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Weak-acid preservatives such as sorbic acid are added to foods to prevent fungal spoilage. The modes of action of weak-acid preservatives are only partially understood and, in this paper, further insight is presented into the mechanisms by which weak acids inhibit the growth of fungi. Uridine-requiring strains of Aspergillus niger were shown to be more sensitive to weak acids (including sorbic, acetic and benzoic acids) than wild-type (WT) strains. In contrast, sensitivity to other, non-acidic, antifungal substances was similar in mutant and WT strains. By complementing a pyr[G.sup.-] strain of A. niger with an intact pyrG gene, WT-like resistance to weak-acid preservatives was restored. Using [14.sup.C]-labelled uridine, sorbic acid was shown to completely inhibit uridine uptake in germinating conidia in a non-competitive manner. It is therefore proposed that the additional weak-acid sensitivity of the pyr[G.sup.-] strains was caused by weak-acid inhibition of uridine uptake. Several other auxotrophic strains of A. niger were screened for sensitivity to acetic, sorbic and decanoic acids. Strains auxotrophic for either adenine or uridine were found to have enhanced sensitivity but, in contrast, amino acid auxotrophs showed resistance comparable to that of the WT. Uridine auxotrophs of Saccharomyces cerevisiae were not more sensitive to weak acids compared to WT strains. In conclusion, this study describes a previously unknown mechanism of action of weak acids against the filamentous fungus A. niger, which may fundamentally affect our understanding of the preservation of food against spoilage fungi.

The authors have previously reported on molecular responses of Aspergillus nidulans to bacterial antifungal metabolites, e.g. bafilomycins and the related concanamycins. These compounds are known inhibitors of V-ATPases and cause dramatic effects on mycelial growth and morphology. In Neurospora crassa, studies have shown that disruption of the gene encoding subunit A of the V-ATPase results in morphological changes and reduced growth similar to those observed after addition of concanamycin. This phenotype, and the fact that this mutation confers resistance to concanamycin, suggests that V-ATPase is the main (or only) target for the antibiotics. However, growth inhibition and morphology changes in, for example, A. nidulans and Penicillium roqueforti are more severe, and thus other targets are possible. In this study, the vmaA gene of A. nidulans, encoding the subunit A of V-ATPase, was disrupted by homologous recombination. The resulting vmaA 1 mutant strain displayed extremely slow growth and failed to produce asexual spores. Furthermore, an altered morphology similar to that caused by addition of V-ATPase inhibitors, i.e. bafilomycin or concanamycin, was observed, indicating that V-ATPase is the main target for the antibiotics also in A. nidulans. The vmaA1 mutant was not viable at pH values above 7 and was highly sensitive to high [Zn.sup.2+] concentrations, in agreement with previous results from studies of Saccharomyces cerevisiae and N. crassa.

Background The disaccharide trehalose is a major component of fungal spores and is released upon germination. Moreover, the sugar is well known for is protective functions, e.g. against thermal stress and dehydration. The properties and synthesis of trehalose have been well investigated in the bakers' yeast Saccharomyces cerevisiae. In filamentous fungi, such knowledge is limited, although several gene products have been identified. Results Using Aspergillus niger as a model fungus, the aim of this study was to provide an overview of all genes involved in trehalose synthesis. This fungus has three potential trehalose-6- phosphate synthase encoding genes, tpsA-C, and three putative trehalose phosphate phosphatase encoding genes, tppA-C, of which two have not previously been identified. Expression of all six genes was confirmed using real-time PCR, and conserved orthologs could be identified in related Aspergilli. Using a two-hybrid approach, there is a strong indication that four of the proteins physically interact, as has previously been shown in S. cerevisiae. When creating null mutants of all the six genes, three of them, ΔtpsA, ΔtppA and ΔtppB, had lower internal trehalose contents. The only mutant with a pronounced morphological difference was ΔtppA, in which sporulation was severely reduced with abnormal conidiophores. This was also the only mutant with accumulated levels of trehalose- 6-phosphate, indicating that the encoded protein is the main phosphatase under normal conditions. Besides ΔtppA, the most studied deletion mutant in this work was ΔtppB. This gene encodes a protein conserved in filamentous Ascomycota. The ΔtppB mutant displayed a low, but not depleted, internal trehalose content, and conidia were more susceptible to thermal stress. Conclusion A. niger contains at least 6 genes putatively involved in trehalose synthesis. Gene expressions related to germination have been quantified and deletion mutants characterized: Mutants lacking tpsA, tppA or tppB have reduced internal trehalose contents. Furthermore, tppA, under normal conditions, encodes the functional trehalose-6-phosphate-phosphatase. [ABSTRACT FROM AUTHOR]

Highlights: [•] Subgroup C-II chitinase genes are induced during fungal interspecific interactions. [•] Subgroup C-II chitinase genes are not induced during interactions with Phytophthora. [•] Deletion of subgroup C-II chitinase genes affects abiotic stress tolerance. [•] Deletion of chiC2-2 reduces the A. nidulans growth inhibitory activity against B. cinerea. [Copyright &y& Elsevier]

Background Food spoilage caused by molds is a severe problem. In food and feed, e.g. dairy products, sourdough bread and silage, lactic acid bacteria are used as starter cultures. Besides lactic and acetic acid, some strains produce other low molecular weight compounds with antifungal activities. One of these metabolites is phenyllactic acid (PLA), well known for its antifungal effect. The inhibitory effect of PLA has only partially been investigated, and the objective of this study was to elucidate in detail the antifungal properties of PLA. Results We investigated the outgrowth of individual conidia from Aspergillus niger, Cladosporium cladosporioides and Penicillium roqueforti, and observed the morphologies of resulting colonies on solid media using different acid concentrations. We found that PLA inhibits molds similar to weak acid preservatives. Furthermore, it has an additional activity: at subinhibitory concentrations, fungal colonies displayed slower radial growth and inhibited sporulation. The L isoform of PLA is a more potent inhibitor than the D form. Increased expression of phiA was observed during PLA treatment. This gene was initially identified as being induced by Streptomyces-produced macrolide antibiotics, and is shown to be a structural protein in developed cells. This suggests that PhiA may act as a general stress protectant in fungi. Conclusion From a food protection perspective, the results of this study support the usage of lactic acid bacteria strains synthesizing PLA as starter cultures in food and feed. Such starter cultures could inhibit spore synthesis, which would be beneficial as many food borne fungi are spread by airborne spores. [ABSTRACT FROM AUTHOR]

Abstract: Glycoside hydrolase family 18 contains hydrolytic enzymes with chitinase or endo-N-acetyl-β-d-glucosaminidase (ENGase) activity, while glycoside hydrolase family 20 contains enzymes with β-N-acetylhexosaminidase (NAGase) activity. Chitinases and NAGases are involved in chitin degradation. Chitinases are phylogenetically divided into three main groups (A, B and C), each further divided into subgroups. In this study, we investigated the functional role of 10 Neurospora crassa genes that encode chitinases, 2 genes that encode ENGases and 1 gene that encode a NAGase, using gene deletion and gene expression techniques. No phenotypic effects were detected for any of the studied group A chitinase gene deletions. Deletion of the B group member chit-1 resulted in reduced growth rate compared with the wild type (WT) strain. In combination with the presence of a predicted glycosylphosphatidylinositol anchor motif in the C-terminal of chit-1, indicating cell wall localization, these data suggest a role in cell wall remodeling during hyphal growth for chit-1. Deletion of the ENGase gene gh18-10 resulted in reduced growth rate compared with WT, increased conidiation, and increased abiotic stress tolerance. In addition, Δgh18-10 strains displayed lower secretion of extracellular proteins compared to WT and reduced levels of extracellular protease activity. The connection between gh18-10 ENGase activity and the endoplasmic reticulum associated protein degradation process, a stringent quality control of glycoprotein maturation, is discussed. N. crassa group C chitinase genes gh18-6 and gh18-8 were both induced during fungal–fungal interactions. However, gh18-6 was only induced during interspecific interactions, while gh18-8 displayed the highest induction levels during self–self interactions. These results provide new information on functional differentiation of fungal chitinases. [Copyright &y& Elsevier]

The fate and behaviour of the seed-applied biocontrol strain Pseudomonas brassicacearum MA250 in a field trial with winter wheat was determined using sequence-characterised amplified region (SCAR) markers. Samples of belowground plant parts from healthy and withered (due to snow mould infection) seedlings were collected approximately one and seven months after sowing, which was performed in early autumn. DNA was extracted from roots and remaining parts of seeds with adhering soil, and the abundance of the strain was determined in quantitative real-time PCR (qPCR) assays. The results show that the introduced strain persisted over the whole trial-period of seven months. On termination of the trial (after seven months) the belowground plant parts of each plant housed 106–107 cells, substantially less than the original approximately 109 cells inoculated onto the seed. In healthy seedlings, there was a shift in cell numbers from seeds to roots between the samplings, suggesting colonisation of the roots during this time. The results show that with sufficient attention given to analytical control measures and the possibility of resident background populations, SCAR markers in combination with qPCR provide valuable information regarding the fate and behaviour of biocontrol micro-organisms under field conditions. [ABSTRACT FROM PUBLISHER]

Abstract: The ability to resist anti-microbial compounds is of key evolutionary benefit to microorganisms. Aspergillus niger has previously been shown to require the activity of a phenylacrylic acid decarboxylase (encoded by padA1) for the decarboxylation of the weak-acid preservative sorbic acid (2,4-hexadienoic acid) to 1,3-pentadiene. It is now shown that this decarboxylation process also requires the activity of a putative 4-hydroxybenzoic acid (3-octaprenyl-4-hydroxybenzoic acid) decarboxylase, encoded by a gene termed ohbA1, and a putative transcription factor, sorbic acid decarboxylase regulator, encoded by sdrA. The padA1, ohbA1 and sdrA genes are in close proximity to each other on chromosome 6 in the A. niger genome and further bioinformatic analysis revealed conserved synteny at this locus in several Aspergillus species and other ascomycete fungi indicating clustering of metabolic function. This cluster is absent from the genomes of A. fumigatus and A. clavatus and, as a consequence, neither species is capable of decarboxylating sorbic acid. [Copyright &y& Elsevier]

Certain species of the fungal genus Trichoderma are potent mycoparasites and are used for biological control of fungal diseases on agricultural crops. In Trichoderma, whole-genome sequencing reveal between 20 and 36 different genes encoding chitinases, hydrolytic enzymes that are involved in the mycoparasitic attack. Sequences of Trichoderma chitinase genes chi18-5, chi18-13, chi18-15 and chi18-17, which all exhibit specific expression during mycoparasitism-related conditions, were determined from up to 13 different taxa and studied with regard to their evolutionary patterns. Two of them, chi18-13 and chi18-17, are members of the B1/B2 chitinase subgroup that have expanded significantly in paralog number in mycoparasitic Hypocrea atroviridis and H. virens. Chi18-13 contains two codons that evolve under positive selection and seven groups of co-evolving sites. Chi18-15 displays a unique codon-usage and contains five codons that evolve under positive selection and three groups of co-evolving sites. Regions of high amino acid variability are preferentially localized to substrate- or product side of the catalytic clefts. Differences in amino acid diversity/conservation patterns between different Trichoderma clades are observed. These observations show that Trichoderma chitinases chi18-13 and chi18-15 evolve in a manner consistent with rapid co-evolutionary interactions and identifies putative target regions involved in determining substrate-specificity. [ABSTRACT FROM AUTHOR]

Abstract: Biological control microorganisms have long been promoted as an alternative to conventional pesticides. Before registration of a microbial biocontrol product for commercial sale, it must be evaluated as regards potential spread and persistence after release. In this study, strainspecific sequence-characterized amplified region (SCAR) markers were developed to monitor the biocontrol candidate strain Pseudomonas brassicacearum MA250, which is effective against snow mould (Microdochium nivale). One SCAR marker, OPA2-73, was used in quantitative real-time PCR (Q-PCR) on samples from a climate chamber experiment in which winter wheat seeds were treated with the bacterium or a chemical control agent, or left untreated. The results showed that MA250 persisted for up to 3 weeks after sowing on the kernel residues and also colonized the roots of treated seedlings. Total MA250 cell numbers on biocontrol treated seedlings after three weeks were approximately 106 cells, compared with the original inoculum of 106–107 cells per seed. Corresponding cell numbers of MA250 on chemically treated and untreated seedlings were below the detection limit. This study shows that SCAR marker OPA2-73 is a specific and sensitive tool for monitoring the biocontrol microorganism MA250 in environmental samples. [Copyright &y& Elsevier]

The biocontrol yeast Pichia anomala J121 can effectively reduce mould growth on moist cereal grains during airtight storage. Practical use of microorganisms requires formulated products that meet a number of criteria. In this study we compared different formulations of P. anomala. The best way to formulate P. anomala was freeze-drying. The initial viability was as high as 80%, with trehalose previously added to the yeast. Freeze-dried products could be stored at temperatures as high as 30 °C for a year, with only a minor decrease in viability. Vacuum-drying also resulted in products with high storage potential, but the products were not as easily rehydrated as freeze-dried samples. Upon desiccating the cells using fluidised-bed drying or as liquid formulations, a storage temperature of 10 °C was required to maintain viability. Dependent on the type of formulation, harvesting of cells at different nutritional stresses affected the initial viabilities, e.g. the initial viability for fluidised-bed-dried cells was higher when the culture was fed with excess glucose, but for freeze-drying it was superior when cells were harvested after depletion of carbon. Using micro-silos we found that the biocontrol activity remained intact after drying, storage and rehydration for all formulations. [ABSTRACT FROM AUTHOR]

We amplified, sequenced and studied the transcriptional regulation of genes of the alcoholic fermentation pathway in the biocontrol and non- Saccharomyces wine yeast, Pichia anomala. Two ADH isogenes, PaADH1 and PaADH2, and one PDC gene, PaPDC1, were amplified from genomic P. anomala DNA by a two-step PCR approach, using degenerated primers against conserved regions of the respective genes for cloning core regions, and PCR-based gene walking for cloning the respective 5′ and 3′-ends. According to sequence analysis, ADH1 and PDC1 are most likely cytoplasmatic proteins, while ADH2 is most probably localized in the mitochondria. PaADH1 was expressed during aerobic growth on glucose, ethanol and succinate, but was nine-fold upregulated in response to oxygen limitation when grown on glucose. The gene seems to be involved in both production and consumption of ethanol. Only low expression of PaADH2 was detected during growth on glucose and ethanol, but it was highly expressed during growth on the non-fermentable carbon source succinate and repressed by the addition of glucose. PaPDC1 was expressed during aerobic growth on glucose and was upregulated four-fold in response to oxygen limitation. PaPDC1 expression was lower in cells grown on ethanol and succinate than on glucose and was up- regulated two- and four-fold, respectively, after glucose addition. Our results demonstrate that transcription of genes of the fermentative pathway is regulated by hypoxia and carbon source but posttranscriptional regulation may play a major role in regulating the metabolic flux. Copyright © 2006 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract: The fungal inhibitory effects of strain Lactobacillus plantarum MiLAB 393, producing broad-spectrum antifungal compounds, were evaluated. A co-cultivation method was set up to monitor effects on fungal growth and protein expression of growing Aspergillus nidulans with L. plantarum MiLAB 393. The effects of inhibitory metabolites produced by L. plantarum MiLAB 393, cyclo(l-Phe¿l-Pro), lactic acid and 3-phenyllactic acid, were also investigated by addition of pure compounds to the growth medium of A. nidulans. The co-cultivation strongly affected the morphology of the fungal mycelium and decreased the biomass to 36% of control. Co-cultivation with Lactobacillus coryniformis MiLAB 123 gave only marginal morphological changes and minor biomass reduction, suggesting specific effects of L. plantarum MiLAB 393. The amount of several A. nidulans-proteins was increased during co-cultivation and by all of the inhibiting substances. This study shows that the growth of A. nidulans is inhibited during co-cultivation with L. plantarum MiLAB 393 and that the expression of fungal proteins is altered. [Copyright &y& Elsevier]

We have previously identified genes and proteins involved in the fungal response to the Streptomyces-produced antibiotics, bafilomycin B1 and concanamycin A, known inhibitors of V-ATPases. Using mRNA differential display we identified an Aspergillus nidulans gene with 30-fold up-regulated expression in the presence of bafilomycin. This gene, here denoted phiA, and its gene product, were further characterized by targeted gene disruption and immunohistochemistry. Phenotypically, the phiA mutation resulted in reduced growth and severely reduced sporulation. The abnormality could be traced to the phialides, which divided several times instead of forming a single flask-shaped cell. The importance of phiA for phialide and conidium development was supported by immunohistochemistry experiments that showed the protein to be mainly present in these two cell types. Attempts to relate phiA to inhibition of V-ATPases did not result in unambiguous conclusions, but suggest the possibility that changed expression of phiA is correlated with growth arrest caused by inhibited V-ATPases. [Copyright &y& Elsevier]

Examines the effect of the antibiotic bafilomycin on Aspergillus nidulans gene expression. Changes in concentrations of corresponding mRNAs of genes; Encoding of ASPND1 glycoprotein by one down-regulated mRNA; Potential application of mRNA differential display to analysis of interactions between bacteria and filamentous fungi.

Bacillus thuringiensis serovar israelensis is a wide-spread soil bacterium affiliated with the B. cereus group (Bcg) and is widely used in biocontrol products applied against mosquito and black fly larvae. For monitoring and quantification of applied B. thuringiensis serovar israelensis and its effect on indigenous B. thuringiensis serovar israelensis and Bcg assemblages, efficient and reliable tools are essential. The abundance and properties of B. thuringiensis serovar israelensis strains in the environment traditionally have been investigated with cultivation-dependent techniques, which are hampered by low sensitivity and the morphological similarity between B. cereus and B. thuringiensis. Currently available PCR-based detection and quantification tools target markers located on plasmids. In this study, a new cultivation-independent PCR-based method for efficient and specific quantification of B. thuringiensis serovar israelensis and Bcg is presented, utilizing two sets of PCR primers targeting the bacterial chromosome. Sequence database searches and empirical tests performed on target and nontarget species, as well as on bulk soil DNA samples, demonstrated that this diagnostic tool is specific for B. thuringiensis serovar israelensis and Bcg. The method will be useful for comparisons of Bcg and B. thuringiensis serovar israelensis abundances in the same samples. Moreover, the effect of B. thuringiensis serovar israelensis-based insecticide application on the total Bcg assemblages, including indigenous populations, can be investigated. This type of information is valuable in risk assessment and policy making for use of B. thuringiensis serovar israelensis in the environment. [ABSTRACT FROM AUTHOR]

Abstract: The focused-ion-beam (FIB) is the method of choice for site-specific sample preparation for Transmission Electron Microscopy (TEM) in material sciences. A lamella can be physically lifted out from a specific region of a bulk specimen with submicrometer precision and thinned to electron transparency for high-resolution imaging in the TEM. The possibility to use this tool in life sciences applications has been limited by the lack of lift-out capabilities at the cryogenic temperatures often needed for biological samples. Conventional cryo-TEM sample preparation is mostly based on ultramicrotomy, a procedure that is not site-specific and known to produce artifacts. Here we demonstrate how a cooled nanomanipulator and a custom-built transfer station can be used to achieve cryo-preparation of TEM samples with the FIB, enabling high-resolution investigation of frozen-hydrated specimens in the TEM. [Copyright &y& Elsevier]

Background: The fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus., Results: We have generated genome sequences for ten novel, highly diverse Aspergillus species and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli., Conclusions: Many aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi.

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms or solutions. We make use of a custom built cryo-transfer station and a modified cryo-SEM preparation chamber. The spores are taken from a culture, plunge-frozen in a liquid nitrogen slush and observed in the cryo-SEM to select a region of interest. A thin lamella is then extracted using the FIB, attached to a TEM grid and subsequently thinned to electron transparency. The grid is transferred to a cryo-TEM holder and into a TEM for high resolution studies. Thanks to the introduction of a cooled nanomanipulator tip and a cryo-transfer station, this protocol is a straightforward adaptation to cryogenic temperature of the routinely used FIB preparation of TEM samples. As such it has the advantages of requiring a small amount of modifications to existing instruments, setups and procedures; it is easy to implement; it has a broad range of applications, in principle the same as for cryo-TEM sample preparation. One limitation is that it requires skillful handling of the specimens at critical steps to avoid or minimize contaminations.

Within ecosystems microorganisms coexist and interact. Knowledge of these interactions is of great importance in the fields of ecology, food production, and medicine. Such interactions often involve the synthesis of antibiotic secondary metabolites. Different kinds of s molecules or direct contacts are other forms of microbial interactions. Recently, modern molecular methods such as microarrays and proteomics have been employed to investigate such interactions. In this chapter, the use of proteomics for studies of microbial interactions is discussed. The choice of experimental setup is dependent on the aims of the specific study. One aspect of competition between microbes can be simulated by treatment of one microbe with antibiotics produced by a competing microbe. A more complicated approach involves cocultivation of the competitors, but in order to reveal species-specific protein patterns it is advisable to keep the organisms separated. Alternative techniques are to monitor alterations in the proteomes between the wild-type and mutant strains. The mutant can be either natural or created using random or targeted mutagenesis. Generally, a proteomic study will reveal proteins with both expected and surprising changes in abundance upon competition, but also previously unknown proteins are likely to be identified. A proteomic approach is usually insufficient to obtain a complete data set describing microbial interactions. Therefore, it is essential to follow up identification of proteins with changed abundance by, e.g., the creation of knockout strains for phenotypic analyses. Despite the limitations, proteomics is a useful method, and an important complement to other approaches for studies of microbial interactions.