Dataset Information

Moving from qPCR to Chip Digital PCR Assays for Tracking of some Fusarium Species Causing Fusarium Head Blight in Cereals.

ABSTRACT: Fusarium Head Blight (FHB) is one of the major diseases affecting small-grain cereals, worldwide spread and responsible for severe yield and quality losses annually. Diagnostic tools, able to track Fusarium species even in the early stages of infection, can contribute to mycotoxins' risk control. Among DNA-based technologies for Fusarium detection, qPCR (single and multiplex assays) is currently the most applied method. However, pathogen diagnostics is now enforced by digital PCR (dPCR), a breakthrough technology that provides ultrasensitive and absolute nucleic acid quantification. In our work, a panel of chip digital PCR assays was developed to quantify Fusarium graminearum, F.culmorum, F. sporotrichioides, F. poae and F. avenaceum. The primers/probes combinations were evaluated on pure fungal samples with cdPCR technique, in comparison with the qPCR approach. Moreover, the cdPCR assays were applied to quantify Fusarium in durum wheat and oat samples, naturally contaminated or spiked with fungal DNA. For a better evaluation of infection level in plants, duplex assays were developed, able to co-amplify both plant and fungal DNA. To the best of our knowledge, this is the first study directed to the application of digital PCR to Fusarium diagnosis in plants.

SUBMITTER: Morcia C

PROVIDER: S-EPMC7564955 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Moving from qPCR to Chip Digital PCR Assays for Tracking of some Fusarium Species Causing Fusarium Head Blight in Cereals.

Morcia Caterina C Tumino Giorgio G Gasparo Giulia G Ceresoli Caterina C Fattorini Chiara C Ghizzoni Roberta R Carnevali Paola P Terzi Valeria V

Microorganisms 20200827 9

Fusarium Head Blight (FHB) is one of the major diseases affecting small-grain cereals, worldwide spread and responsible for severe yield and quality losses annually. Diagnostic tools, able to track Fusarium species even in the early stages of infection, can contribute to mycotoxins' risk control. Among DNA-based technologies for Fusarium detection, qPCR (single and multiplex assays) is currently the most applied method. However, pathogen diagnostics is now enforced by digita ...[more]

PMID: 32867286

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Project description:IntroductionFusarium avenaceum causing Fusarium seedling blight (FSB) and Fusarium head blight (FHB) on barley is associated with economic losses of crop yield and quality, and the accumulation of mycotoxins including the enniatins (ENNs) A, A1, B and B1. Although F. avenaceum is the main producer of ENNs, studies on the ability of isolates to cause severe Fusarium diseases or produce mycotoxins in barley are limited.MethodsIn this work, we investigated the aggressiveness of nine isolates of F. avenaceum to two cultivars of malting barley, Moonshine and Quench, and defined their ENN mycotoxin profiles in in vitro and in planta experiments. We assessed and compared the severity of FSB and FHB caused by these isolates to disease severity by F. graminearum, F. tricinctum and F. poae. Quantitative real-time polymerase chain reaction and Liquid Chromatography Tandem Mass Spectrometry assays were used to quantify pathogen DNA and mycotoxin accumulation, respectively, in barley heads.ResultsIsolates of F. avenaceum were equally aggressive to barley stems and heads and caused the most severe FSB symptoms resulting in up to 55% reductions of stem and root length. Fusarium graminearum caused the most severe FHB disease, followed by the isolates of F. avenaceum with the most aggressive F. avenaceum isolates capable of causing similar bleaching of barley heads as F. avenaceum. Fusarium avenaceum isolates produced ENN B as the predominant mycotoxin, followed by ENN B1 and A1 in vitro. However, only the most aggressive isolates produced ENN A1 in planta and none produced ENN A or beauvericin (BEA) either in planta or in vitro.DiscussionThe capacity of F. avenaceum isolates to produce ENNs was related to the accumulation of pathogen DNA in barley heads, whilst FHB severity was related to the synthesis and accumulation of ENN A1 in planta. Cv. Moonshine was significantly more resistant than Quench to FSB or FHB, caused by any Fusarium isolate, and to the accumulation of pathogen DNA, ENNs or BEA. In conclusion, aggressive F. avenaceum isolates are potent ENN producers causing severe FSB and FHB with ENN A1 requiring further investigation as potential virulence factor for F. avenaceum in cereals.

Project description:Fusarium head blight (FHB) is one of the most important diseases of wheat, causing yield losses and mycotoxin contamination of harvested grain. A complex of different toxigenic Fusarium species is responsible for FHB and the composition and predominance of species within the FHB complex are determined by meteorological and agronomic factors. In this study, grain of three different susceptible winter wheat cultivars from seven locations in northern Germany were analysed within a five-year survey from 2013 to 2017 by quantifying DNA amounts of different species within the Fusarium community as well as deoxynivalenol (DON) and zearalenone (ZEA) concentrations. Several Fusarium species co-occur in wheat grain samples in all years and cultivars. F. graminearum was the most prevalent species, followed by F. culmorum, F. avenaceum and F. poae, while F. tricinctum and F. langsethiae played only a subordinate role in the FHB complex in terms of DNA amounts. In all cultivars, a comparable year-specific quantitative occurrence of the six detected species and mycotoxin concentrations were found, but with decreased DNA amounts and mycotoxin concentrations in the more tolerant cultivars, especially in years with higher disease pressure. In all years, similar percentages of DNA amounts of the six species to the total Fusarium DNA amount of all detected species were found between the three cultivars for each species, with F. graminearum being the most dominant species. Differences in DNA amounts and DON and ZEA concentrations between growing seasons depended mainly on moisture factors during flowering of wheat, while high precipitation and relative humidity were the crucial meteorological factors for infection of wheat grain by Fusarium. Highly positive correlations were found between the meteorological variables precipitation and relative humidity and DNA amounts of F. graminearum, DON and ZEA concentrations during flowering, whereas the corresponding correlations were much weaker several days before (heading) and after flowering (early and late milk stage).

Project description:Fusarium head blight (FHB) is a major cereal disease caused by a complex of Fusarium species. These species vary in importance depending on climatic conditions, agronomic factors or host genotype. In addition, Fusarium species can release toxic secondary metabolites. These mycotoxins constitute a significant food safety concern as they have health implications in both humans and animals. The Fusarium species involved in FHB differ in their pathogenicity, ability to produce mycotoxins, and fungicide sensitivity. Accurate and exhaustive identification of Fusarium species in planta is therefore of great importance. In this study, using a new set of primers targeting the EF1? gene, the diversity of Fusarium species on cereals was evaluated using Illumina high-throughput sequencing. The PCR amplification parameters and bioinformatic pipeline were optimized with mock and artificially infected grain communities and further tested on 65 field samples. Fusarium species were retrieved from mock communities and good reproducibility between different runs or PCR cycle numbers was be observed. The method enabled the detection of as few as one single Fusarium-infected grain in 10,000. Up to 17 different Fusarium species were detected in field samples of barley, durum and soft wheat harvested in France. This new set of primers enables the assessment of Fusarium diversity by high-throughput sequencing on cereal samples. It provides a more exhaustive picture of the Fusarium community than the currently used techniques based on isolation or species-specific PCR detection. This new experimental approach may be used to show changes in the composition of the Fusarium complex or to detect the emergence of new Fusarium species as far as the EF1? sequence of these species show a sufficient amount of polymorphism in the portion of sequence analyzed. Information on the distribution and prevalence of the different Fusarium species in a given geographical area, and in response to various environmental factors, is of great interest for managing the disease and predicting mycotoxin contamination risks.

Project description:Head blight or scab caused by Fusarium graminearum (FG), once ranked as a minor disease in wheat, is now emerging as one of the economically important diseases in India. The present study represents the first in-depth population genetic analysis of the FG from the northern wheat belt of India. In this study, multiple conserved gene sequences comprised of β-tubulin (TUB), translation elongation factor 1-α (TEF), and histone-3 (HIS) regions were used for multi-locus phylogenetic analysis of 123 geographically distinct F. graminearum isolates collected from four different states (Haryana (HR), Punjab (PB), Rajasthan (RJ) and West Bengal (WB)) of India. The phylogenetic and haplotype analysis showed the presence of thirty haplotypes in all the analyzed populations. The haplotypic diversity in the RJ population (Hd = 0.981) was higher than in the HR (Hd = 0.972), PB (Hd = 0.965) and WB population (Hd = 0.962). Recombination events (Rm = 12) and mutation events (485) were also detected. Analysis of molecular variance (AMOVA) indicated that genetic diversity was exclusively due to the differences within populations. The haplotype network was widely dispersed and not associated with specific populations, as a single common haplotype was not detected. The PB population contained both unique (H9, H10 and H11) and shared haplotypes (27 haplotypes) in a higher number in comparison to other geographical locations. Except for haplotype H22 (contains highly aggressive isolates), there was no specific linkage noticed between the isolate aggressiveness and haplotype. The concatenated sequences of all the three genes demonstrated a low level of genetic differentiation (Fst = -0.014 to 0.02) in the analyzed population. Positive values for the neutrality tests in PB, HR and RJ reveal a balancing selection mechanism behind the FG population structure. The WB population showed both positive and negative values of neutrality indices, indicating the role of both population expansion as well as balancing selection in structuring the FG population.

Dataset Information

Moving from qPCR to Chip Digital PCR Assays for Tracking of some Fusarium Species Causing Fusarium Head Blight in Cereals.

Publications

Moving from qPCR to Chip Digital PCR Assays for Tracking of some <i>Fusarium</i> Species Causing <i>Fusarium</i> Head Blight in Cereals.

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