Screening of Mycotoxigenic Fungi in Barley and Barley Malt (Hordeum vulgare L.) Using Real-Time PCR-A Comparison between Molecular Diagnostic and Culture Technique

Filamentous fungi have a crucial impact on the food safety and technological quality of malting barley. Commonly used techniques for the detection of seed-borne fungi are based on cultivation and identification by morphological criteria. In contrast, this study established a quantitative real-time polymerase chain reaction (PCR) assay based on SYBR green technology for the detection and quantification of black fungal species (Alternaria spp., Epicoccum nigrum, Cladosporium cladosporioides, Penicillium verrucosum and Aspergillus niger) on brewing barley and compares it with the traditional cultivation technique and visual assessment. To screen the fungal spectrum over different barley varieties and harvest years, naturally infected samples of malting barley and corresponding malts (Hordeum vulgare L.) were analyzed over four consecutive years (2018-2021), grown under different climatic conditions in Germany. Alternaria and Cladosporium spp. DNA were present in all examined barley samples, even without visible contamination. In contrast, detection via culture-based methods does not reliably cover all species. Molecular analysis showed that there was less fungal biomass after malting, by 58.57% in the case of A. alternata, by 28.27% for Cladosporium spp. and by 12.79% for Epicoccum nigrum. Correlation analysis showed no causal relationship between fungal DNA and the number of black kernels. The qPCR provides a highly sensitive and time-saving screening method for detecting latent fungal infections in brewing grains to identify batches that are potentially highly contaminated with toxigenic fungi.

Automated summary

A quantitative real-time PCR assay based on SYBR Green technology was established for the detection and quantification of black fungal species on brewing barley. The study found that Alternaria and Cladosporium spp. DNA were present in barley samples even without visible contamination. Molecular analysis showed a decrease in fungal biomass after malting. The qPCR method provides a highly sensitive and time-saving screening method for detecting latent fungal infections in brewing grains.