The environmental stress sensitivities of pathogenic Candida species, including Candida auris, and implications for their spread in the hospital setting.

Candida auris is an emerging pathogenic yeast of significant clinical concern because of its frequent intrinsic resistance to fluconazole and often other antifungal drugs and the high mortality rates associated with systemic infections. Furthermore, C. auris has a propensity for persistence and transmission in health care environments. The reasons for this efficient transmission are not well understood, and therefore we tested whether enhanced resistance to environmental stresses might contribute to the ability of C. auris to spread in health care environments. We compared C. auris to other pathogenic Candida species with respect to their resistance to individual stresses and combinations of stresses. Stress resistance was examined using in vitro assays on laboratory media and also on hospital linen. In general, the 17 C. auris isolates examined displayed similar degrees of resistance to oxidative, nitrosative, cationic and cell wall stresses as clinical isolates of C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. krusei, C. guilliermondii, C. lusitaniae and C. kefyr. All of the C. auris isolates examined were more sensitive to low pH (pH 2, but not pH 4) compared to C. albicans, but were more resistant to high pH (pH 13). C. auris was also sensitive to low pH, when tested on contaminated hospital linen. Most C. auris isolates were relatively thermotolerant, displaying significant growth at 47°C. Furthermore, C. auris was relatively resistant to certain combinations of combinatorial stress (e.g., pH 13 plus 47°C). Significantly, C. auris was sensitive to the stress combinations imposed by hospital laundering protocol (pH > 12 plus heat shock at >80°C), suggesting that current laundering procedures are sufficient to limit the transmission of this fungal pathogen via hospital linen.

The genome of the emerging barley pathogen Ramularia collo-cygni.

BACKGROUND: Ramularia collo-cygni is a newly important, foliar fungal pathogen of barley that causes the disease Ramularia leaf spot. The fungus exhibits a prolonged endophytic growth stage before switching life habit to become an aggressive, necrotrophic pathogen that causes significant losses to green leaf area and hence grain yield and quality. RESULTS: The R. collo-cygni genome was sequenced using a combination of Illumina and Roche 454 technologies. The draft assembly of 30.3 Mb contained 11,617 predicted gene models. Our phylogenomic analysis confirmed the classification of this ascomycete fungus within the family Mycosphaerellaceae, order Capnodiales of the class Dothideomycetes. A predicted secretome comprising 1053 proteins included redox-related enzymes and carbohydrate-modifying enzymes and proteases. The relative paucity of plant cell wall degrading enzyme genes may be associated with the stealth pathogenesis characteristic of plant pathogens from the Mycosphaerellaceae. A large number of genes associated with secondary metabolite production, including homologs of toxin biosynthesis genes found in other Dothideomycete plant pathogens, were identified. CONCLUSIONS: The genome sequence of R. collo-cygni provides a framework for understanding the genetic basis of pathogenesis in this important emerging pathogen. The reduced complement of carbohydrate-degrading enzyme genes is likely to reflect a strategy to avoid detection by host defences during its prolonged asymptomatic growth. Of particular interest will be the analysis of R. collo-cygni gene expression during interactions with the host barley, to understand what triggers this fungus to switch from being a benign endophyte to an aggressive necrotroph.

Parents lend a helping hand to their offspring in plant defence.

Plants under attack by pathogens and pests can mount a range of inducible defences, encompassing both chemical and structural changes. Although few reports exist, it appears that plants responding to pathogen or herbivore attack, or chemical defence elicitors, may produce progeny that are better able to defend themselves against attack, compared with progeny from unthreatened or untreated plants. To date, all research on transgenerational effects of biotic stress has been conducted on dicotyledenous plants. We examined the possibility that resistance induced by application of chemical defence elicitors to the monocot plant barley, could be passed on to the progeny. Plants were treated with acibenzolar-S-methyl (ASM) or saccharin, and grain harvested at maturity. Germination was unaffected in seed collected from plants treated with saccharin, while germination was reduced significantly in seed collected from ASM-treated plants. The subsequent growth of the seedlings was not significantly different in any of the treatments. However, plants from parents treated with both ASM or saccharin exhibited significantly enhanced resistance to infection by Rhynchosporium commune, despite not being treated with elicitor themselves. These data hint at the possibility of producing disease-resistant plants by exposing parent plants to chemical elicitors.

Cultivar Effects on the Expression of Induced Resistance in Spring Barley.

The influence of host genotype on the expression of induced resistance was examined in several cultivars of spring barley (Hordeum vulgare). Induced resistance was activated using a combination of elicitors (acibenzolar- S-methyl, ß-aminobutyric acid, and cis-jasmone) shown in previous work to induce resistance effectively in barley. The barley cultivars examined were Cellar, Chalice, Decanter, Oxbridge, Tipple, Troon, and Westminster, which differed in their genetic resistance to two major pathogens of barley, Rhynchosporium secalis and Blumeria graminis f. sp. hordei. Controlled-environment studies showed that, although the elicitor combination reduced levels of R. secalis in all but one cultivar, the magnitude of the reduction differed among cultivars. Similar results were obtained in field experiments in 2007, 2008, and 2009, although there was inconsistency in cultivar effects between years, with the elicitor providing disease control in some cultivars in some years and not others. Use of the elicitor combination produced no significant effect on grain yield compared with untreated plants in most cases, although significant increases in grain yield were obtained with the elicitor treatment in two cultivars in 2007 and one cultivar in 2009. Analysis of the defense-related enzyme cinnamyl alcohol dehydrogenase in leaf samples from the field experiment in 2007 showed that activity of the enzyme was already high prior to elicitor application, although activity was increased further in one cultivar following elicitor treatment. It is possible, therefore, that these plants were already induced. Further work is required to confirm this and to determine whether prior induction has any bearing on the variable disease control obtained from elicitors in spring barley.

The quality of barley husk-caryopsis adhesion is not correlated with caryopsis cuticle permeability.

Adhesion of the barley husk to the underlying caryopsis requires the development of a cuticular cementing layer on the caryopsis surface. Differences in adhesion quality among genotypes have previously been correlated with cementing layer composition, which is thought to influence caryopsis cuticle permeability, the hypothesised mechanism of adhesion mediation. It is not yet known whether differences in adhesion quality among genotypes are determined by changes in caryopsis cuticle permeability. We examined changes in candidate cementing layer biosynthetic and regulatory genes to investigate the genetic mechanisms behind husk adhesion quality. We used both commercially relevant UK malting cultivars and older European lines to ensure phenotypic diversity in adhesion quality. An ethylene responsive transcription factor (NUD) is required for the development of the cementing layer. To examine correlations between gene expression, cementing layer permeability and husk adhesion quality we also treated cultivars with ethephon (2-chloroethylphosphonic acid) which breaks down to ethylene, and silver thiosulphate which inhibits ethylene reception, and measured caryopsis cuticle permeability. Differential adhesion qualities among genotypes are not determined by NUD expression during development of the cementing material alone, but could result from differences in biosynthetic gene expression during cementing layer development in response to longer-term NUD expression patterns. Altered caryopsis cuticle permeability does result in altered adhesion quality, but the correlation is not consistently positive or negative. Cuticle permeability is therefore not the mechanism that determines husk adhesion quality, but is likely a consequence of the required cuticular compositional changes that determine adhesion.

Diagnosis of Ramularia collo-cygni and Rhynchosporium spp. in Barley.

Ramularia leaf spot and Rhynchosporium leaf scald are two of the major diseases of barley crops in cooler temperate countries. The methods below are aimed at the identification and quantification of fungal DNA in leaf samples but can also be used for pathogen detection from seed or DNA extracted from environmental samplers. The methods describe in detail two individual quantitative PCR tests. The successful multiplexing of assays will lead to faster throughput of samples.

Control of foliar pathogens of spring barley using a combination of resistance elicitors.

The ability of the resistance elicitors acibenzolar-S-methyl (ASM), ß-aminobutyric acid (BABA), cis-jasmone (CJ), and a combination of the three products, to control infection of spring barley by Rhynchosporium commune was examined under glasshouse conditions. Significant control of R. commune was provided by ASM and CJ, but the largest reduction in infection was obtained with the combination of the three elicitors. This elicitor combination was found to up-regulate the expression of PR-1b, which is used as a molecular marker for systemic acquired resistance (SAR). However, the elicitor combination also down-regulated the expression of LOX2, a gene involved in the biosynthesis of jasmonic acid (JA). In field experiments over 3 consecutive years, the effects of the elicitor combination were influenced greatly by crop variety and by year. For example, the elicitor combination applied on its own provided significant control of powdery mildew (Blumeria graminis f.sp. hordei) and R. commune in 2009, whereas no control on either variety was observed in 2007. In contrast, treatments involving both the elicitor combination and fungicides provided disease control and yield increases which were equal to, and in some cases better than that provided by the best fungicide-only treatment. The prospects for the use of elicitor plus fungicide treatments to control foliar pathogens of spring barley in practice are discussed.

Inducibility studies with the arbuscular mycorrhizal fungus Glomus mosseae 3-phosphoglycerate kinase (PGK) gene promoter.

Arbuscular mycorrhizal (AM) fungi are a multifaceted group of mutualistic symbionts that are common to terrestrial ecosystems. The role of AM fungi within processes related to carbon (C) dynamics of the plant-root-soil system are of global significance. An understanding of the C metabolism of AM fungi within the symbiotic and asymbiotic stages of their lifecycle is a necessary pre-requisite to understanding the mechanisms of C movement. This investigation studies the regulation of the Glomus mosseae (Gm) 3-phosphoglycerate kinase ( PGK) gene promoter by different C sources. The suitability of studying the GmPGK promoter (P(GmPGK)) within Saccharomyces cerevisiae was confirmed by complementation of a S. cerevisiae pgk (-) mutant with an expression vector carrying the P(GmPGK) driving the expression of the S. cerevisiae PGK gene. The inducibility of the P(GmPGK) to different C sources was studied by creating a P(GmPGK)-luciferase fusion, which was introduced into a S. cerevisiae expression vector and used to transform S. cerevisiae. The P(GmPGK)-luciferase fusion was tested for expression within S. cerevisiae by RT-PCR analysis and was shown to be expressing the luciferase gene. C upshift studies were completed to evaluate the inducibility of this promoter by different C sources. The sources raffinose, glycerol and glucose significantly induced the P(GmPGK), whereas the sources galactose, succinate and sucrose had the lowest transcriptional responses. Semi-quantitative RT-PCR was used to establish gene expression patterns of GmPGK during spore germination; and up-regulation of the GmPGK gene was observed.