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Updated: Mar 6, 2026
Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays
Published on: April 23, 2012
Quantitative molecular diagnostic assays of grain washes for Claviceps purpurea are correlated with visual
Alexia Comte1, Tom Gräfenhan2, Matthew G Links1,3
1Agriculture and Agri-Food Canada Saskatoon Research and Development Centre, Saskatoon, Saskatchewan, Canada.
Abstract:
We examined the epiphytic microbiome of cereal grain using the universal barcode chaperonin-60 (cpn60). Microbial community profiling of seed washes containing DNA extracts prepared from field-grown cereal grain detected sequences from a fungus identified only to Class Sordariomycetes. To identify the fungal sequence and to improve the reference database, we determined cpn60 sequences from field-collected and reference strains of the ergot fungus, Claviceps purpurea. These data allowed us to identify this fungal sequence as deriving from C. purpurea, and suggested that C. purpurea DNA is readily detectable on agricultural commodities, including those for which ergot was not identified as a grading factor. To get a sense of the prevalence and level of C. purpurea DNA in cereal grains, we developed a quantitative PCR assay based on the fungal internal transcribed spacer (ITS) and applied it to 137 samples from the 2014 crop year. The amount of Claviceps DNA quantified correlated strongly with the proportion of ergot sclerotia identified in each grain lot, although there was evidence that non-target organisms were responsible for some false positives with the ITS-based assay. We therefore developed a cpn60-targeted loop-mediated isothermal amplification assay and applied it to the same grain wash samples. The time to positive displayed a significant, inverse correlation to ergot levels determined by visual ratings. These results indicate that both laboratory-based and field-adaptable molecular diagnostic assays can be used to detect and quantify pathogen load in bulk commodities using cereal grain washes.
Insights
We identified the ergot fungus Claviceps purpurea on cereal grains using the chaperonin-60 (cpn60) gene. Molecular assays effectively detect and quantify this pathogen in agricultural commodities.
Area of Science:
- Agricultural Science
- Mycology
- Molecular Biology
Background:
- The epiphytic microbiome of cereal grains can harbor plant pathogens.
- Ergot fungus (Claviceps purpurea) poses a threat to cereal grain quality and safety.
- Accurate detection methods are crucial for managing ergot contamination in agricultural commodities.
Purpose of the Study:
- To identify a fungal sequence detected on cereal grains using the chaperonin-60 (cpn60) barcode.
- To develop and validate molecular assays for detecting and quantifying Claviceps purpurea DNA in cereal grains.
- To assess the prevalence and load of C. purpurea on agricultural commodities.
Main Methods:
- Microbial community profiling using cpn60 gene sequencing.
- Determination of cpn60 sequences from reference strains of Claviceps purpurea.
- Development and application of a quantitative PCR (qPCR) assay targeting the fungal internal transcribed spacer (ITS) region.
- Development and application of a cpn60-targeted loop-mediated isothermal amplification (LAMP) assay.
Main Results:
- The fungal sequence detected on cereal grains was identified as Claviceps purpurea.
- Quantitative PCR assay results correlated strongly with visual ergot sclerotia levels, but showed some false positives.
- The cpn60-targeted LAMP assay demonstrated a significant inverse correlation with ergot levels, indicating its utility for pathogen quantification.
- Both laboratory-based and field-adaptable molecular assays can detect and quantify pathogen load in bulk commodities via cereal grain washes.
Conclusions:
- Molecular methods, including cpn60 sequencing and qPCR, can identify and quantify Claviceps purpurea on cereal grains.
- The cpn60-targeted LAMP assay offers a promising field-adaptable tool for detecting ergot contamination.
- Molecular diagnostics are valuable for assessing pathogen load in agricultural commodities, even when ergot is not a primary grading factor.
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