Airborne Conidia Research Articles

Plant Growth Promotion and Plant Disease Suppression Induced by Bacillus amyloliquefaciens Strain GD4a.

Botrytis cinerea, the causative agent of gray mold disease (GMD), invades plants to obtain nutrients and disseminates through airborne conidia in nature. Bacillus amyloliquefaciens strain GD4a, a beneficial bacterium isolated from switchgrass, shows great potential in managing GMD in plants. However, the precise mechanism by which GD4a confers benefits to plants remains elusive. In this study, an A. thaliana-B. cinerea-B. amyloliquefaciens multiple-scale interaction model was used to explore how beneficial bacteria play essential roles in plant growth promotion, plant pathogen suppression, and plant immunity boosting. Arabidopsis Col-0 wild-type plants served as the testing ground to assess GD4a's efficacy. Additionally, bacterial enzyme activity and targeted metabolite tests were conducted to validate GD4a's potential for enhancing plant growth and suppressing plant pathogens and diseases. GD4a was subjected to co-incubation with various bacterial, fungal, and oomycete pathogens to evaluate its antagonistic effectiveness in vitro. In vivo pathogen inoculation assays were also carried out to investigate GD4a's role in regulating host plant immunity. Bacterial extracellular exudate (BEE) was extracted, purified, and subjected to untargeted metabolomics analysis. Benzocaine (BEN) from the untargeted metabolomics analysis was selected for further study of its function and related mechanisms in enhancing plant immunity through plant mutant analysis and qRT-PCR analysis. Finally, a comprehensive model was formulated to summarize the potential benefits of applying GD4a in agricultural systems. Our study demonstrates the efficacy of GD4a, isolated from switchgrass, in enhancing plant growth, suppressing plant pathogens and diseases, and bolstering host plant immunity. Importantly, GD4a produces a functional bacterial extracellular exudate (BEE) that significantly disrupts the pathogenicity of B. cinerea by inhibiting fungal conidium germination and hypha formation. Additionally, our study identifies benzocaine (BEN) as a novel small molecule that triggers basal defense, ISR, and SAR responses in Arabidopsis plants. Bacillus amyloliquefaciens strain GD4a can effectively promote plant growth, suppress plant disease, and boost plant immunity through functional BEE production and diverse gene expression.

Self-Cleaning Biomimetic Surfaces—The Effect of Microstructure and Hydrophobicity on Conidia Repellence

Modification of surface structure for the promotion of food safety and health protection is a technology of interest among many industries. With this study, we aimed specifically to develop a tenable solution for the fabrication of self-cleaning biomimetic surface structures for agricultural applications such as post-harvest packing materials and greenhouse cover screens. Phytopathogenic fungi such as Botrytis cinerea are a major concern for agricultural systems. These molds are spread by airborne conidia that contaminate surfaces and infect plants and fresh produce, causing significant losses. The research examined the adhesive role of microstructures of natural and synthetic surfaces and assessed the feasibility of structured biomimetic surfaces to easily wash off fungal conidia. Soft lithography was used to create polydimethylsiloxane (PDMS) replications of Solanum lycopersicum (tomato) and Colocasia esculenta (elephant ear) leaves. Conidia of B. cinerea were applied to natural surfaces for a washing procedure and the ratios between applied and remaining conidia were compared using microscopy imaging. The obtained results confirmed the hypothesis that the dust-repellent C. esculenta leaves have a higher conidia-repellency compared to tomato leaves which are known for their high sensitivities to phytopathogenic molds. This study found that microstructure replication does not mimic conidia repellency found in nature and that conidia repellency is affected by a mix of parameters, including microstructure and hydrophobicity. To examine the effect of hydrophobicity, the study included measurements and analyses of apparent contact angles of natural and synthetic surfaces including activated (hydrophilic) surfaces. No correlation was found between the surface apparent contact angle and conidia repellency ability, demonstrating variation in washing capability correlated to microstructure and hydrophobicity. It was also found that a microscale sub-surface (tomato trichromes) had a high conidia-repelling capability, demonstrating an important role of non-superhydrophobic microstructures.

Mitochondrial Reactive Oxygen Species Enhance Alveolar Macrophage Activity against Aspergillus fumigatus but Are Dispensable for Host Protection.

ABSTRACTAspergillus fumigatus is the most common cause of mold pneumonia worldwide, and a significant cause of infectious morbidity and mortality in immunocompromised individuals. The oxidative burst, which generates reactive oxidative species (ROS), plays a pivotal role in host defense against aspergillosis and induces regulated cell death in Aspergillus conidia, the infectious propagules. Beyond the well-established role of NADP (NADPH) oxidase in ROS generation by neutrophils and other innate effector cells, mitochondria represent a major ROS production site in many cell types, though it is unclear whether mitochondrial ROS (mtROS) contribute to antifungal activity in the lung. Following A. fumigatus infection, we observed that innate effector cells, including alveolar macrophages (AMs), monocyte-derived dendritic cells (Mo-DCS), and neutrophils, generated mtROS, primarily in fungus-infected cells. To examine the functional role of mtROS, specifically the H2O2 component, in pulmonary host defense against A. fumigatus, we infected transgenic mice that expressed a mitochondrion-targeted catalase. Using a reporter of fungal viability during interactions with leukocytes, mitochondrial H2O2 (mtH2O2) was essential for optimal AM, but not for neutrophil phagocytic and conidiacidal activity in the lung. Catalase-mediated mtH2O2 neutralization did not lead to invasive aspergillosis in otherwise immunocompetent mice and did not shorten survival in mice that lack NADPH oxidase function. Collectively, these studies indicate that mtROS-associated defects in AM antifungal activity can be functionally compensated by the action of NADPH oxidase and by nonoxidative effector mechanisms during murine A. fumigatus lung infection.IMPORTANCE Aspergillus fumigatus is a fungal pathogen that causes invasive disease in humans with defects in immune function. Airborne conidia, the infectious propagules, are ubiquitous and inhaled on a daily basis. In the respiratory tree, conidia are killed by the coordinated actions of phagocytes, including alveolar macrophages, neutrophils, and monocyte-derived dendritic cells. The oxidative burst represents a central killing mechanism and relies on the assembly of the NADPH oxidase complex on the phagosomal membrane. However, NADPH oxidase-deficient leukocytes have significant residual fungicidal activity in vivo, indicating the presence of alternative effector mechanisms. Here, we report that murine innate immune cells produce mitochondrial reactive oxygen species (mtROS) in response to fungal interactions. Neutralizing the mtROS constituent hydrogen peroxide (H2O2) via a catalase expressed in mitochondria of innate immune cells substantially diminished fungicidal properties of alveolar macrophages, but not of other innate immune cells. These data indicate that mtH2O2 represent a novel AM killing mechanism against Aspergillus conidia. mtH2O2 neutralization is compensated by other killing mechanisms in the lung, demonstrating functional redundancy at the level of host defense in the respiratory tree. These findings have important implications for the development of host-directed therapies against invasive aspergillosis in susceptible patient populations.

The Pathogenesis of Aspergillus fumigatus, Host Defense Mechanisms, and the Development of AFMP4 Antigen as a Vaccine

Aspergillus fumigatus is one of the ubiquitous fungi with airborne conidia, which accounts for most aspergillosis cases. In immunocompetent hosts, the inhaled conidia are rapidly eliminated. However, immunocompromised or immunodeficient hosts are particularly vulnerable to most Aspergillus infections and invasive aspergillosis (IA), with mortality from 50% to 95%. Despite the improvement of antifungal drugs over the last few decades, the therapeutic effect for IA patients is still limited and does not provide significant survival benefits. The drawbacks of antifungal drugs such as side effects, antifungal drug resistance, and the high cost of antifungal drugs highlight the importance of finding novel therapeutic and preventive approaches to fight against IA. In this article, we systemically addressed the pathogenic mechanisms, defense mechanisms against A. fumigatus, the immune response, molecular aspects of host evasion, and vaccines’ current development against aspergillosis, particularly those based on AFMP4 protein, which might be a promising antigen for the development of anti-A. fumigatus vaccines.

Identification and evaluation of the main risk periods of Botrytis cinerea infection on grapevine based on phenology, weather conditions and airborne conidia

AbstractIn the present study, a new method for a decision-support system for fungicide administration against the pathogen Botrytis cinerea in vineyards was developed based on Integrated Pest Management principles which identified an infection risk before the appearance of disease symptoms. The proposed method is based on the combination of (i) the phenological observations of the main susceptible stages to infection, (ii) the airborne spores monitoring, (iii) the forecasting of the suitable meteorological conditions for B. cinerea spore germination during the subsequent 4–6 days after the spore detection. Aerobiological, phenological and meteorological analyses were carried out using data from 2008 to 2015 in a vineyard of Northwestern Spain. Aerobiological spore data were obtained using a Lanzoni VPPS-2000 pollen-spore trap. Phenological observations were conducted on 22 plants of Treixadura cultivar following the BBCH (Biologische Bundesanstalt für Land und Forstwirtschaft, Bundessortenamt und CHemische Industrie) scale. The Magarey generic fungal model was applied for the identification of the main meteorological suitable periods for infection within the susceptible phenological stages of flowering and ripening of berries. Our results showed that climatic conditions favoured fungal development during flowering, although a higher incidence of B. cinerea infection risk-periods occurred during the prior-to-harvest stage of ripening of berries, the most susceptible phenological stage to B. cinerea infection obtained by the proposed methodology. This approach enables more precise targeting in pesticide spraying and reduction in pesticide application from 4–5 to 2–3 times per year at our commercial study. It also illustrates the real-world benefits of integrated disease risk modelling.

Dual Transcriptome and Metabolic Analysis of Vitis vinifera cv. Pinot Noir Berry and Botrytis cinerea During Quiescence and Egressed Infection

Botrytis cinerea is an important necrotroph in vineyards. Primary infections are mostly initiated by airborne conidia from overwintered sources around bloom, then the fungus remains quiescent from bloom till maturity and egresses at ripeness. We previously described in detail the process of flower infection and quiescence initiation. Here, we complete the characterization studying the cross-talk between the plant and the fungus during pathogen quiescence and egression by an integrated transcriptomic and metabolic analysis of the host and the pathogen. Flowers from fruiting cuttings of the cv. Pinot Noir were inoculated with a GFP-labeled strain of B. cinerea at full cap-off stage, and molecular analyses were carried out at 4 weeks post inoculation (wpi, fungal quiescent state) and at 12 wpi (fungal pre-egression and egression states). The expressed fungal transcriptome highlighted that the fungus remodels its cell wall to evade plant chitinases besides undergoing basal metabolic activities. Berries responded by differentially regulating genes encoding for different PR proteins and genes involved in monolignol, flavonoid, and stilbenoid biosynthesis pathways. At 12 wpi, the transcriptome of B. cinerea in the pre-egressed samples showed that virulence-related genes were expressed, suggesting infection process was initiated. The egressed B. cinerea expressed almost all virulence and growth related genes that enabled the pathogen to colonize the berries. In response to egression, ripe berries reprogrammed different defense responses, though futile. Examples are activation of membrane localized kinases, stilbene synthases, and other PR proteins related to SA and JA-mediated responses. Our results indicated that hard-green berries defense program was capable to hamper B. cinerea growth. However, ripening associated fruit cell wall self-disassembly together with high humidity created the opportunity for the fungus to egress and cause bunch rot.

Characterization of favorable weather conditions with powdery mildew disease incidence on pumpkin

Weather variables were characterized for the powdery mildew disease caused by Sphaerotheca fuliginea on pumpkin crop. The relationship between airborne conidia and incidence of powdery mildew on pumpkin was studied. Disease incidence data was recorded on weekly basis and correlated with prevailing environmental conditions. All the environmental factors showed positive relationship with disease incidence except wind speed that exhibited negative correlation. Understanding of favorable environment is helpful to use appropriate management approaches.

Improved classification accuracy of powdery mildew infection levels of wine grapes by spatial-spectral analysis of hyperspectral images

BackgroundHyperspectral imaging is an emerging means of assessing plant vitality, stress parameters, nutrition status, and diseases. Extraction of target values from the high-dimensional datasets either relies on pixel-wise processing of the full spectral information, appropriate selection of individual bands, or calculation of spectral indices. Limitations of such approaches are reduced classification accuracy, reduced robustness due to spatial variation of the spectral information across the surface of the objects measured as well as a loss of information intrinsic to band selection and use of spectral indices. In this paper we present an improved spatial-spectral segmentation approach for the analysis of hyperspectral imaging data and its application for the prediction of powdery mildew infection levels (disease severity) of intact Chardonnay grape bunches shortly before veraison.ResultsInstead of calculating texture features (spatial features) for the huge number of spectral bands independently, dimensionality reduction by means of Linear Discriminant Analysis (LDA) was applied first to derive a few descriptive image bands. Subsequent classification was based on modified Random Forest classifiers and selective extraction of texture parameters from the integral image representation of the image bands generated. Dimensionality reduction, integral images, and the selective feature extraction led to improved classification accuracies of up to 0.998pm 0.003 for detached berries used as a reference sample (training dataset). Our approach was validated by predicting infection levels for a sample of 30 intact bunches. Classification accuracy improved with the number of decision trees of the Random Forest classifier. These results corresponded with qPCR results. An accuracy of 0.87 was achieved in classification of healthy, infected, and severely diseased bunches. However, discrimination between visually healthy and infected bunches proved to be challenging for a few samples, perhaps due to colonized berries or sparse mycelia hidden within the bunch or airborne conidia on the berries that were detected by qPCR.ConclusionsAn advanced approach to hyperspectral image classification based on combined spatial and spectral image features, potentially applicable to many available hyperspectral sensor technologies, has been developed and validated to improve the detection of powdery mildew infection levels of Chardonnay grape bunches. The spatial-spectral approach improved especially the detection of light infection levels compared with pixel-wise spectral data analysis. This approach is expected to improve the speed and accuracy of disease detection once the thresholds for fungal biomass detected by hyperspectral imaging are established; it can also facilitate monitoring in plant phenotyping of grapevine and additional crops.

Fungicide Efficacy Against Botrytis cinerea at Different Positions on Grape Shoots

Fungicide efficacy at different positions on grape shoots was determined on vinelets inoculated with Botrytis cinerea. To ensure proper fungicide coverage, vinelets (table grape cultivar Dauphine, wine grape cultivar Merlot noir), prepared from cuttings, were sprayed in a spray chamber at the recommended dosages with iprodione, pyrimethanil, cyprodinil/fludioxonil and fenhexamid. Vinelets for the control treatment were left unsprayed. After 24 h the vinelets were dusted with dry, airborne conidia in a settling ower to reduce the sporadic occurrence of the pathogen on shoots and incubated for 24 h at high relative humidity (±93 % ) to promote infection. Fungicide efficacy was determined by observing intact vinelets for symptom expression at nodes, internodes, leaf blades, petioles and inflorescences, and by determining surface colonisation (shoots left unsterile) and penetration (surfacedisinfested shoots) by isolation studies. Symptoms of B. cinerea decay did not develop on sprayed and unsprayed vinelets that were kept in dry chambers during the 2-week observation period. The pathogen, however, developed from the isolated parts. On the unsprayed vinelets for both cultivars B. cinerea incidences recorded on two media and the two sterility regimes were significantly higher on leaf blades than on the petioles, shoot pieces and inflorescences. The isolation studies showed that the different fungicides were highly and nearly equally efficient in reducing superficial B. cinerea and latent infection at the various positions. In the case of leaf blades B. cinerea incidence was significantly reduced by each fungicide on both cultivars. This was not the case for the other parts, which yielded B. cinerea at low incidences under the two sterility regimes. The study showed that the fungicides used in this study, if applied properly to shoots at the pre-bloom stage, should effectively reduce the amount of B. cinerea in leaves and completely prevent the infection of nodes, internodes and inflorescences.

Epidemiology of Botrytis cinerea on Grape: Wound Infection by Dry, Airborne Conidia

This study describes the infection of fresh wounds on berries exposed to freshly deposited airborne Botrytis cinerea conidia, and on berries carrying previously deposited conidia and germlings (latent infections). Grapes at bunch closure and at the mature (harvest) stage, as well as mature, cold-stored grapes, were used. The grapes were dusted with dry conidia in a settling tower. The inocula were subjected to conditions commonly encountered by the pathogen in grape bunches: dry conidia on dry berries under dry conditions, dry conidia on dry berries under high relative humidity, and dry conidia exposed to a film of water on the berry surface. The mean number of wounds that yielded B. cinerea decay 14 days after inoculation was calculated. Fluorescence microscopy revealed that conidia occurred evenly as single cells on the grape berry surface and seldom landed at the wound periphery. They remained dormant on dry berries, but germinated freely on the unbroken skin and at the wound periphery on moist and wet berries. In the case of berries inoculated at bunch closure and harvest stages, wounds were not infected by conidia deposited on berries four days prior to wounding. This finding indicated that, following adhesion and the first stages of growth, the pathogen did not survive for extended periods on surfaces of immature and mature grape berries. Freshly deposited dry conidia were needed to infect the wounds. The freshly deposited conidia furthermore needed free water, and not high humidity or wound exudates, to infect the fresh wounds. Proportions of wounds infected were extremely low. According to these findings, this mode of infection should not contribute to a gradual build-up of secondary inoculum and to B. cinerea epiphytotics in the vineyard. The previously and freshly deposited conidia both infected wounds made on cold-stored mature berries. Of the two inocula, freshly deposited conidia were more successful in causing wound infection. Therefore, in the event of wounding, postharvest decay would be caused primarily by conidia occurring on the grape berry surface, and not by mycelia in the berry skin.

Fitness on Grape Berries of Botrytis cinerea Isolates Belonging to Different Dicarboximide Sensitivity Classes

Seasonal fluctuations in the frequency of dicarboximide-resistant Botrytis cinerea isolates in Western Cape vineyards suggest a reduced fitness of these isolates. In this study fitness of sensitive, ultra-low-level and low-level dicarboximide-resistant isolates of B. cinerea were compared on grape berries. Conidia were dispersed as single cells on berry surfaces from airborne inoculum in a settling tower or deposited as clusters in water droplets. Investigations were conducted at two potential infection courts on dicarboximide treated and untreated berries, namely the intact cuticle and wounds. Surface sterilisation, isolation and freezing techniques were used to determine surface colonisation and penetration by airborne inoculum. Decay incidence, severity and sporulation incidence were determined following inoculation with conidial clusters. The different tests indicated that germlings of dicarboximide-sensitive and -resistant isolates had similar surface-colonising abilities of dicarboximide-free berries. However, sensitive strains penetrated significantly more often. Fitness decreased with an increase in the level of dicarboximide resistance. Iprodione caused a drastic disturbance in the ratio of different dicarboximide sensitivity classes that occupied the berry surface and allowed the development of germlings of predominantly resistant isolates, but with few successful infections. Significantly higher levels of infection and proliferation of dicarboximide-resistant isolates on sprayed or unsprayed berries were facilitated by wounding or the termination of host resistance (freezing). According to these findings, these modes of infection should not contribute to a gradual build-up of inoculum of either dicarboximide-sensitive or -resistant isolates. Trends by airborne conidia described here suggest that another primary infection event in the vineyard, most likely floral infection and subsequent debris colonisation, should largely regulate the dynamics between dicarboximide-sensitive and -resistant isolates in B. cinerea populations on grapevine.

Airborne inoculum of Fusarium oxysporum f. sp. cucumerinum

Fusarium oxysporum f. sp. cucumerinum is the fungal pathogen responsible for Fusarium vascular wilt of cucumber. In Australia, disease management in soilless greenhouse cucumbers is limited by the understanding of the disease cycle, in particular the risk associated with infection of pruning wounds by airborne propagules. Aerial dissemination of the pathogen in response to temperature and relative humidity was investigated using a specific and sensitive quantitative real-time PCR assay. Both macroconidia and microconidia were identified as airborne propagules and a potential relationship between fluctuation in relative humidity and spore release was found, however, it appears that crop disturbance may also influence conidia liberation. Experimental inoculation of stem wounds with conidia however, failed to establish infections. These results suggest that aerial inoculum propagates and disseminates the pathogen, however airborne spores are deposited on the substrate surface and infection occurs primarily through the root. We conclude that while airborne conidia are a risk to infection of cucumber plants in soilless greenhouse crops, resistant rootstocks are likely to provide good protection against this pathogen.

Spatial Pattern of Strawberry Powdery Mildew (Podosphaera aphanis) and Airborne Inoculum.

The relationship between strawberry powdery mildew and airborne conidium concentration (ACC) of Podosphaera aphanis was studied using data collected from 2006 to 2009 in 15 fields, and spatial pattern was described using 2 years of airborne inoculum and disease incidence data collected in fields planted with the June-bearing strawberry (Fragaria × ananassa) cultivar Jewel. Disease incidence, expressed as the proportion of diseased leaflets, and ACC were monitored in fields divided into 3 × 8 grids containing 24 100 m2 quadrats. Variance-to-mean ratio, index of dispersion, negative binomial distribution, Poisson distribution, and binomial and beta-binomial distributions were used to characterize the level of spatial heterogeneity. The relationship between percent leaf area diseased and daily ACC was linear, while the relationship between ACC and disease incidence followed an exponential growth curve. The V/M ratios were significantly greater than 1 for 100 and 96% of the sampling dates for ACC sampled at 0.35 m from the ground (ACC0.35m) and for ACC sampled at 1.0 m from the ground (ACC1.0m), respectively. For disease incidence, the index of dispersion D was significantly greater than 1 for 79% of the sampling dates. The negative binomial distribution fitted 86% of the data sets for both ACC1.0m and ACC0.35m. For disease incidence data, the beta-binomial distribution provided a good fit of 75% of the data sets. Taylor's power law indicated that, for ACC at both sampling heights, heterogeneity increased with increasing mean ACC, whereas the binary form of the power law suggested that heterogeneity was not dependent on the mean for disease incidence. When the spatial location of each sampling location was taken into account, Spatial Analysis by Distance Indices showed low aggregation indices for both ACCs and disease incidence, and weak association between ACC and disease incidence. Based on these analyses, it was found that the distribution of strawberry powdery mildew was weakly aggregated. Although a higher level of heterogeneity was observed for airborne inoculum, the heterogeneity was low with no distinct foci, suggesting that epidemics are induced by well-distributed inoculum. This low level of heterogeneity allows mean airborne inoculum concentration to be estimated using only one sampler per field with an overall accuracy of at least 0.841. The results obtained in this study could be used to develop a sampling scheme that will improve strawberry powdery mildew risk estimation.

Transcriptome Profiling of Botrytis cinerea Conidial Germination Reveals Upregulation of Infection-Related Genes during the Prepenetration Stage

Botrytis cinerea causes gray mold on a great number of host plants. Infection is initiated by airborne conidia that invade the host tissue, often by penetration of intact epidermal cells. To mimic the surface properties of natural plant surfaces, conidia were incubated on apple wax-coated surfaces, resulting in rapid germination and appressorium formation. Global changes in gene expression were analyzed by microarray hybridization between conidia incubated for 0 h (dormant), 1 h (pregermination), 2.5 h (postgermination), 4 h (appressoria), and 15 h (early mycelium). Considerable changes were observed, in particular between 0 h and 1 h. Genes induced during germination were enriched in those genes encoding secreted proteins, including lytic enzymes. Comparison of wild-type and a nonpathogenic MAP kinase mutant (bmp1) revealed marked differences in germination-related gene expression, in particular related to secretory proteins. Using promoter-GFP reporter strains, we detected a strictly germination-specific expression pattern of a putative chitin deacetylase gene (cda1). In contrast, a cutinase gene (cutB) was found to be expressed only in the presence of plant lipids, in a developmentally less stringent pattern. We also identified a coregulated gene cluster possibly involved in secondary metabolite synthesis which was found to be controlled by a transcription factor also encoded in this cluster. Our data demonstrate that early conidial development in B. cinerea is accompanied by rapid shifts in gene expression that prepare the fungus for germ tube outgrowth and host cell invasion.

A sticky situation: extracellular DNA shapes Aspergillus fumigatus biofilms

Multicellular stages play an important role for microbial communities as well as during infections. For one of the major fungal pathogens, the environmental mold Aspergillus fumigatus, an interesting and relevant morphotype beyond the conidial and hyphal stages has gained increasing attention: the multicellular biofilm. Such hyphal communities were first described for A. fumigatus in 2007 (Mowat et al., 2007) and are nowadays recognized to be relevant in the clinic (Muller et al., 2011). A. fumigatus is environmentally omnipresent and its airborne conidia are constantly inhaled to become eliminated by the functional host immune system. In case of immune response failure, these infectious propagules germinate and invade the infected tissue to cause forms of aspergillosis (Dagenais and Keller, 2009). Treatment options for this disease are still inadequate, based on the limited number of antifungal drugs and their side effects. Resistance of A. fumigatus to antifungal treatment is influenced by several factors, among them biofilm formation (Seidler et al., 2008). In these structures, a dense network of hyphae becomes embedded in an extracellular matrix (ECM) that is mainly composed of polysaccharides. The ECM of such biofilms grown in vitro further contains melanin, proteins like hydrophobins or antigens, polyols, and monosaccharides (Beauvais et al., 2007). A. fumigatus biofilms are also formed in vivo during invasive aspergillosis or aspergilloma and have been characterized to slightly differ from their in vitro counterparts with respect to ECM composition (Loussert et al., 2010). Recent evidence has identified extracellular DNA (eDNA) as important component of A. fumigatus biofilms, either derived from fungal autolysis (Rajendran et al., 2013) or, as elegantly demonstrated in a recent publication by Brakhage, Hillmann, and colleagues, when externally supplied (Shopova et al., 2013). Probably supported by a high affinity of nucleic acids to the cell wall of A. fumigatus (Jochl et al., 2009) that is based on electrostatic interactions, extrinsic eDNA promotes adhesion of the conidia in the initial phase of biofilm formation, triggers polysaccharide formation and becomes incorporated in the biofilm, thereby shaping its overall structure. Accordingly, DNA contributes to the structural integrity of mature A. fumigatus biofilms, a finding that has been described for several bacterial biofilm formers before (Flemming and Wingender, 2010) and that highlights the structural conservation of microbial biofilms. Considering that A. fumigatus commonly colonizes patients suffering from cystic fibrosis and that the mucus of such patients contains extrinsic DNA at high concentrations that may stem from of neutrophil extracellular traps (NETs) (Brinkmann et al., 2004; Bruns et al., 2010), which are released by a special form of cell death termed NETosis (Remijsen et al., 2011; Almyroudis et al., 2013), from necrotic tissue, or from competing microorganisms, the formation of biofilms is of special significance in this particular host group. In the light of the clinical as well as industrial relevance of Aspergillus biofilms (Ramage et al., 2011), the newly presented data and insights not only sharpen our image of this multicellular stage within the Aspergillus lifestyle but also provide perspective for improvement and new avenues in antifungal therapy or biotechnological applications.

State of the art diagnostic of mold diseases: a practical guide for clinicians

The epidemiology of fungal diseases changed, and molds have been increasingly associated with high mortality in severe immunocompromised patients. Invasive mold diseases may originate from the airborne conidia through inhalation or inoculation in skin fissures associated with indwelling catheters, wounds, burns, or onychomycosis. The diagnosis and treatment of fungal diseases is problematic and raises considerable challenges. Diagnosis of invasive mold diseases includes several methodologies, of which the most commonly used are the cultural methods, antigen testing, nucleic acid detection, and radiological imaging. Galactomannan and (1 → 3)-β-D-glucan detection significantly improved mold diagnosis in the last decade. Several molecular strategies have been proposed over the years but no consensus was achieved for standardized protocols or cut-off values. Recently, the first commercially available molecular assay for detection of Aspergillus was tested and the results were highly reproducible. In addition, blood cultures may also be helpful for invasive aspergillosis by following a novel procedure for the recovery of Aspergillus spp. from blood cultures. The association of distinct diagnostic methods, particularly molecular tests, galactomannan, and/or (1 → 3)-β-D-glucan detection, may provide earlier and more sensitive diagnosis of mold diseases and be indicative for early antifungal treatment. Accurate routine use of diagnostic tests can be cost-effective for laboratories and be of great value to patients.

Innate Immunity and the Role of Epithelial Barrier During Aspergillus fumigatus Infection

Fungi are the most important eukaryotic infective agents in Europe which largely overpass parasite infections. Total number of people dying of fungal infection is increasing and this trend is likely to continue due to the increase in immunosuppressive treatments. The opportunistic pathogen Aspergillus fumigatus (Af) is a saprophytic filamentous fungus that can cause invasive pulmonary diseases in immuno-compromised hosts. In veterinary medicine aspergillosis is also a recurrent problem since it infects various species, birds are particularly susceptible. It propagates through airborne conidia (spores), which are inhaled into the small airways where they may germinate and initiate an infection. The host epithelium has permanent contact with the environment and a multitude of diverse microorganisms, resulting in a network of the host’s defense mechanisms. Pathogens use various strategies to invade epithelial barriers, to exploit eukaryotic host function to their own benefit and disseminate throughout the host using the epithelium as a reservoir. The current revue will discuss the ways how epithelial and innate immunity cells can contlol Af infection. We will focus on Af strategies for the host’s invasion, antifungal innate immune response and antimicrobial activities of the respiratory epithelial cells.

A new risk indicator for botrytis leaf blight of onion caused by Botrytis squamosa based on infection efficiency of airborne inoculum

Botrytis leaf blight (BLB) caused by Botrytis squamosa is a major leaf disease of onion. Various forecasting systems have been developed to help growers manage the disease. To improve forecasting reliability, the influence of temperature and wetness duration on B. squamosa infection was quantified by inoculating onion leaves with a conidial suspension and incubating them under various combinations of temperature (10–30°C) and leaf wetness duration (0–84 h). Infection was measured as the number of lesions per cm2 of leaf and converted to the proportion of maximum infection (PMI). Regardless of leaf wetness duration, only a few lesions developed at 30°C and the number of lesions increased as the temperature rose from 10 to 20°C but decreased at 25°C. Between 10 and 25°C the number of lesions per cm2 of leaf area increased gradually with increasing leaf wetness duration from 12 to 72 h. Relative infection was modelled as a function of both temperature and wetness duration using a modified version of the Weibull equation, which provided a precise description of the response of B. squamosa (R2 = 0·88). To facilitate field validation, receiving operating characteristic curve analysis was performed to determine the accuracy of various sets of criteria for establishing the length of an infection event based on field weather data. The total number of leaf wetness and RH >90% hours over a 72 h period was the best criterion, regardless of the wetness interruption pattern (sensitivity = 90·91, specificity = 84·62, area under the receiving operating curve = 0·878). The model describing the relationship of PMI to temperature and leaf wetness duration, and field observations on airborne conidium concentration (ACC) were used to calculate the risk of infection (RIBLB) as RIBLB = PMI × ACC. In 2009 and 2010, this risk index was compared to the observed rate of BLB progress (RateBLB+5 days) during the following 5 days. There was a linear relationship between RIBLB and RateBLB+5 days indicating that this new risk indicator was reliable for predicting the risk of BLB development. These findings will help to improve the timing of fungicide applications for BLB management.

Polymerase Chain Reaction Assay for Rapid, Sensitive Detection, and Identification of Colletotrichum gloeosporioides causing Greater Yam Anthracnose

Anthracnose caused by Colletotrichum gloeosporioides is an economically important disease which affects greater yam (Dioscorea alata L.) worldwide. Apart from airborne conidia, the pathogen propagules surviving in soil and planting material are the major sources of inoculum. A nested PCR assay has been developed for specific detection of C. gloeosporioides in soil and planting material. In conventional (single-round) PCR, the limit of detection was 20 pg, whereas in nested PCR the detection limit increased to 0.2 pg of DNA. The primers designed were found to be highly specific and could be used for accurate identification of the pathogen up to species level. The protocol was standardized for detection of the pathogen in artificially and naturally infected field samples.

Comparison of monitoring based indicators for initiating fungicide spray programs to control Botrytis leaf blight of onion

Botrytis leaf blight (BLB) of onion is a well-documented disease and several management tools are available. However, achieving good control with minimum use of fungicides is time consuming and requires dedication on the part of growers. In this study, three indicators for the initiation of fungicide spray programs were compared. The study was conducted in 2008, 2009 and 2010 at commercial onion farms located in the muck soil area southwest of Montreal. The indicators evaluated were: 1) first airborne Botrytis squamosa conidia detected; 2) cumulative airborne conidium concentration (ACC) of 15 conidia m −3; 3) first lesion caused by B. squamosa detected. The interval between subsequent fungicide applications was determined by taking into account sporulation potential, airborne conidium concentration and lesion density. Reliability of the indicators was evaluated on the basis of observed maximum disease level, area under the disease progress curve and rate of disease progress. Initiation of the fungicide spray program when the first airborne B. squamosa conidia were detected resulted in the lowest values for maximum disease level and area under the disease progress curve, followed by initiation when a cumulative concentration of 15 conidia m −3 was reached, and by initiation when the first lesion was detected. However, there was no significant difference in rate of disease progress, calculated using data from fields where spray programs were initiated based on the different indicators. The critical disease level for curative fungicide applications (ten lesions per leaf) was not reached when the spray program was initiated upon detection of the first conidia; was reached a few days after bulb initiation for a spray program initiated at a cumulative ACC of 15 conidia m −3; and was reached prior to bulb initiation for the spray program initiated when the first lesion was detected. These results suggest that to avoid yield reduction, BLB should be managed such that the disease does not reach the exponential phase. This was achieved by initiating the fungicide spray program based on first conidia detected.