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.
Highlights
Fusarium Head Blight (FHB) is one of the major diseases affecting small-grain cereals, it is worldwide spread and responsible for severe yield and quality losses annually.Several fungal species, mainly of the Fusarium genus, have been identified as the etiological agents of such a disease
The fungal DNA stocks were initially quantified with Qubit and the same dilutions of F. sporotrichioides, F. graminearum, F. culmorum, F. poae and F. avenaceum DNA were amplified with both qPCR and chip digital PCR (cdPCR) techniques
In this work we propose four cdPCR assays for detection and quantification of mycotoxigenic
Summary
Fusarium Head Blight (FHB) is one of the major diseases affecting small-grain cereals, it is worldwide spread and responsible for severe yield and quality losses annually. Mainly of the Fusarium genus, have been identified as the etiological agents of such a disease. Most of the species associated with FHB, in advantageous environmental conditions, invade the ear of the cereals and produce toxic secondary metabolites—mycotoxins—that contaminate the grain. FHB, compromises the yield and the grain safety and quality due to the accumulation of mycotoxins in infected kernels. Depending on species and chemotypes, Fusarium can produce A and B trichothecenes: type A trichothecenes include highly toxic mycotoxins, such as T-2 and HT-2, type B trichothecenes include, among others, deoxynivalenol (DON), nivalenol (NIV) and acetyl-NIV. Fumonisins, zearalenone, beauvericin and enniatin B can accumulate in cereal grains after
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