convex to plano-convex, light brown to strong brown umbonate pileus, clavate to slightly thickened fibrillose stipe and broadly ellipsoid to sub-amygdaliform, ornamented basidiospores with suraphilar depression. Maximum likelihood analysis inferred from nrITS data clusters the new species within the Hinnulei clade of Cortinarius supported by a strong bootstrap value.
The genus Inocybe from cedar dominating forests of Pakistan
- S. Jabeen
- A. Khalid
Inocybe is a large genus of mushroom-forming ectomycorrhizal fungi with more than 700 species distributed worldwide. The number is increasing considerably with the exploration of tropical and southern temperate areas. Many species have been reported from Asian continent. From Pakistan, 22 species of Inocybe have been reported so far. During the field survey to study the ectomycorrhizal fungal communities associated with Cedrus deodara from Pakistan, several fungal taxa have been identified using morphological and molecular techniques. Inocybe has been found to be the most diverse ectomycorrhizal genus. It is represented by 20 species, among these 6 species have been found in the form of basidiomata while 13 were found in association with root as ectomycorrhizae. One species has been represented from the above ground and also identified from the host root in the form of morphotype. Among these, four species as basidiomata are previously undescribed while from the belowground data, 12 were identified as operational taxonomic units and given the names with running numeric. This data gives an overview about the species diversity of the genus Inocybe from Pakistan. Identification of operational taxonomic units suggests the presence of quite a large number of undescribed Inocybe species which were not represented by above ground fruiting bodies.
Multigene phylogenetic analyses and morphology reveals three new species within section Rimosae (Inocybe) from western Himalayas, Pakistan
- M. Saba
- D. Pfister
- A. Khalid
- P. Matheny
Inocybe (Inocybaceae) is a highly diverse ectomycorrhizal genus among the gilled mushrooms (Agaricales) comprising some 750 known taxa worldwide. However, a considerable number of taxa have yet to be formally described. Inocybe has a widespread distribution and is found commonly in temperate areas and to a lesser extent in the tropics. The objective of the present work is to collect, describe, identify using morphological and multigene phylogenetic approach, and enumerate diversity of Inocybe species associated with pines in Pakistan. During fungal surveys conducted between 2012-2014, several species of genus Inocybe were collected from pine dominating forests of western Himalayas, Pakistan. These were documented morphologically and phylogenetically. Gene regions examined during this study include the complete ITS region (ITS1 - 5.8S - ITS2) of the nuclear ribosomal RNA gene, the first ca. 900 bp of the 25S rRNA gene that encodes the nuclear large subunit of ribosomal RNA (nLSU), and the mitochondrial small subunit of ribosomal DNA (mtSSU). Three different data matrices for phylogenetic analysis were prepared: (i) Maximum Likelihood (ML) analysis of combined datasets of ITS+LSU+mtSSU of Pseudosperma clade (ii) ML analysis of nLSU region of I. rimosa s.l. (iii) Maximum Parsimony (MP) analysis of I. rimosa s.l. using combined datasets of ITS+LSU+mtSSU. Both morphological and phylogenetical analyses validate the occurrence of three new species, I. brunneoumbonata, I. pinophila, and I. triacicularis. These species shared an alliance with the Pseudosperma clade (sect. Rimosae), one of the seven major clades in the Inocybaceae. Species of the Pseudosperma clade are typically characterized by a rimose pileus; furfuraceous to furfuraceous-fibrillose stipe; absence of metuloids and pleurocystidia; smooth, elliptical to indistinctly phaseoliform basidiospores, and cylindrical to clavate cheilocystidia. Comprehensive morphological and microscopic descriptions, illustrations, and phylogenetic affiliations of the studied taxa are provided. The new species are differentiated from their close relatives by basidiospore size and fruit body coloration. Combined sequence data from ITS, nLSU-rRNA, and mtSSU-RNA gene regions also confirmed the novelty of species and their placement within the Pseudosperma clade. All three newly described taxa likely share an ecological association with pine species. This is the first attempt to describe new taxa of Inocybe in clade Pseudosperma from Pakistan based on combined morphological and multi-gene phylogenetic approaches.
Evidence of speciation in North American and European populations of Thelephora cuticularis
- D. Smid
- J. Kout
- L. Quijada
- M. Sadlikova
Fungi in the genus Thelephora are macroscopically characterized by their purplish-brownish , tough or slightly fleshy, erect fruitbodies. Microscopically, they have brownish, angular, and echinulate spores, and a monomitic hyphal system, usually with clamped hyphae. One species in the genus, Thelephora cuticularis, is a rather small, black, water-absorbent fungus, easily recognizable by its non-angular spores, the absence of clamp connections, and the cyanescence of its subhymenium in KOH. Because of these morphological features, it is not easily confused with any other species in the genus Thelephora. The type of T. cuticularis was collected in Ohio (United States) in 1844 and described by Berkeley in 1847. Since then, it has been found in both Europe and North America, but is considered rare on both continents. There are very few recent reports, with most records consisting of old collections deposited in North American herbaria. Morphological and molecular methods have been used to study several specimens of Thelephora cuticularis from Europe and North America. Our preliminary study reveals that there is evidence for splitting T. cuticularis into two species, one found in North America and the second restricted to Europe. The external morphology of both species is similar, but their spores are slightly different in size, shape, and spine length. Additionally, the two species have different ecologies. The American specimens are described as truly lignicolous, while European specimens have been collected on bare soil or litter debris in hardwood forests. Our results confirm the biogeographic separation of the populations from North America and Europe. The specific epithet T. cuticularis belongs to the American species and so a new name will be proposed for the European species.
Worldwide eco-evolutionary dynamics of the Peltigera-Nostoc symbiosis
- N. Magain
- J. Miadlikowska
- P. Chagnon
- C. Truong
- C. Pardo De La Hoz
- L. Cornet
- D. Baurain
- R. Darnajoux
- I. Medeiros
- J. Lu
- E. Sérusiaux
- F. Lutzoni
All species of lichen-forming fungi (mycobionts) in the genus Peltigera (Ascomycota, Lecanoromycetes) associate with cyanobacteria of the genus Nostoc, either as a main or secondary photosynthetic and nitrogen-fixing partner (photobiont). A thorough phylogenetic revision of the fungal genus, based on phylogenomic approaches and several species delimitation analyses, revealed the presence of ~150 species, of which about half are new to science. In addition, we inferred the phylogeny of Nostoc associating with Peltigera, using a phylogenomic approach, and genotyped the Nostoc symbionts associating with Peltigera species across the entire genus. This provided a unique opportunity to use the Peltigera-Nostoc symbiosis as a model system to study patterns of association (including specificity) and diversification rates geographically and through time. We used this phylogenetic framework to study the evolution of ecological networks, including factors such as interaction dependency, symmetry, and nestedness. We compared the eco-evolutionary dynamics among the main clades of Peltigera, and the observed patterns in the context of ecological preferences of each species, the type of interactions (Nostoc as main or secondary partner) and dispersal mode (sexual or asexual propagules). This study reveals common trends at the genus level, such as the higher specialization and dependency of the mycobiont, but also differences within and among clades, including drastic differences in specificity.
Species delimitation in the Usnea cornuta aggregate (Parmeliaceae, lichenized Ascomycota)
- A. Gerlach
- Z. Toprak
- Y. Naciri
- E. Caviró
- R. Borges Da Silveira
- P. Clerc
The Usnea cornuta aggregate, as defined here, includes U. brasiliensis (Zahlbr.) Motyka, U. cornuta Körb. and U. dasaea Stirt. These species are mainly corticolous, with an erect-shrubby life form, composed of more or less inflated branches constricted at their point of attachment and relatively small, punctiform soralia usually covered with isidiomorphs. The relative thickness of the cortex, medulla and axis (CMA) is of the cornuta-type or the brasiliensis-type. However, recent studies on some South American Usnea taxa revealed that this group is based on homoplasious characters. The increasing availability of DNA sequences data provides much deeper insight in our understanding of the various biological processes responsible for species formation and their consequences on the biodiversity. However, complexity in the evolutionary mechanisms, as for example the incomplete lineage sorting (ILS), can obscure the signals for the recognition of evolutionary distinct lineages, in particular in the recently diverged groups of organisms. Therefore evaluation of the evidences from diverse sources may be critical for the accurate assessment of the species boundaries in such groups. The coalescence model takes ILS into account, and a derived multi species coalescent framework is now implemented in several softwares used for species delimitation based on DNA sequences data with or without the requirement of defining putative species a priori. The objective of our study is to investigate how many clades are hidden in the U. cornuta aggr. and if they can be characterized by morphological, anatomical or chemical characters. For that purpose, we provide the first multi species coalescent model based species delimitation for the Neotropical Usnea species. Based on the ITS rDNA, two protein-coding genes RPB1 and Mcm7, we estimated the species tree under the multi species coalescent model in a Bayesian framework using the STACEY method. We used DNA sequences, chemical, geographic, and morphological data from 156 specimens, and applied an integrative approach to define species boundaries with a particular focus on the cosmopolitan species group U. cornuta aggr. (n = 82 specimens). These specimens span over a broad geographical area including Brazil, Costa Rica, Ecuador, France, Peru, Portugal, Spain, United Kingdom and USA. Our results indicate that the Usnea cornuta aggr. represents a complex of nine strongly supported lineages correlated with secondary chemistry, as well as two unsupported groups (possibly including Usnea cornuta s. str.) and three singletons making a total of at least 14 distinct lineages. This shows that the diversity in the U. cornuta aggr., especially in Brazil, was so far underestimated. Further investigations are on going to show whether this genetic diversity correlates or not with morphology and anatomy.
Thyriothecial fungi and fossil evidence of Dothideomycetes
- L. Le Renard
- M. Berbee
- R. Stockey
Critical reinterpretations of morphological characters in fossil fungi have the potential to improve estimates of the geological timing of fungal radiations. Some of the best fungal fossils are thyriothecia. A thyriothecium is a minute ascomatal type, with a flat scutellum, a shield-like upper surface, with a distinctive cell pattern that is formed by a sequence of hyphal branching and septation. The fossil record rarely provides diagnostic characters such as asci but the diversity of fossil scutella offers great potential to calibrate ages of extant fungal lineages. However, both DNA sequence data for genera of thyriothecia-forming fungi and illustrations of scutella remain sparse in public databases. We present an updated phylogeny of thyriothecial Dothideomycetes based on 4251 positions for 320 taxa, contributing new LSU and SSU rDNA sequence data for 14 thyriothecial fungi. We define thyriothecia as flattened, superficial ascomata lacking a differentiated lower wall, with varied scutellum patterns, many, but not all, radiate. We coded character states for taxa including 60 thyriothecial species and then estimated ancestral character states over the Bayesian posterior distribution of topologies from our dataset to account for the phylogenetic uncertainty. Radiate thyriothecia were only found in Class Dothideomycetes, where they seem to have evolved independently at least five times. Clades containing thyriothecia are mostly folicolous and caulicolous, and it remains unclear whether these ascomata were derived from perithecioid or apothecioid ancestors. Genera traditionally recognized in the order Asterinales were polyphyletic and formed two independent clades (Asterinales and Asterotexiales). Morphological distinction between thyriothecia of these orders was impossible because they share a combination of scutellum and mycelium characters. In contrast, the most typical members of Microthyriales and Aulographaceae were readily recognizable from the organization of cells at their scutellum margins, patterns of septation and branching of the scutellum hyphae. We used our newly defined characters to interpret published fossil thyriothecia. We show which of the lineages now sampled for DNA are represented in the fossil record of fungi and present potential Dothideomycetes calibration points available in fossil occurrences of thyriothecia. A phylogenetically congruent distribution of character states among modern thyriothecial clades will help to improve interpretation of fossil material, and will increase the precision of Ascomycota age estimates.
Exploring the diversity of secondary metabolite clusters in Dothideomycete fungi
- E. Gluck-Thaler
- K. Bushley
- S. Haridas
- I. Grigoriev
- P. Crous
- J. Spatafora
- J. Slot
Quantifying genotypic diversity is essential for understanding and predicting how organisms adapt to their environment, and in fungi, perhaps no process is as central to adaptation as metabolism. As heterotrophs, fungal growth and survival is necessarily contingent on their ability to extract carbon and energy from a particular niche. Fungi are also adept chemical engineers who synthesize broad arrays of specialized or secondary metabolites (SMs) that shape their interactions with surrounding organisms. For example, many plant pathogens produce toxic SMs to kill their hosts, while plant saprotrophs produce antimicrobial SMs to help them compete for available resources. Although fungi have historically been an important source of economically and medically important SMs (e.g., penicillin), relatively little research has been devoted to studying patterns in the diversity of SMs they produce. Consequently, we lack a comparative framework for understanding both the drivers of fungal metabolic diversity, and the dynamics between metabolic evolution and ecological adaptation. New fungal SM pathways are usually encoded in “gene clusters” made up of neighboring genes in fungal genomes, which facilitates their identification and characterization through whole genome sequencing. Here, we conducted a systematic analysis of SM gene clusters in the largest group of fungi, the Dothideomycetes, in order to evaluate the diversity of their SM gene clusters, and to assess the potential for novel SM discovery within this group. We first developed new bioinformatic algorithms for refining gene cluster prediction that we used to query a database of 101 Dothideomycete genomes. We identified 459 unique types of clusters, of which only 6% produce known SMs, and found that even closely related species harbor distinct SM cluster repertoires. We then used network analyses to identify genes within these clusters that have played an outsized role in the evolution of SM biosynthesis, and whose presence in these clusters can be used to improve future gene cluster detection methods. Next, we used comparative phylogenetic methods to identify clusters with strong signatures of selection that are present in fungal decomposers and pathogens, and that may encode pathways underlying specific plant-associated lifestyles. Finally, based on the phylogenetic distributions of the clusters we detected, we identified specific lineages within the Dothideomycetes that are projected to harbor the greatest diversity of SM clusters, thus providing a valuable roadmap to direct future SM sampling and characterization efforts.
Mediterranean biodiversity hotspot harbours extraordinary diversity of soil yeasts
- A. Yurkov
- A. Pontes
- O. Roehl
- C. Carvalho
- J. Sampaio
Species richness and endemism of land plants and animals are important characteristics of a biodiversity hotspot. These parameters were also used to estimate the global diversity of Fungi, including millions of yet undiscovered species. Hence, biodiversity assessments are important to provide more accurate estimations and sampling in areas considered as biodiversity hotspots. Due to small cell size and enigmatic appearance, biodiversity assessments of microscopic fungi become are generally difficult and become laborious in the case of endophytes and soil-borne species. Here, we provide the first comprehensive inventory of soil yeasts in the Mediterranean biome, which is also the leading biodiversity hotspot for vascular plants and vertebrates in Europe. Studies in the Mediterranean biome included a natural forest vegetation and cultural landscape. The cultural landscape was an agro-silvo-pastoral system with Mediterranean oaks and grassland known as Montado (Portugal) and Dehesa (Spain). Diverse forested sites of Serra da Arrábida Natural Park (Portugal) represented the Mediterranean forests, woodlands, and scrub biome. In sclerophyll scrublands, both cultivation experiments and the subsequent species richness estimations suggest the highest species richness values of soil yeasts reported to date. These values far exceed those reported for other forest soils in Europe using either cultivation or culture-independent techniques. Remarkably, about a third of isolated yeasts represented new species, some of which have been recently described. Precipitation level, above ground biomass and plant projective cover under sclerophyll forest vegetation had strong impact on yeast diversity and on community structure and lower precipitation resulted in an increased number of rare species and decreased evenness values along the gradient from humid broadleaf forests to dry scrublands. In agreement with previous observations made in temperate climate, managed agro-silvo-pastoral system was characterised by lower species diversity. This observation further suggests extraordinary value of natural, unmanaged and low-managed, forests for biodiversity conservation of animals, plants and fungi, including saprobic free-living micromycetes. The Mediterranean forests, woodlands, and scrub biome is not limited to the Mediterranean region. Similar plant formations represented by sclerophyll shrublands are known as maquis in the Mediterranean Basin, chaparral in California, matorral in Chile, fynbos in South Africa, and mallee and kwongan shrublands in Australia. Many of these regions have been proposed for World Heritage status. Both high species richness values and high proportion of potential new species makes studies in natural biotopes in the Mediterranean climate of high priority for mycologists and conservation biologists.
Population variation in post-harvest rot Rhizopus stolonifer
- S. Masonjones
- J. Stajich
Globally postharvest diseases contributes to one third of food losses. Rhizopus stolonifer, a Mucoromycota fungus, causes postharvest soft rot in fruits and vegetables. Control is typically focused on quick harvesting, packing at cool temperatures combined with treatment of fungicides. However, many fungicides are not approved for direct use on fruits and understanding mechanisms of resistance development is limited. We are phenotyping and sequencing ~250 geographically and substrate diverse R. stolonifer strains. Strains have been cultured from California and Florida strawberries, from almond hulls from California orchards, from other California fruit farms. A subset are collected from around the world and obtained from culture stocks from the USDA-NRRL collection. Fungicide resistance varies among isolates with reduced sensitivity to Fludioxonil in some strains. Linear growth rates in race tubes at 12C, 23C, and 30C show significant differences among strains that originate from varied climates or isolation substrates. Whole genome sequencing is being performed on 250 strains from this collection and analysis of patterns of genetic variation was performed to test for evidence of population structure, demography, and association of genotype to phenotype, particularly fungicide resistance. Preliminary data from a geographically diverse subset of 100 samples identified more than ~70,000 SNPs across the 38 Mb genome. Variants were analyzed to scan for highly diverse gene loci, evidence of directional selection, or insertion/deletions of transposable elements. The strains group into 3 main clades, but no phylogeographic pattern has emerged yet. Initial GWAS show 3 loci potentially linked to fungicide resistance. Further sequencing will test robustness of these potential sub-populations, examine genotype and phenotype correlations, and identify genetic loci with signatures of rapid evolutionary change.
Combining phylogenetic and ecological evidence for achieving one fungus-one name based taxonomy of Hypocreaceae (Hypocreales, Ascomycota)
- K. Põldmaa
The ascomycete order Hypocreales is characterised by remarkable diversity of lifestyles, habitats and substrata with the best known members representing ubiquitous biotrophs – pathogens and parasites of plants and insects as well as endophytes. However, it is also the group most rich in mycoparasitic or fungicolous fungi, many of which inhabit not only mushrooms but fruitbodies of various taxonomic and ecological groups of fungi. They range from obligate parasites to facultative saprotrophs as well as from biotrophs to necrotrophs characteised by a wide array of secondary metabolites. Majority of such taxa belong to the Hypocreaceae, including Hypomyces and Trichoderma but also their satellite genera, that comprise sexually and/or asexually reproducing species. The present study aimed at phylogeny-based taxonomic revision of the family to discontinue the use of dual or even triple names currenty in use for the mostly pleomorphic species. A multigene dataset was created for a wide array of fungicolous Hypocreaceae and analyzed together with sequences of closely related taxa. The resulting phylogenetic reconstruction enabled to present a hypotheses about the evolution of this intricate group of organisms, focusing on the main shifts in the nutritional mode, lifestyle and specialization to hosts from various lineages of ectomycorrhizal vs saprotrophic Basidiomycota and Ascomycota. Evidence about the phylogeny and ecology was used for proposing taxonomic rearrangements in the group where quarter of a hundred generic names are available and several lineages, distinguished in the new phylogenetic hypothesis, await for being described. Possible solutions for sorting out the genus-level taxonomy according to the one fungus-one name principle, while considering both teleomorph- and anamorph-typified names, are presented for discussion. Advantages and limitations of the official barcode marker of fungi in building species hypotheses and identifying isolates/specimens will likewise be discussed.