Effect of solute and matric potential on growth rate of fungal species isolated from cheese

The effect of water potential ( J w ) on the growth of 15 fungal species isolated from cheeses was analysed. The species, identified mainly by analysis of DNA sequences, belonged to genera Penicillium , Geotrichum , Mucor , Aspergillus , Microascus and Talaromyces . Particularly, the effect of matric potential ( J m ), and ionic (NaCl) and non-ionic (glycerol) solute potentials ( J s ) on growth rate was studied. The response of strains was highly dependent on the type of J w . For J s , clear profiles for optimal, permissive and marginal conditions for growth were obtained, and differences in growth rate were achieved comparing NaCl and glycerol for most of the species. Conversely, a sustained growth was obtained for J m in all the strains, with the exception of Aspergillus pseudoglaucus , whose growth increased proportionally to the level of water stress. Our results might help to understand the impact of environmental factors on the ecophysiology and dynamics of fungal populations associated to cheeses.

Fungal biodeterioration of colour cinematographic films of the cultural heritage of Cuba

Until recently, cinematographic film was largely cellulose-triacetate-based. However, this material is highly susceptible to biodeterioration, thus placing historic film collections, an important part of the cultural heritage of many countries, at risk. In the present study, samples taken from several biodeteriorated color cinematographic films belonging to the collection of the Cuban Institute for Cinematographic Industry and Arts (ICAIC) were investigated. Infrared spectroscopy showed that all films were of the same composition, i.e., a gelatin emulsion coating one side of a cellulose-triacetate-based film support. The films were analyzed by environmental scanning electron microscopy and scanning electron microscopy to determine the degree of biodeterioration and the type of colonizing microorganisms. Significant fungal colonization was found on both sides of the films in all samples, with a higher concentration of fungi on the gelatin emulsion side. Epifluorescence microscopy of fluorochrome-dyed films demonstrated that some of the fungi were still active, indicating that the films under study, and probably others at the ICAIC, are at risk of further deterioration. Fungi were identified by molecular biology techniques. The fungi mainly responsible for the observed biodeterioration were those belonging to the genera Aspergillus and Cladosporium, although other genera, such as Microascus and Penicillium, were identified as well. In accordance with the findings described herein, the existing guidelines for the prevention and control of film biodeterioration are discussed.

Effect of solute and matric potential on growth rate of fungal species isolated from cheese

The effect of water potential ( J w ) on the growth of 15 fungal species isolated from cheeses was analysed. The species, identi fi ed mainly by analysis of DNA sequences, belonged to genera Penicillium, Geotrichum, Mucor , Aspergillus , Microascus and Talaromyces . Particularly, the effect of matric potential ( J m ), and ionic (NaCl) and non-ionic (glycerol) solute potentials ( J s ) on growth rate was studied. The response of strains was highly dependent on the type of J w . For J s, clear profiles for optimal, permissive and marginal conditions for growth were obtained, and differences in growth rate were achieved comparing NaCl and glycerol for most of the species. Conversely, a sustained growth was obtained for J m in all the strains, with the exception of Aspergillus pseudoglaucus, whose growth increased proportionally to the level of water stress. Our results might help to understand the impact of environmental factors on the ecophysiology and dynamics of fungal populations associated to cheeses.

Analysis of the role of the Spitzenkörper in fungal morphogenesis by computer simulation of apical branching in Aspergillus niger

High-resolution video microscopy, image analysis, and computer simulation were used to study the role of the Spitzenkörper (Spk) in apical branching of ramosa-1, a temperature-sensitive mutant of Aspergillus niger. A shift to the restrictive temperature led to a cytoplasmic contraction that destabilized the Spk, causing its disappearance. After a short transition period, new Spk appeared where the two incipient apical branches emerged. Changes in cell shape, growth rate, and Spk position were recorded and transferred to the fungus simulator program to test the hypothesis that the Spk functions as a vesicle supply center (VSC). The simulation faithfully duplicated the elongation of the main hypha and the two apical branches. Elongating hyphae exhibited the growth pattern described by the hyphoid equation. During the transition phase, when no Spk was visible, the growth pattern was nonhyphoid, with consecutive periods of isometric and asymmetric expansion; the apex became enlarged and blunt before the apical branches emerged. Video microscopy images suggested that the branch Spk were formed anew by gradual condensation of vesicle clouds. Simulation exercises where the VSC was split into two new VSCs failed to produce realistic shapes, thus supporting the notion that the branch Spk did not originate by division of the original Spk. The best computer simulation of apical branching morphogenesis included simulations of the ontogeny of branch Spk via condensation of vesicle clouds. This study supports the hypothesis that the Spk plays a major role in hyphal morphogenesis by operating as a VSC—i.e., by regulating the traffic of wall-building vesicles in the manner predicted by the hyphoid model.

The roles of zinc and copper sensing in fungal pathogenesis

Open Access funded by Wellcome Trust Acknowledgements ERB is funded by the BBSRC (BB/M014525/1). DW is supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (Grant Number 102549/Z/13/Z). We additionally acknowledge the MRC and University of Aberdeen for funding (MR/N006364/1) and the Wellcome Trust Strategic Award for Medical Mycology and Fungal Immunology (097377/Z/11/Z). Finally, we acknowledge FungiDB and the Candida Genome Database [ 56 and 57].

Host-Imposed Copper Poisoning Impacts Fungal Micronutrient Acquisition during Systemic Candida albicans Infections

This work was supported by the European Research Council (http://erc.europa.eu/: STRIFE Advanced Grant ERC-2009-AdG-249793). A.J.P.B. was also supported by the UK Biotechnology and Biological Research Council (www.bbsrc.ac.uk: Research Grants BB/F00513X/1, BB/K017365/1), the UK Medical Research Council (www.mrc.ac.uk: Programme Grant MR/M026663/1; Centre Grant MR/ N006364/1), and the Wellcome Trust (www.wellcome.ac.uk: Strategic Award 097377)

A Fungal Kinesin Required for Organelle Motility, Hyphal Growth, and Morphogenesis

A gene (NhKIN1) encoding a kinesin was cloned from Nectria haematococca genomic DNA by polymerase chain reaction amplification, using primers corresponding to conserved regions of known kinesin-encoding genes. Sequence analysis showed that NhKIN1 belongs to the subfamily of conventional kinesins and is distinct from any of the currently designated kinesin-related protein subfamilies. Deletion of NhKIN1 by transformation-mediated homologous recombination caused several dramatic phenotypes: a 50% reduction in colony growth rate, helical or wavy hyphae with reduced diameter, and subcellular abnormalities including withdrawal of mitochondria from the growing hyphal apex and reduction in the size of the Spitzenkörper, an apical aggregate of secretory vesicles. The effects on mitochondria and Spitzenkörper were not due to altered microtubule distribution, as microtubules were abundant throughout the length of hyphal tip cells of the mutant. The rate of spindle elongation during anaphase B of mitosis was reduced 11%, but the rate was not significantly different from that of wild type. This lack of a substantial mitotic phenotype is consistent with the primary role of the conventional kinesins in organelle motility rather than mitosis. Our results provide further evidence that the microtubule-based motility mechanism has a direct role in apical transport of secretory vesicles and the first evidence for its role in apical transport of mitochondria in a filamentous fungus. They also include a unique demonstration that a microtubule-based motor protein is essential for normal positioning of the Spitzenkörper, thus providing a new insight into the cellular basis for the aberrant hyphal morphology.

Involvement of the Octadecanoid Pathway and Protein Phosphorylation in Fungal Elicitor-Induced Expression of Terpenoid Indole Alkaloid Biosynthetic Genes in Catharanthus roseus

Two key genes in terpenoid indole alkaloid biosynthesis, Tdc and Str, encoding tryptophan decarboxylase and strictosidine synthase, respectively, are coordinately induced by fungal elicitors in suspension-cultured Catharanthus roseus cells. We have studied the roles of the jasmonate biosynthetic pathway and of protein phosphorylation in signal transduction initiated by a partially purified elicitor from yeast extract. In addition to activating Tdc and Str gene expression, the elicitor also induced the biosynthesis of jasmonic acid. The jasmonate precursor α-linolenic acid or methyl jasmonate (MeJA) itself induced Tdc and Str gene expression when added exogenously . Diethyldithiocarbamic acid, an inhibitor of jasmonate biosynthesis, blocked both the elicitor-induced formation of jasmonic acid and the activation of terpenoid indole alkaloid biosynthetic genes. The protein kinase inhibitor K-252a abolished both elicitor-induced jasmonate biosynthesis and MeJA-induced Tdc and Str gene expression. Analysis of the expression of Str promoter/gusA fusions in transgenic C. roseus cells showed that the elicitor and MeJA act at the transcriptional level. These results demonstrate that the jasmonate biosynthetic pathway is an integral part of the elicitor-triggered signal transduction pathway that results in the coordinate expression of the Tdc and Str genes and that protein kinases act both upstream and downstream of jasmonates.

PLS1, a gene encoding a tetraspanin-like protein, is required for penetration of rice leaf by the fungal pathogen Magnaporthe grisea

We describe in this study punchless, a nonpathogenic mutant from the rice blast fungus M. grisea, obtained by plasmid-mediated insertional mutagenesis. As do most fungal plant pathogens, M. grisea differentiates an infection structure specialized for host penetration called the appressorium. We show that punchless differentiates appressoria that fail to breach either the leaf epidermis or artificial membranes such as cellophane. Cytological analysis of punchless appressoria shows that they have a cellular structure, turgor, and glycogen content similar to those of wild type before penetration, but that they are unable to differentiate penetration pegs. The inactivated gene, PLS1, encodes a putative integral membrane protein of 225 aa (Pls1p). A functional Pls1p-green fluorescent protein fusion protein was detected only in appressoria and was localized in plasma membranes and vacuoles. Pls1p is structurally related to the tetraspanin family. In animals, these proteins are components of membrane signaling complexes controlling cell differentiation, motility, and adhesion. We conclude that PLS1 controls an appressorial function essential for the penetration of the fungus into host leaves.

A novel clade of protistan parasites near the animal-fungal divergence.

Sequences of nuclear-encoded small-subunit rRNA genes have been determined for representatives of the enigmatic genera Dermocystidium, Ichthyophonus, and Psorospermium, protistan parasites of fish and crustaceans. The small-subunit rRNA genes from these parasites and from the "rosette agent" (also a parasite of fish) together form a novel, statistically supported clade. Phylogenetic analyses demonstrate this clade to diverge near the animal-fungal dichotomy, although more precise resolution is problematic. In the most parsimonious and maximally likely phylogenetic frameworks inferred from the most stably aligned sequence regions, the clade constitutes the most basal branch of the metazoa; but within a limited range of model parameters, and in some analyses that incorporate less well-aligned sequence regions, an alternative topology in which it diverges immediately before the animal-fungal dichotomy was recovered. Mitochondrial cristae of Dermocystidium spp. are flat, whereas those of Ichthyophonus hoferi appear tubulovesiculate. These results extend our understanding of the types of organisms from which metazoa and fungi may have evolved.

Extensive alteration of fungal gene transcript accumulation and elevation of G-protein-regulated cAMP levels by a virulence-attenuating hypovirus.

Persistent infection of the chestnut blight fungus Cryphonectria parasitica with the prototypic hypovirus CHVI-713 results in attenuation of fungal virulence (hypo-virulence) and reduced accumulation of the GTP-binding (G) protein a subunit CPG-1. Transgenic cosuppression of CPG-1 accumulation in the absence of virus infection also confers hypovirulence. We now report the use of mRNA differential display to examine the extent to which virus infection alters fungal gene transcript accumulation and to assess the degree to which modification of CPG-1 signal transduction contributes to this alteration. More than 400 PCR products were identified that either increased (296 products) or decreased (127 products) in abundance as a result of virus infection. Significantly, 65% of these products exhibited similar changes as a result of CPG-1 cosuppression in the absence of virus infection. We also report that both virus infection and CPG-1 cosuppression elevate cAMP levels 3- to 5-fold. Additionally, it was possible to mimic the effect of virus infection and CPG-1 cosuppression on transcript accumulation for representative fungal genes by drug-induced elevation of cAMP levels. These results strengthen and extend previous indications that hypovirus infection causes a significant and persistent alteration of fungal gene expression/transcript accumulation. They further show that this alteration is primarily mediated through modification of the CPG-1 signaling pathway and suggest that, similar to mammalian Gi alpha subunits, CPG-1 functions as a negative modulator of adenylyl cyclase. Finally, these results suggest a role for G-protein-regulated cAMP accumulation in hypovirus-mediated alteration of fungal gene expression.

The terminal Paleozoic fungal event: evidence of terrestrial ecosystem destabilization and collapse.

Because of its prominent role in global biomass storage, land vegetation is the most obvious biota to be investigated for records of dramatic ecologic crisis in Earth history. There is accumulating evidence that, throughout the world, sedimentary organic matter preserved in latest Permian deposits is characterized by unparalleled abundances of fungal remains, irrespective of depositional environment (marine, lacustrine, fluviatile), floral provinciality, and climatic zonation. This fungal event can be considered to reflect excessive dieback of arboreous vegetation, effecting destabilization and subsequent collapse of terrestrial ecosystems with concomitant loss of standing biomass. Such a scenario is in harmony with predictions that the Permian-Triassic ecologic crisis was triggered by the effects of severe changes in atmospheric chemistry arising from the rapid eruption of the Siberian Traps flood basalts.