Measurement of the effectiveness of Clonostachys rosea in reducing Fusarium biomass on wheat straw.

The survival and growth of plant pathogens on crop residues are key factors facilitating the dynamics of crop diseases. Spores (e.g., perithecia, and chlamydospores) and mycelium of pathogenic fungi overwinter on harvest residues, such as straw, and serve as initial inoculum infecting crops in the next growing season. Therefore, targeting overwintering fungi is essential to attaining effective disease control. Beneficial microorganisms offer advantages in controlling pathogens through their ability to colonize and exploit different environmental niches. In this study, we applied qPCR assays to explore the biocontrol performance of locally isolated strains of Clonostachys against various Fusarium pathogens. We proved that prior colonization of wheat straw by Fusarium spp. can be effectively reduced by Clonostachys rosea. We demonstrated that the efficiency of C. rosea to reduce Fusarium inoculum appears to remain at a similar level for most studied strains regardless of the target pathogen and the level of colonization of substrates by pathogens. Efficient performance of local C. rosea strains identifies possible targets for future strategies to control Fusarium diseases in cereals. Our study also highlights the challenge in sequence-based determination of C. rosea, which is crucial for the efficient selection of beneficial strains for biocontrol purposes.

Whole-genome single nucleotide polymorphism analysis for typing the pandemic pathogen Fusarium graminearum sensu stricto.

Recent improvements in microbiology and molecular epidemiology were largely stimulated by whole- genome sequencing (WGS), which provides an unprecedented resolution in discriminating highly related genetic backgrounds. WGS is becoming the method of choice in epidemiology of fungal diseases, but its application is still in a pioneer stage, mainly due to the limited number of available genomes. Fungal pathogens often belong to complexes composed of numerous cryptic species. Detecting cryptic diversity is fundamental to understand the dynamics and the evolutionary relationships underlying disease outbreaks. In this study, we explore the value of whole-genome SNP analyses in identification of the pandemic pathogen Fusarium graminearum sensu stricto (F.g.). This species is responsible for cereal diseases and negatively impacts grain production worldwide. The fungus belongs to the monophyletic fungal complex referred to as F. graminearum species complex including at least sixteen cryptic species, a few among them may be involved in cereal diseases in certain agricultural areas. We analyzed WGS data from a collection of 99 F.g. strains and 33 strains representing all known cryptic species belonging to the FGSC complex. As a first step, we performed a phylogenomic analysis to reveal species-specific clustering. A RAxML maximum likelihood tree grouped all analyzed strains of F.g. into a single clade, supporting the clustering-based identification approach. Although, phylogenetic reconstructions are essential in detecting cryptic species, a phylogenomic tree does not fulfill the criteria for rapid and cost-effective approach for identification of fungi, due to the time-consuming nature of the analysis. As an alternative, analysis of WGS information by mapping sequence data from individual strains against reference genomes may provide useful markers for the rapid identification of fungi. We provide a robust framework for typing F.g. through the web-based PhaME workflow available at EDGE bioinformatics. The method was validated through multiple comparisons of assembly genomes to F.g. reference strain PH-1. We showed that the difference between intra- and interspecies variability was at least two times higher than intraspecific variation facilitating successful typing of F.g. This is the first study which employs WGS data for typing plant pathogenic fusaria.

Development of a Highly Sensitive FcMito qPCR Assay for the Quantification of the Toxigenic Fungal Plant Pathogen Fusarium culmorum.

Fusarium culmorum is a ubiquitous, soil-borne fungus (ascomycete) causing foot and root rot and Fusarium head blight on cereals. It is responsible for yield and quality losses as well as grain contamination with mycotoxins, which are a potential health hazard. An extremely sensitive mitochondrial-based qPCR assay (FcMito qPCR) for quantification of F. culmorum was developed in this study. To provide specificity, the FcMito assay was successfully validated against 85 F. culmorum strains and 53 isolates of 30 other fungal species. The assay efficiency and sensitivity were evaluated against different F. culmorum strains with various amounts of pure fungal DNA and in the presence of background wheat DNA. The results demonstrated the high efficiency of the assay (97.2⁻106.0%, R²-values > 0.99). It was also shown that, in the presence of background DNA, 0.01 pg of fungal template could be reliably quantified. The FcMito assay was used to quantify F. culmorum DNA using 108 grain samples with different trichothecene levels. A significant positive correlation was found between fungal DNA quantity and the total trichothecene content. The obtained results showed that the sensitivity of the FcMito assay was much higher than the nuclear-based qPCR assay for F. culmorum.

Development of an FgMito assay: A highly sensitive mitochondrial based qPCR assay for quantification of Fusarium graminearum sensu stricto.

An ascomycete fungus, Fusarium graminearum sensu stricto (s.s.), is the major cause of Fusarium head blight (FHB), a devastating disease of cereals worldwide. The fungus contaminates crops with mycotoxins, which pose a serious threat to food and feed safety. In this study, we developed a highly sensitive mitochondrial based qPCR assay (FgMito qPCR) for quantification of F. graminearum s.s. To ensure high sensitivity of the assay, primers and a Minor-groove binding (MGB) probe were designed based on multi-copy mitochondrial DNA. The FgMito assay was successfully validated against a range of geographically diverse F. graminearum s.s. strains to ensure uniformity of the assay at an intraspecific level, as well as with other fungal species to ensure specificity. The assay was further evaluated in terms of efficiency and sensitivity against a test panel of different F. graminearum s.s. strains with various levels of pure fungal DNA and in the presence of wheat background DNA. The results showed a high efficiency of the assay developed, ranging from 93% to 101% with r(2)-values of >0.99. We further showed that three low concentrations of fungal template 2 pg, 0.6 pg and 0.2 pg could be reliably quantified in the presence of wheat background DNA. The FgMito assay was used to quantify F. graminearum s.s. DNA on 65 field samples from a range of hosts with defined levels of trichothecenes. We revealed a significant positive correlation between fungal DNA quantity and the sum of trichothecenes. Lastly, we showed a higher sensitivity of the FgMito assay than the nuclear based qPCR assay for F. graminearum s.s. by comparing Ct-values from both assays.