Search

Fusarium head blight (FHB) is a major disease worldwide on cultivated cereals, caused by several Fusarium species. FHB can cause not only yield reduction but also accumulation of mycotoxins in the grain contaminating the food supply. Much of the earlier research has focused on Fusarium pathogenesis, conditions required for disease development and toxin accumulation, and FHB management. However, the Fusarium community composition within the micro-habitat of a single diseased wheat head in the field has had limited investigation. Similarly, the relationship between the Fusarium community structure and mycotoxin accumulation within diseased heads remains unclear. In the present study, we investigated the Fusarium community in diseased heads sampled from different geographical sites in China. Several sites in Shandong province formed a transitional region which contained highly variable profiles of Fusarium OTUs, where a single diseased head could contain more than 10 Fusarium OTUs. Mycotoxin accumulation was independent of geographical properties, however, deoxynivalenol, 15-acetyldeoxynivalenol and zearalenone concentrations showed a significant negative correlation with Fusarium diversity on diseased heads while a significant positive correlation between nivalenol concentration and Fusarium diversity was observed. Taken together, the Fusarium OTU diversity within diseased heads in the field significantly influences mycotoxin accumulation, providing an important point to consider in FHB disease management and mycotoxin research.

Fusarium oxysporum f. sp. vasinfectum race 4 (FOV4) is a devastating fungus pathogen that causes Fusarium wilt in both domesticated cotton species, Gossypium hirsutum (Upland) and G. barbadense (Pima). Greenhouse and field-based pathogenicity assays can be a challenge because of nonuniform inoculum levels, the presence of endophytes, and varying environmental factors. Therefore, an in vitro coculture system was designed to support the growth of both domesticated cotton species and FOV4 via an inert polyphenolic foam substrate with a liquid medium. A Fusarium wilt–susceptible Pima cotton cultivar, G. barbadense ‘GB1031’; a highly resistant Pima cotton cultivar, G. barbadense ‘DP348RF’; and a susceptible Upland cotton cultivar, G. hirsutum ‘TM-1’, were evaluated for 30 days during coculture with FOV4 in this foam-based system. Thirty days after inoculation, disease symptoms were more severe in both susceptible cultivars, which displayed higher percentages of foliar damage, and greater plant mortality than observed in ‘DP348RF’, the resistant Pima cotton cultivar. This foam-based in vitro system may be useful for screening cotton germplasm for resistance to a variety of fungus pathogens and may facilitate the study of biotic interactions in domesticated cotton species under controlled environmental conditions.

The

Fusarium avenaceum is a filamentous fungus commonly associated with plants and soil. It is a causal agent of Fusarium head blight (FHB) on maize and small-grain cereals and blights on other plant species, and is one of the very few fungal species known to have ice nucleation activity (i.e., it catalyzes ice formation). Here, we report the draft genome of the ice-nucleation-active F. avenaceum strain F156N33 isolated from the atmosphere above Virginia. The genome assembly is 41,175,306 bp long, consists of 214 contigs, and is predicted to encode 11,233 proteins, which were annotated using RNA-sequencing data obtained from the same strain.

Ice nucleation activity (INA) is the capacity of certain particles to catalyze ice formation at temperatures higher than the temperature at which pure water freezes. INA impacts the ratio of liquid to frozen cloud droplets and, therefore, the formation of precipitation and Earth’s radiative balance. Some Fusarium strains secrete ice-nucleating particles (INPs); they travel through the atmosphere and may thus contribute to these atmospheric processes. Fusarium INPs were previously found to consist of proteinaceous aggregates. Here, we determined that in F. avenaceum, the proteins forming these aggregates are smaller than 5 nm and INA is higher after growth at low temperatures and varies among strains. Leveraging these findings, we used comparative genomics and transcriptomics to identify candidate INA genes. Ten candidate INA genes that were predicted to encode secreted proteins were present only in the strains that produced the highest number of INPs. In total, 203 candidate INA genes coding for secreted proteins were induced at low temperatures. Among them, two genes predicted to encode hydrophobins stood out because hydrophobins are small, secreted proteins that form aggregates with amphipathic properties. We discuss the potential of the candidate genes to encode INA proteins and the next steps necessary to identify the molecular basis of INA in F. avenaceum. Published version

International audience; Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. Previously (Geiser et al. 2013; Phytopathology 103:400-408. 2013), the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani Species Complex (FSSC). Subsequently, this concept was challenged by one research group (Lombard et al. 2015 Studies in Mycology 80: 189-245) who proposed dividing Fusarium into seven genera, including the FSSC as the genus Neocosmospora, with subsequent justification based on claims that the Geiser et al. (2013) concept of Fusarium is polyphyletic (Sandoval-Denis et al. 2018; Persoonia 41:109-129). Here we test this claim, and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species recently described as Neocosmospora were recombined in Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural and practical taxonomic option available.

A group of aggressive pathogens have evolved to colonize the plant xylem. In this vascular tissue, where water and nutrients are transported from the roots to the rest of the plant, pathogens must be able to thrive under acropetal xylem sap flow and scarcity of nutrients while having direct contact only with predominantly dead cells. Nevertheless, a few bacteria have adapted to exclusively live in the xylem, and various pathogens may colonize other plant niches without causing symptoms unless they reach the xylem. Once established, the pathogens modulate its physicochemical conditions to enhance their growth and virulence. Adaptation to the restrictive lifestyle of the xylem leads to genome reduction in xylem-restricted bacteria, as they have a higher proportion of pseudogenes in their genome. The basis of xylem adaptation is not completely understood; therefore, a need still exists for model systems to advance the knowledge on this topic.

Characterization of a gene of interest frequently relies on generation of a mutant as a critical component. Transformation to disrupt a gene has been previously accomplished by several methods in Fusarium oxysporum. Here we provide a detailed method to generate a gene mutation mediated by a CRISPR/Cas9 ribonucleoprotein (RNP) complex. The Cas9 RNP cleaves the DNA at the target site, and during DNA repair integration of a dominant selectable marker is incorporated via homologous recombination generating the desired gene disruption.

Plants produce low molecular weight compounds with antimicrobial activity in response to microbial attack termed phytoalexins. The first phytoalexin identified was (+) pisatin from pea, and several fungi are able to detoxify pisatin to a less inhibitory compound, including F. oxysporum f. sp. pisi. This detoxification is catalyzed by demethylation of the compound (termed pisatin demethylase activity, or PDA) by the cytochrome P450, Pda. Here we detail two procedures to assess PDA using radiolabeled [

Plants produce low molecular weight compounds with antimicrobial activity in response to microbial attack termed phytoalexins. The first phytoalexin identified was (+) pisatin from pea, and several fungi are able to detoxify pisatin to a less inhibitory compound, including F. oxysporum f. sp. pisi. This detoxification is catalyzed by demethylation of the compound (termed pisatin demethylase activity, or PDA) by the cytochrome P450, Pda. Here we detail two procedures to assess PDA using radiolabeled [14C]pisatin as a substrate and monitoring activity using a scintillation counter.

F. oxysporum is a notorious filamentous pathogenic fungus that causes serious problems in agriculture and animal/human health. Knowing how the fungus interacts throughout the course of an infection is necessary to propose an effective control strategy, and consequently the manipulation of the F. oxysporum genome is essential to investigate the molecular interplay between the host and fungus. To facilitate assessing protein quantification and subcellular localization, we developed a simple, economical CRISPR/Cas9-mediated endogenous gene tagging (EGT) system based on two different strategies, homology-independent targeted integration (HITI) and homology-dependent recombination integration (HDRI). Reporter genes, including GFP and LacZ, can be inserted at the N- or C-terminus of an endogenous gene of interest at the original chromosomal locus, allowing partial characterization of the gene function.

F. oxysporum is a notorious filamentous pathogenic fungus that causes serious problems in agriculture and animal/human health. Knowing how the fungus interacts throughout the course of an infection is necessary to propose an effective control strategy, and consequently the manipulation of the F. oxysporum genome is essential to investigate the molecular interplay between the host and fungus. To facilitate assessing protein quantification and subcellular localization, we developed a simple, economical CRISPR/Cas9-mediated endogenous gene tagging (EGT) system based on two different strategies, homology-independent targeted integration (HITI) and homology-dependent recombination integration (HDRI). Reporter genes, including GFP and LacZ, can be inserted at the N- or C-terminus of an endogenous gene of interest at the original chromosomal locus, allowing partial characterization of the gene function.

Characterization of a gene of interest frequently relies on generation of a mutant as a critical component. Transformation to disrupt a gene has been previously accomplished by several methods in Fusarium oxysporum. Here we provide a detailed method to generate a gene mutation mediated by a CRISPR/Cas9 ribonucleoprotein (RNP) complex. The Cas9 RNP cleaves the DNA at the target site, and during DNA repair integration of a dominant selectable marker is incorporated via homologous recombination generating the desired gene disruption.

Fusarium oxysporum f. sp. vasinfectum is an important plant pathogen responsible for vascular wilt disease on cotton. Members of this group are known to carry supernumerary chromosomes that encode virulence factors. We sequenced the genomes of five F. oxysporum f. sp. vasinfectum isolates, including the genome of a representative of the highly virulent genotype race 4, at a high coverage to assemble reference-quality genomes. These genomes provide a necessary resource for comparative genomic analyses to identify genes or genome features that are involved in pathogenicity on cotton and may ultimately be used to identify improved management strategies.

Fungal infections are a worldwide problem associated with high morbidity and mortality. There are relatively few antifungal agents, and resistance has emerged within these pathogens for the newest antifungal drugs. As the fungal cell wall is critical for growth and development, it is one of the most important targets for drug development. In this review, the currently available cell wall inhibitors and suitable drug candidates for the treatment of fungal infections are explored. Future studies of the fungal cell wall and compounds that have detrimental effects on this important outer structural layer could aid in antifungal drug discovery and lead to the development of alternative cell wall inhibitors to fill gaps in clinical therapies for difficult-to-treat fungal infections.

Widely distributed in various environmental niches, filamentous fungi play an important role in industry, drug development, and plant/animal health. Manipulation of the genome and the coding sequences are essential for a better understanding of the function of genes and their regulation, but traditional genetic approaches in some filamentous fungi are either inefficient or nonfunctional. The rapid development and wide implementation of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats /(CRISPR)-associated protein-9 nuclease) technology for various model and non-model organisms has provided the initial framework to adapt this gene editing technology for filamentous fungi. In this review, an overview of the CRISPR/Cas9 tools and strategies that have been developed for different filamentous fungi is presented, including integration of the CAS9 gene into the genome, transient expression of Cas9/sgRNA, the AMA1-based plasmid approach, and the Cas9 RNP method. The various applications of CRISPR/Cas9 technology in filamentous fungi that have been implemented are explored, with particular emphasis on gene disruption/deletion and precise genome modification through gene tagging and alteration in gene regulation. Potential challenges that are confronted when developing a CRISPR/Cas9 system for filamentous fungi are also discussed such as the nuclear localization sequence for the CAS9 gene, potential off-target effects, and highly efficient transformation methods. Overcoming these obstacles may further facilitate wide application of this technology. As a simple, economical, and powerful tool, CRISPR/Cas9 systems have the potential for future implementation into many molecular aspects of filamentous fungi., Graphical Abstract Unlabelled Image

Reactive oxygen species (ROS) produced by hosts serve as a general defense mechanism against various pathogens. At the interaction site between the host and pathogen, host cells rapidly accumulate high concentrations of ROS, called the oxidative burst, that damage and kill the invading microbes. However, successful pathogens usually survive in a high ROS environment and have evolved strategies to overcome these detrimental effects. Here we characterized the biological function of the extracellular superoxide dismutase (SOD) FoSod5 fromFusarium oxysporumf. sp.vasinfectum.FoSOD5is strongly up-regulated during infection of cotton, and a ΔFoSOD5mutant was significantly reduced in virulence on cotton. Purified 6 × His-FoSod5 could significantly inhibit the reduction of NBT and WST-1, indicating that FoSod5 was a functional SOD protein. Based on CRISPR/Cas9 technology, several different FoSod5 variants were generated and used to assess the secretion, expression, and subcellular localization of FoSod5 inF. oxysporum. The subcellular localization of FoSod5 is altered under different environmental conditions. During normal growth conditions, FoSod5 was primarily localized to the phialides; however, in a nutrient-limited environment, FoSod5 was localized to a wide array of fungal structures including the septum and cell wall. FoSod5 is an alkaline-induced glycosylphosphatidylinositol (GPI) protein and the GPI anchor was required for proper protein subcellular localization. The multiple mechanisms fungi utilize to tolerate the oxidative burst is indicative of the importance of this plant defense response; however, the presence of a conserved extracellular SOD in many phytopathogenic fungi suggests tolerance to ROS is initiated prior to the ROS entering the fungal cell.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the

Reactive oxygen species (ROS) produced by hosts serve as a general defense mechanism against various pathogens. At the interaction site between the host and pathogen, host cells rapidly accumulate high concentrations of ROS, called the oxidative burst, that damage and kill the invading microbes. However, successful pathogens usually survive in a high ROS environment and have evolved strategies to overcome these detrimental effects. Here we characterized the biological function of the extracellular superoxide dismutase (SOD) FoSod5 from Fusarium oxysporum f. sp. vasinfectum. FoSOD5 is strongly up-regulated during infection of cotton, and a ΔFoSOD5 mutant was significantly reduced in virulence on cotton. Purified 6 × His-FoSod5 could significantly inhibit the reduction of NBT and WST-1, indicating that FoSod5 was a functional SOD protein. Based on CRISPR/Cas9 technology, several different FoSod5 variants were generated and used to assess the secretion, expression, and subcellular localization of FoSod5 in F. oxysporum. The subcellular localization of FoSod5 is altered under different environmental conditions. During normal growth conditions, FoSod5 was primarily localized to the phialides; however, in a nutrient-limited environment, FoSod5 was localized to a wide array of fungal structures including the septum and cell wall. FoSod5 is an alkaline-induced glycosylphosphatidylinositol (GPI) protein and the GPI anchor was required for proper protein subcellular localization. The multiple mechanisms fungi utilize to tolerate the oxidative burst is indicative of the importance of this plant defense response; however, the presence of a conserved extracellular SOD in many phytopathogenic fungi suggests tolerance to ROS is initiated prior to the ROS entering the fungal cell.

Fusarium oxysporum is a cross-kingdom fungal pathogen that infects plants and humans. Horizontally transferred lineage-specific (LS) chromosomes were reported to determine host-specific pathogenicity among phytopathogenic F. oxysporum. However, the existence and functional importance of LS chromosomes among human pathogenic isolates are unknown. Here we report four unique LS chromosomes in a human pathogenic strain NRRL 32931, isolated from a leukemia patient. These LS chromosomes were devoid of housekeeping genes, but were significantly enriched in genes encoding metal ion transporters and cation transporters. Homologs of NRRL 32931 LS genes, including a homolog of ceruloplasmin and the genes that contribute to the expansion of the alkaline pH-responsive transcription factor PacC/Rim1p, were also present in the genome of NRRL 47514, a strain associated with Fusarium keratitis outbreak. This study provides the first evidence, to our knowledge, for genomic compartmentalization in two human pathogenic fungal genomes and suggests an important role of LS chromosomes in niche adaptation., Zhang, Yang et al. compare a Fusarium oxysporum isolate obtained clinically to a phytopathogenic strain to examine transfer of lineage-specific chromosomes in determining host specificity. They find four unique lineage-specific chromosomes that seem to contribute to fungal adaptation to human hosts.

This article is to alert medical mycologists and infectious disease specialists of recent name changes of medically important species of the filamentous mold Fusarium. Fusarium species can cause localized and life-threating infections in humans. Of the 70 Fusarium species that have been reported to cause infections, close to one-third are members of the Fusarium solani species complex (FSSC), and they collectively account for approximately two-thirds of all reported Fusarium infections., This article is to alert medical mycologists and infectious disease specialists of recent name changes of medically important species of the filamentous mold Fusarium. Fusarium species can cause localized and life-threating infections in humans. Of the 70 Fusarium species that have been reported to cause infections, close to one-third are members of the Fusarium solani species complex (FSSC), and they collectively account for approximately two-thirds of all reported Fusarium infections. Many of these species were recently given scientific names for the first time by a research group in the Netherlands, but they were misplaced in the genus Neocosmospora. In this paper, we present genetic arguments that strongly support inclusion of the FSSC in Fusarium. There are potentially serious consequences associated with using the name Neocosmospora for Fusarium species because clinicians need to be aware that fusaria are broadly resistant to the spectrum of antifungals that are currently available.

Background Protoporphyrinogen oxidase (PPO) with two isoforms, chloroplast-targeted (PPO1) and mitochondrial-targeted (PPO2), catalyzes a step in the biosynthesis of chlorophyll and heme. PPO1 and PPO2 are herbicide target sites of PPO-inhibiting herbicides. Target-site mutations conferring resistance to PPO inhibitors have all thus far been in PPO2. Oxadiazon is a unique PPO inhibitor utilized for preemergence Eleusine indica control. In this research, we evaluated the response of two previously confirmed oxadiazon-resistant and susceptible E. indica biotypes to other PPO inhibitors and identified the resistance mechanism in two oxadiazon-resistant E. indica biotypes. Results Two E. indica biotypes were resistant to oxadiazon, but not to other structurally unrelated PPO inhibitors, such as lactofen, flumioxazin and sulfentrazone. A novel mutation A212T was identified in the chloroplast-targeted PPO1, conferring resistance to oxadiazon in a heterologous expression system. Computational structural modeling provided a mechanistic explanation for reduced herbicide binding to the variant protein: the presence of a methyl group of threonine 212 changes the PPO1 active site and produces repulsive electrostatic interactions that repel oxadiazon from the binding pocket. Conclusion The novel A212T mutation in PPO1 conferring resistance specifically to PPO inhibitor oxadiazon was characterized. This is the first evidence of the direct role of PPO1 in the PPO mode of action, and the first evidence of evolved resistance in PPO1. © 2019 Society of Chemical Industry.

Phosphorus (P) is considered a scarce macronutrient for plants in most tropical soils. The application of rock phosphate (RP) has been used to fertilize crops, but the amount of P released is not always at a necessary level for the plant. An alternative to this problem is the use of Phosphorus Solubilizing Microorganisms (PSM) to release P from chemically unavailable forms. This study compared the P sorption capacity of soils (the ability to retain P, making it unavailable for the plant) and the profile of organic acids (OA) produced by fungal isolates and the in vitro solubilization efficiency of RP. Trichoderma and Aspergillus strains were assessed in media with or without RP and different soils (Andisol, Alfisol, Vertisol). The type and amount of OA and the amount of soluble P were quantified, and according to our data, under the conditions tested, significant differences were observed in the OA profiles and the amount of soluble P present in the different soils. The efficiency to solubilize RP lies in the release of OAs with low acidity constants independent of the concentration at which they are released. It is proposed that the main mechanism of RP dissolution is the production of OAs.

Fusarium oxysporum is an economically important pathogen that widely exists in the environment and is capable of causing serious problems in crop production and animal/human health. One important step for characterization of a fungal protein with an unknown function is to determine its subcellular localization within the cell. To facilitate the study of important functional regulators or key virulence factors, we developed a CRISPR/Cas9-mediated endogenous gene tagging (EGT) system based on two different strategies, homology-independent targeted integration (HITI) and homology-dependent recombination integration (HDRI). The HITI strategy was able to facilitate integration of a large DNA fragment, ∼8 kb in length, into the genome of F. oxysporum at the sgRNA cleavage site, and was used to insert a C-terminal 3×sGFP tag to the FoCHS5 gene and a N-terminal mCherry tag to the FoSSO2 gene. The HDRI strategy was used to tag the paralogous gene, FoSSO1, with a C-terminal mCherry marker. FoChs5-3×sGFP localized to conidia, some septa, and fungal tips. A majority of the FoSso1-mCherry was distributed in the conidia, septum, and hyphae that were distal from the fungal tips. While FoSso1-mCherry showed a very weak fluorescent signal at the fungal tips, mCherry-FoSso2 accumulated in the plasma membrane of conidia, germlings, fungal tips, hyphae, and phialides, suggesting FoSSO1 and FoSSO2 are regulated differently during fungal development. These results indicate this EGT system is efficient and can be another molecular tool to resolve the function(s) of proteins and infection strategies of F. oxysporum.

The Fusarium oxysporum species complex (FOSC) is an economically important group of pathogenic filamentous fungi that are able to infect both animals and plants. Reverse genetic techniques, including gene disruption/deletion methods, to study these fungi are available although limitations exist resulting in decreased efficiency. Herein we describe a gene editing system developed using a F. oxysporum-optimized Cas9 ribonucleoprotein (RNP) and protoplast transformation method. The Cas9 protein and sgRNA were assembled to form a stable RNP in vitro and this complex was transferred into fungal protoplasts for gene editing with PEG-mediated transformation. In order to determine if the Cas9 RNP system is functional in the FOSC protoplasts and assess the efficacy of the system, two genes, URA5 and URA3, were selected for targeted disruption generating uracil auxotroph mutants that are resistant to 5-fluoroorotic acid, 5-FOA. In addition, a gene in a secondary metabolite biosynthetic cluster, the ortholog of BIK1, was mutated using this system and the maximum efficiency of this gene disruption was about 50%. Further analysis of the bik1 mutant confirmed that this polyketide synthase was involved in the synthesis of the red pigment, bikaverin. The mutants generated in this study displayed the strong expected phenotypes, demonstrating this F. oxysporum-optimized CRISPR/Cas9 system is stable and can efficiently disrupt the genes of interest.

Summary Members of the Fusarium solani species complex (FSSC) are capable of causing disease in many agriculturally important crops. The genomes of some of these fungi include supernumerary chromosomes that are dispensable and encode host-specific virulence factors. In addition to genomics, this review summarizes the known molecular mechanisms utilized by members of the FSSC in establishing disease. Taxonomy Kingdom Fungi; Phylum Ascomycota; Class Sordariomycetes; Order Hypocreales; Family Nectriaceae; Genus Fusarium. Host range Members of the FSSC collectively have a very broad host range, and have been subdivided previously into formae speciales. Recent phylogenetic analysis has revealed that formae speciales correspond to biologically and phylogenetically distinct species. Disease symptoms Typically, FSSC causes foot and/or root rot of the infected host plant, and the degree of necrosis correlates with the severity of the disease. Symptoms on above-ground portions of the plant can vary greatly depending on the specific FSSC pathogen and host plant, and the disease may manifest as wilting, stunting and chlorosis or lesions on the stem and/or leaves. Control Implementation of agricultural management practices, such as crop rotation and timing of planting, can reduce the risk of crop loss caused by FSSC. If available, the use of resistant varieties is another means to control disease in the field. Useful websites http://genome.jgi-psf.org/Necha2/Necha2.home.html

Francisella tularensis is a highly infectious Gram-negative intracellular pathogen that causes tularemia. Because of its potential as a bioterrorism agent, there is a need for new therapeutic agents. We therefore developed a whole-animal Caenorhabditis elegans - F. tularensis pathosystem for high-throughput screening to identify and characterize potential therapeutic compounds. We found that the C. elegans p38 mitogen-activate protein (MAP) kinase cascade is involved in the immune response to F. tularensis , and we developed a robust F. tularensis -mediated C. elegans killing assay with a Z′ factor consistently of >0.5, which was then utilized to screen a library of FDA-approved compounds that included 1,760 small molecules. In addition to clinically used antibiotics, five FDA-approved drugs were also identified as potential hits, including the anti-inflammatory drug diflunisal that showed anti- F. tularensis activity in vitro . Moreover, the nonsteroidal anti-inflammatory drug (NSAID) diflunisal, at 4× MIC, blocked the replication of an F. tularensis live vaccine strain (LVS) in primary human macrophages and nonphagocytic cells. Diflunisal was nontoxic to human erythrocytes and HepG2 human liver cells at concentrations of ≥32 μg/ml. Finally, diflunisal exhibited synergetic activity with the antibiotic ciprofloxacin in both a checkerboard assay and a macrophage infection assay. In conclusion, the liquid C. elegans - F. tularensis LVS assay described here allows screening for anti- F. tularensis compounds and suggests that diflunisal could potentially be repurposed for the management of tularemia.

Fusarium species is a ubiquitous fungus that causes opportunistic infections. We present 26 cases of invasive fusariosis categorized according to the European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria of fungal infections. All cases (20 proven and 6 probable) were treated from January 2000 until January 2010. We also review 97 cases reported since 2000. The most important risk factors for invasive fusariosis in our patients were compromised immune system, specifically lung transplantation (n = 6) and hematologic malignancies (n = 5), and burns (n = 7 patients with skin fusariosis), while the most commonly infected site was the skin in 11 of 26 patients. The mortality rates among our patients with disseminated, skin, and pulmonary fusariosis were 50%, 40%, and 37.5%, respectively. Fusarium solani was the most frequent species, isolated from 49% of literature cases. Blood cultures were positive in 82% of both current study and literature patients with disseminated fusariosis, while the remaining 16% had 2 noncontiguous sites of infection but negative blood cultures. Surgical removal of focal lesions was effective in both current study and literature cases. Skin lesions in immunocompromised patients should raise the suspicion for skin or disseminated fusariosis. The combination of medical monotherapy with voriconazole or amphotericin B and surgery in such cases is highly suggested.

In this letter, we advocate recognizing the genus Fusarium as the sole name for a group that includes virtually all Fusarium species of importance in plant pathology, mycotoxicology, medicine, and basic research. This phylogenetically guided circumscription will free scientists from any obligation to use other genus names, including teleomorphs, for species nested within this clade, and preserve the application of the name Fusarium in the way it has been used for almost a century. Due to recent changes in the International Code of Nomenclature for algae, fungi, and plants, this is an urgent matter that requires community attention. The alternative is to break the longstanding concept of Fusarium into nine or more genera, and remove important taxa such as those in the F. solani species complex from the genus, a move we believe is unnecessary. Here we present taxonomic and nomenclatural proposals that will preserve established research connections and facilitate communication within and between research communities, and at the same time support strong scientific principles and good taxonomic practice Fil: Geiser, David M.. State University of Pennsylvania; Estados Unidos Fil: Aoki, Takayuki. National Institute of Agrobiological Sciences; Japón Fil: Bacon, Charles W.. Toxicology and Mycotoxin Research; Grecia Fil: Baker, Scott E.. Pacific Northwest National Laboratory; Estados Unidos Fil: Bhattacharyya, Madan K.. University of Iowa; Estados Unidos Fil: Chulze, Sofia Noemi. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Scandiani, María Mercedes. Laboratorio Agrícola Río Paraná. San Pedro; Argentina Fil: Ploetz, Randy C.. University of Florida; Estados Unidos Fil: Proctor, Robert H.. NCAUR-ARS-USDA; Estados Unidos Fil: Rehner, Stephen A.. United States Department of Agriculture; Estados Unidos Fil: Robert, Vincent A. R. G.. Fungal Biodiversity Center; Países Bajos Fil: Rooney, Alejandro P.. NCAUR-ARS-USDA; Estados Unidos Fil: Salleh, Baharuddin bin. University of Malaysia; Malasia Fil: Scauflaire, Jonathan. Université Catholique de Louvain; Bélgica Fil: Short, Dylan P. G.. University of California; Estados Unidos Fil: Steenkamp, Emma. University of Pretoria; Sudáfrica Fil: Suga, Haruhisa. Gifu University; Japón Fil: Summerell, Brett A.. The Royal Botanic Garden Sydney; Australia Fil: Sutton, Deanna A.. University of Texas; Estados Unidos Fil: Thrane, Ulf. Technical University of Denmark; Dinamarca Fil: Trail, Francis. Michigan State University; Estados Unidos Fil: Van Diepeningen, Anne. Fungal Biodiversity Center; Países Bajos Fil: VanEtten, Hans D.. University of Arizona; Estados Unidos Fil: Viljoen, Altus. University of Stellenbosch; Sudáfrica Fil: Waalwijk, Cees. Wageningen University; Países Bajos Fil: Ward, Todd J.. NCAUR-ARS-USDA. Pretoria; Sudáfrica Fil: Wingfield, Michael J.. University of Pretoria; Sudáfrica Fil: Xu, Jin-Rong. Purdue University; Estados Unidos Fil: Yang, Xiao-Bing. University of Iowa; Estados Unidos Fil: Yli-Mattila, Tapani. University of Turku; Finlandia Fil: Zhang, Ning. Rutgers University; Estados Unidos

Aim: Caffeic acid (3,4-dihydroxycinnamic acid) phenethyl ester (CAPE), the major constituent of propolis, is able to increase the survival of the nematode Caenorhabditis elegans after infection with the fungal pathogen Candida albicans. Results: CAPE increases the expression of several antimicrobial proteins involved in the immune response to C. albicans. Structural derivatives of CAPE were synthesized to identify structure–activity relationships and decrease metabolic liability, ultimately leading to a compound that has similar efficacy, but increased in vivo stability. The CED-10(Rac-1)/PAK1 pathway was essential for immunomodulation by CAPE and was a critical component involved in the immune response to fungal pathogens. Conclusion: Caenorhabditis elegans is an efficient heterologous host to evaluate immunomodulatory compounds and identify components of the pathway(s) involved in the mode of action of compounds.

The intrinsic resistance of Enterococcus faecium to ceftriaxone and cefepime (here referred to as “cephalosporins”) is reliant on the presence of class A penicillin-binding proteins (Pbps) PbpF and PonA. Mutants lacking these Pbps exhibit cephalosporin susceptibility that is reversible by exposure to penicillin and by selection on cephalosporin-containing medium. We selected two cephalosporin-resistant mutants (Cro1 and Cro2) of class A Pbp-deficient E. faecium CV598. Genome analysis revealed changes in the serine-threonine kinase Stk in Cro1 and a truncation in the associated phosphatase StpA in Cro2 whose respective involvements in resistance were confirmed in separate complementation experiments. In an additional effort to identify proteins linked to cephalosporin resistance, we performed tandem affinity purification using Pbp5 as bait in penicillin-exposed E. faecium; these experiments yielded a protein designated Pbp5-associated protein (P5AP). Transcription of the P5AP gene was increased after exposure to penicillin in wild-type strains and in Cro2 and suppressed in Cro2 complemented with the wild-type stpA. Transformation of class A Pbp-deficient strains with the plasmid-carried P5AP gene conferred cephalosporin resistance. These data suggest that Pbp5-associated cephalosporin resistance in E. faecium devoid of typical class A Pbps is related to the presence of P5AP, whose expression is influenced by the activity of the serine-threonine phosphatase/kinase system., IMPORTANCE β-Lactam antibiotics remain our most effective therapies against susceptible Gram-positive bacteria. The intrinsic resistance of Enterococcus faecium to β-lactams, particularly to cephalosporins, therefore represents a major limitation of therapy. Although the primary mechanism of resistance to β-lactams in E. faecium is the presence of low-affinity monofunctional transpeptidase (class B) penicillin-binding protein Pbp5, the interaction of Pbp5 with other proteins is fundamental to maintain a resistant phenotype. The present work identifies a novel, previously uncharacterized, protein that interacts with Pbp5, whose expression increases in conjunction with stimuli that increase resistance to cephalosporins, and that confers increased resistance to cephalosporins when overexpressed. P5AP may represent a promising new target, inhibition of which could restore cephalosporin susceptibility to E. faecium.

Fusariosis is an emerging infectious complication of immune deficiency, but models to study this infection are lacking. The use of the soil nematode Caenorhabditis elegans as a model host to study the pathogenesis of Fusarium spp. was investigated. We observed that Fusarium conidia consumed by C. elegans can cause a lethal infection and result in more than 90% killing of the host within 120 hours, and the nematode had a significantly longer survival when challenged with Fusarium proliferatum compared to other species. Interestingly, mycelium production appears to be a major contributor in nematode killing in this model system, and C. elegans mutant strains with the immune response genes, tir-1 (encoding a protein containing a TIR domain that functions upstream of PMK-1) and pmk-1 (the homolog of the mammalian p38 MAPK) lived significantly shorter when challenged with Fusarium compared to the wild type strain. Furthermore, we used the C. elegans model to assess the efficacy and toxicity of various compounds against Fusarium. We demonstrated that amphotericin B, voriconazole, mancozeb, and phenyl mercury acetate significantly prolonged the survival of Fusarium-infected C. elegans, although mancozeb was toxic at higher concentrations. In conclusion, we describe a new model system for the study of Fusarium pathogenesis and evolutionarily preserved host responses to this important fungal pathogen.

The pea pathogen Fusarium oxysporum f. sp. pisi is able to detoxify pisatin produced as a defense response by pea, and the gene encoding this detoxification mechanism, FoPDA1, was 82% identical to the cytochrome P450 pisatin demethylase PDA1 gene in Nectria haematococca. A survey of F. oxysporum f. sp. pisi isolates demonstrated that, as in N. haematococca, the PDA gene of F. oxysporum f. sp. pisi is generally located on a small chromosome. In N. haematococca, PDA1 is in a cluster of pea pathogenicity (PEP) genes. Homologs of these PEP genes also were found in the F. oxysporum f. sp. pisi isolates, and PEP1 and PEP5 were sometimes located on the same small chromosomes as the FoPDA1 homologs. Transforming FoPDA1 into a pda– F. oxysporum f. sp. lini isolate conferred pda activity and promoted pathogenicity on pea to some transformants. Different hybridization patterns of FoPDA1 were found in F. oxysporum f. sp. pisi but these did not correlate with the races of the fungus, suggesting that races within this forma specialis arose independently of FoPDA1. FoPDA1 also was present in the formae speciales lini, glycines, and dianthi of F. oxysporum but they had mutations resulting in nonfunctional proteins. However, an active FoPDA1 was present in F. oxysporum f. sp. phaseoli and it was virulent on pea. Despite their evolutionary distance, the amino acid sequences of FoPDA1 of F. oxysporum f. sp. pisi and F. oxysporum f. sp. phaseoli revealed only six amino acid differences, consistent with a horizontal gene transfer event accounting for the origin of these genes.

The fungal plant pathogen Nectria haematococca MPVI produces a cytochrome P450 that is responsible for detoxifying the phytoalexin pisatin, produced as a defense mechanism by its host, garden pea. In this study, we demonstrate that this fungus also produces a specific ATP-binding cassette (ABC) transporter, NhABC1, that enhances its tolerance to pisatin. In addition, although both mechanisms individually contribute to the tolerance of pisatin and act as host-specific virulence factors, mutations in both genes render the fungus even more sensitive to pisatin and essentially nonpathogenic on pea. NhABC1 is rapidly induced after treatment with pisatin in vitro and during infection of pea plants. Furthermore, NhABC1 was able to confer tolerance to the phytoalexin rishitin, produced by potato. NhABC1 appears to be orthologous to GpABC1 of the potato pathogen Gibberella pulicaris and, along with MoABC1 from Magnaporthe oryzae, resides in a phylogenetically related clade enriched with ABC transorters involved in virulence. We propose that NhABC1 and the cytochrome P450 may function in a sequential manner in which the energy expense from pisatin efflux by NhABC1 releases the repression of the cytochrome P450, ultimately allowing pisatin tolerance by two mechanisms. These results demonstrate that a successful pathogen has evolved multiple mechanisms to overcome these plant antimicrobial compounds.

Fusarium is a fungal pathogen of immunosuppressed lung transplant patients associated with a high mortality in those with severe and persistent neutropenia. The principle portal of entry for Fusarium species is the airways, and lung involvement almost always occurs among lung transplant patients with disseminated infection. In these patients, the immunoprotective mechanisms of the transplanted lungs are impaired, and they are, therefore, more vulnerable to Fusarium infection. As a result, fusariosis occurs in up to 32% of lung transplant patients. We studied fusariosis in 6 patients following lung transplantation who were treated at Massachusetts General Hospital during an 8-year period and reviewed 3 published cases in the literature. Cases were identified by the microbiology laboratory and through discharge summaries. Patients presented with dyspnea, fever, nonproductive cough, hemoptysis, and headache. Blood tests showed elevated white blood cell counts with granulocytosis and elevated inflammatory markers. Cultures of Fusarium were isolated from bronchoalveolar lavage, blood, and sputum specimens.Treatments included amphotericin B, liposomal amphotericin B, caspofungin, voriconazole, and posaconazole, either alone or in combination. Lung involvement occurred in all patients with disseminated disease and it was associated with a poor outcome. The mortality rate in this group of patients was high (67%), and of those who survived, 1 patient was treated with a combination of amphotericin B and voriconazole, 1 patient with amphotericin B, and 1 patient with posaconazole. Recommended empirical treatment includes voriconazole, amphotericin B or liposomal amphotericin B first-line, and posaconazole for refractory disease. High-dose amphotericin B is recommended for treatment of most cases of fusariosis. The echinocandins (for example, caspofungin, micafungin, anidulafungin) are generally avoided because Fusarium species have intrinsic resistance to them. Treatment should ideally be based on the Fusarium isolate, susceptibility testing, and host-specific factors. Prognosis of fusariosis in the immunocompromised is directly related to a patient's immune status. Prevention of Fusarium infection is recommended with aerosolized amphotericin B deoxycholate, which also has activity against other important fungi.

Fluconazole is the first line of therapy for the management of candidiasis. However, fluconazole-resistant strains pose an emerging challenge in everyday clinical practice. In this study, we sought to determine whether cumulative length of hospital stay (CLOS) is a predictive factor for the acquisition of non-susceptible Candida strains to fluconazole. Thirty-three critically ill emergency surgery patients with 56 Candida isolates were enrolled in this prospective study. We divided our isolates according to their minimum inhibitory concentration (MIC) to fluconazole using 8 mcg/ml as a cutoff. We then compared the two groups with respect to basic demographics, antifungal agents prescribed, number of wide-spectrum antibiotics, duration of central venous catheter placement, elapsed time to positive culture, duration of prior hospital stay, and length of hospital stay. Non-susceptible fluconazole samples belonged to patients with a significantly longer prior hospital stay and a longer CLOS (P = 0.02 and 0.01, respectively). The difference between the 2 groups regarding non-albicans strains was statistically significant (P

Members of the F usarium solani species complex (FSSC) are capable of causing disease in many agriculturally important crops. The genomes of some of these fungi include supernumerary chromosomes that are dispensable and encode host‐specific virulence factors. In addition to genomics, this review summarizes the known molecular mechanisms utilized by members of the FSSC in establishing disease. TAXONOMY: Kingdom Fungi; Phylum Ascomycota; Class Sordariomycetes; Order Hypocreales; Family Nectriaceae; Genus F usarium. HOST RANGE: Members of the FSSC collectively have a very broad host range, and have been subdivided previously into formae speciales. Recent phylogenetic analysis has revealed that formae speciales correspond to biologically and phylogenetically distinct species. DISEASE SYMPTOMS: Typically, FSSC causes foot and/or root rot of the infected host plant, and the degree of necrosis correlates with the severity of the disease. Symptoms on above‐ground portions of the plant can vary greatly depending on the specific FSSC pathogen and host plant, and the disease may manifest as wilting, stunting and chlorosis or lesions on the stem and/or leaves. CONTROL: Implementation of agricultural management practices, such as crop rotation and timing of planting, can reduce the risk of crop loss caused by FSSC. If available, the use of resistant varieties is another means to control disease in the field. USEFUL WEBSITES: http://genome.jgi‐psf.org/Necha2/Necha2.home.html

Several nonvertebrate hosts has been used to investigate virulence traits and therapies for several bacterial and fungal pathogens. This chapter details the use of heterologous hosts for the study of Cryptococcus neoformans. A striking finding was the correlation between known mammalian virulence factors such as the capsule, melanin, and phospholipase and their necessity for survival in amoebae. Many of the assays used when working with macrophages can be modified for use with Acanthamoeba castellanii, including killing assays, trypan blue staining, and phagocytosis assays. Although many virulence traits have shown some concordance in their requirement for both mammalian and protozoan virulence, there are some discrepancies. Two assays have been developed using Caenorhabditis elegans as a host to study C. neoformans. The first assay, termed the ‘’killing assay’’, was initially utilized to identify C. neoformans mutants reduced in virulence. Another assay has been developed based on the observation of smaller nematode brood size when infected with pathogenic C. neoformans. Mutants of Pka1 or Gpa1 (the α subunit) are reduced in virulence in C. elegans, similar to the observed virulence in mice studies. Acapsular mutants also displayed attenuated virulence in Dictyostelium discoideum and Galleria mellonella infection assays, although they remain virulent in D. melanogaster infection assays. Since the Cryptococcus genus includes a large set of organisms, all of which are also presumably under ameboid predator selection in soils and are able to survive in their environments, the avirulence of most species represents the absence of a complete trait set for animal hosts.