Determination of the LOQ in real-time PCR by receiver operating characteristic curve analysis: application to qPCR assays for Fusarium verticillioides and F. proliferatum

Real-time PCR (qPCR) is the principal technique for the quantification of pathogen biomass in host tissue, yet no generic methods exist for the determination of the limit of quantification (LOQ) and the limit of detection (LOD) in qPCR. We suggest using the Youden index in the context of the receiver operating characteristic (ROC) curve analysis for this purpose. The LOQ was defined as the amount of target DNA that maximizes the sum of sensitivity and specificity. The LOD was defined as the lowest amount of target DNA that was amplified with a false-negative rate below a given threshold. We applied this concept to qPCR assays for Fusarium verticillioides and Fusarium proliferatum DNA in maize kernels. Spiked matrix and field samples characterized by melting curve analysis of PCR products were used as the source of true positives and true negatives. On the basis of the analysis of sensitivity and specificity of the assays, we estimated the LOQ values as 0.11 pg of DNA for spiked matrix and 0.62 pg of DNA for field samples for F. verticillioides. The LOQ values for F. proliferatum were 0.03 pg for spiked matrix and 0.24 pg for field samples. The mean LOQ values correspond to approximately eight genomes for F. verticillioides and three genomes for F. proliferatum. We demonstrated that the ROC analysis concept, developed for qualitative diagnostics, can be used for the determination of performance parameters of quantitative PCR

Effect of Fungal Colonization of Wheat Grains with Fusarium spp. on Food Choice, Weight Gain and Mortality of Meal Beetle Larvae (Tenebrio molitor).

Species of Fusarium have significant agro-economical and human health-related impact by infecting diverse crop plants and synthesizing diverse mycotoxins. Here, we investigated interactions of grain-feeding Tenebrio molitor larvae with four grain-colonizing Fusarium species on wheat kernels. Since numerous metabolites produced by Fusarium spp. are toxic to insects, we tested the hypothesis that the insect senses and avoids Fusarium-colonized grains. We found that only kernels colonized with F. avenaceum or Beauveria bassiana (an insect-pathogenic fungal control) were avoided by the larvae as expected. Kernels colonized with F. proliferatum, F. poae or F. culmorum attracted T. molitor larvae significantly more than control kernels. The avoidance/preference correlated with larval feeding behaviors and weight gain. Interestingly, larvae that had consumed F. proliferatum- or F. poae-colonized kernels had similar survival rates as control. Larvae fed on F. culmorum-, F. avenaceum- or B. bassiana-colonized kernels had elevated mortality rates. HPLC analyses confirmed the following mycotoxins produced by the fungal strains on the kernels: fumonisins, enniatins and beauvericin by F. proliferatum, enniatins and beauvericin by F. poae, enniatins by F. avenaceum, and deoxynivalenol and zearalenone by F. culmorum. Our results indicate that T. molitor larvae have the ability to sense potential survival threats of kernels colonized with F. avenaceum or B. bassiana, but not with F. culmorum. Volatiles potentially along with gustatory cues produced by these fungi may represent survival threat signals for the larvae resulting in their avoidance. Although F. proliferatum or F. poae produced fumonisins, enniatins and beauvericin during kernel colonization, the larvae were able to use those kernels as diet without exhibiting increased mortality. Consumption of F. avenaceum-colonized kernels, however, increased larval mortality; these kernels had higher enniatin levels than F. proliferatum or F. poae-colonized ones suggesting that T. molitor can tolerate or metabolize those toxins.peerReviewe

Semi‐polar root exudates in natural grassland communities

Abstract In the rhizosphere, plants are exposed to a multitude of different biotic and abiotic factors, to which they respond by exuding a wide range of secondary root metabolites. So far, it has been unknown to which degree root exudate composition is species‐specific and is affected by land use, the local impact and local neighborhood under field conditions. In this study, root exudates of 10 common grassland species were analyzed, each five of forbs and grasses, in the German Biodiversity Exploratories using a combined phytometer and untargeted liquid chromatography‐mass spectrometry (LC‐MS) approach. Redundancy analysis and hierarchical clustering revealed a large set of semi‐polar metabolites common to all species in addition to species‐specific metabolites. Chemical richness and exudate composition revealed that forbs, such as Plantago lanceolata and Galium species, exuded more species‐specific metabolites than grasses. Grasses instead were primarily affected by environmental conditions. In both forbs and grasses, plant functional traits had only a minor impact on plant root exudation patterns. Overall, our results demonstrate the feasibility of obtaining and untargeted profiling of semi‐polar metabolites under field condition and allow a deeper view in the exudation of plants in a natural grassland community

Selective feeding behavior of larvae on fungi-colonized kernels.

<p>(A) <i>F. culmorum</i> and (B) <i>F. proliferatum</i> colonized kernels are shown. Larvae preferred feeding on the inner parts of the kernels infested with <i>F. culmorum</i> and on the outer parts of the kernels infested with <i>F. proliferatum</i>.</p

Reactions of larvae to fungi-colonized wheat kernels.

<p>(A) Boxplot of relative preference or avoidance reactions of larvae within 20 min towards fungi-colonized wheat kernels. N = 360. (B) Boxplot of cumulative weight gain of each 10 larvae per data point within 24 h on colonized wheat kernels with in total N = 1,800. (C) Relative survival rate of larvae on colonized kernels was determined within 15 days. Results are shown as mean values ± CI at 95% levels with N = 360. Significant differences are indicated by letters or by asterisks.</p

Identification of ABC Transporter Genes of <i>Fusarium graminearum</i> with Roles in Azole Tolerance and/or Virulence

<div><p><i>Fusarium graminearum</i> is a plant pathogen infecting several important cereals, resulting in substantial yield losses and mycotoxin contamination of the grain. Triazole fungicides are used to control diseases caused by this fungus on a worldwide scale. Our previous microarray study indicated that 15 ABC transporter genes were transcriptionally upregulated in response to tebuconazole treatment. Here, we deleted four ABC transporter genes in two genetic backgrounds of <i>F. graminearum</i> representing the DON (deoxynivalenol) and the NIV (nivalenol) trichothecene chemotypes. Deletion of <i>FgABC3</i> and <i>FgABC4</i> belonging to group I of ABC-G and to group V of ABC-C subfamilies of ABC transporters, respectively, considerably increased the sensitivity to the class I sterol biosynthesis inhibitors triazoles and fenarimol. Such effects were specific since they did not occur with any other fungicide class tested. Assessing the contribution of the four ABC transporters to virulence of <i>F. graminearum</i> revealed that, irrespective of their chemotypes, deletion mutants of <i>FgABC1</i> (ABC-C subfamily group V) and <i>FgABC3</i> were impeded in virulence on wheat, barley and maize. Phylogenetic context and analyses of mycotoxin production suggests that <i>FgABC3</i> may encode a transporter protecting the fungus from host-derived antifungal molecules. In contrast, <i>FgABC1</i> may encode a transporter responsible for the secretion of fungal secondary metabolites alleviating defence of the host. Our results show that ABC transporters play important and diverse roles in both fungicide resistance and pathogenesis of <i>F. graminearum</i>.</p></div

Survival of larvae feeding on mycelium of diverse <i>Fusarium</i> species grown on PDA.

<p>Survival curves of larvae reared on PDA plates covered with mycelium of diverse <i>Fusarium</i> species were significantly reduced when compared to controls (Cox regression model, χ² = 35.53; d.f. = 8; P<0.001; N = 560). In addition, survival curve on mycelium of the entomopathogen <i>B. bassiana</i> is shown as positive control.</p