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.

[1]  T. Gagkaeva,et al.  Natural Occurrence of Alternaria Fungi and Associated Mycotoxins in Small-Grain Cereals from The Urals and West Siberia Regions of Russia , 2021, Toxins.

[2]  A. Patriarca,et al.  Alternaria in malting barley: Characterization and distribution in relation with climatic conditions and barley cultivars. , 2021, International journal of food microbiology.

[3]  A. Andolfi,et al.  The Genus Cladosporium: A Rich Source of Diverse and Bioactive Natural Compounds , 2021, Molecules.

[4]  M. Rychlik,et al.  Analysis of 13 Alternaria mycotoxins including modified forms in beer , 2021, Mycotoxin Research.

[5]  R. Krska,et al.  Raised concerns about the safety of barley grains and straw: A Swiss survey reveals a high diversity of mycotoxins and other fungal metabolites , 2021, Food Control.

[6]  Jolanta Wawrzyniak,et al.  Prediction of fungal infestation in stored barley ecosystems using artificial neural networks , 2021 .

[7]  M. Balendres,et al.  Epicoccum species: ubiquitous plant pathogens and effective biological control agents , 2021, European Journal of Plant Pathology.

[8]  J. Wawrzyniak.  Model of Fungal Development in Stored Barley Ecosystems as a Prognostic Auxiliary Tool for Postharvest Preservation Systems , 2021, Food and Bioprocess Technology.

[9]  N. Magan,et al.  Toxigenic Fungi and Mycotoxins in a Climate Change Scenario: Ecology, Genomics, Distribution, Prediction and Prevention of the Risk , 2020, Microorganisms.

[10]  T. Kulik,et al.  Promising Perspectives for Detection, Identification, and Quantification of Plant Pathogenic Fungi and Oomycetes through Targeting Mitochondrial DNA , 2020, International journal of molecular sciences.

[11]  A. Crisci,et al.  Occurrence and Co-Occurrence of Mycotoxins in Cereal-Based Feed and Food , 2020, Microorganisms.

[12]  C. Riccardi,et al.  Fusarubin and Anhydrofusarubin Isolated from A Cladosporium Species Inhibit Cell Growth in Human Cancer Cell Lines , 2019, Toxins.

[13]  V. Krstanović,et al.  Multi-(myco)toxins in Malting and Brewing By-Products , 2019, Toxins.

[14]  L. Rocha,et al.  Toxigenic Alternaria species: impact in cereals worldwide , 2018, Current Opinion in Food Science.

[15]  D. Mares,et al.  Wheat black point: Role of environment and genotype , 2018, Journal of Cereal Science.

[16]  A. Sant’Ana,et al.  Real-time PCR-based method for rapid detection of Aspergillus niger and Aspergillus welwitschiae isolated from coffee. , 2018, Journal of microbiological methods.

[17]  Elbelt Sonia,et al.  Optimized real time QPCR assays for detection and quantification of Fusarium and Microdochium species involved in wheat head blight as defined by MIQE guidelines , 2018, bioRxiv.

[18]  K. Hudec,et al.  Fungal infection of malt barley kernels in Slovak Republic , 2018 .

[19]  K. Hudec,et al.  Correlation between black point symptoms and fungal infestation and seedling viability of wheat kernels. , 2018 .

[20]  L. Pezo,et al.  Impact of Alternaria spp. and Alternaria Toxins on Quality of Spelt Wheat , 2018 .

[21]  A. Krasauskas.  Fungi in malting barley grain and malt production , 2017 .

[22]  Muneera D F Alkahtani,et al.  Molecular heterogeneity in the 18s DNA gene of Alternaria sp. and Fusarium sp. producing mycotoxins in rice and maize grains , 2017, Saudi journal of biological sciences.

[23]  A. Okorski,et al.  Real time PCR: a good tool to estimate mycotoxin contamination in pig diets , 2017 .

[24]  T. Becker,et al.  Fusarium Species on Barley Malt: Is Visual Assessment an Appropriate Tool for Detection? , 2017 .

[25]  T. Dumonceaux,et al.  Quantitative molecular diagnostic assays of grain washes for Claviceps purpurea are correlated with visual determinations of ergot contamination , 2017, PloS one.

[26]  M. Gastl,et al.  Fate of Fusarium Toxins during Brewing. , 2017, Journal of agricultural and food chemistry.

[27]  I. Draz,et al.  Pathogens associated with wheat black-point disease and responsibility in pathogenesis , 2016 .

[28]  M. Gastl,et al.  Fate of Fusarium Toxins during the Malting Process. , 2016, Journal of agricultural and food chemistry.

[29]  A. Brillowska-Dąbrowska,et al.  PCR and real-time PCR assays to detect fungi of Alternaria alternata species. , 2015, Acta biochimica Polonica.

[30]  T. Kulik,et al.  Quantification of Alternaria, Cladosporium, Fusarium and Penicillium verrucosum in Conventional and Organic Grains by qPCR , 2015 .

[31]  M. Rychlik,et al.  Assessment and introduction of quantitative resistance to Fusarium head blight in elite spring barley. , 2013, Phytopathology.

[32]  T. García,et al.  PCR-based assay for the detection of Alternaria species and correlation with HPLC determination of altenuene, alternariol and alternariol monomethyl ether production in tomato products , 2012 .

[33]  E. Arendt,et al.  Fundamental study on the influence of Fusarium infection on quality and ultrastructure of barley malt. , 2012, International journal of food microbiology.

[34]  A. Dalcero,et al.  Mycobiota and mycotoxins in malted barley and brewer’s spent grain from Argentinean breweries , 2011, Letters in applied microbiology.

[35]  Nelson Lima,et al.  How will climate change affect mycotoxins in food , 2010 .

[36]  R. Musetti,et al.  DNA-Dependent Detection of the Grapevine Fungal Endophytes Aureobasidium pullulans and Epicoccum nigrum. , 2009, Plant disease.

[37]  V. Beneš,et al.  The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. , 2009, Clinical chemistry.

[38]  N. H. Spliid,et al.  Real-time PCR for quantification of eleven individual Fusarium species in cereals. , 2009, Journal of microbiological methods.

[39]  M. Schmidt-Heydt,et al.  Comprehensive molecular system to study the presence, growth and ochratoxin A biosynthesis of Penicillium verrucosum in wheat , 2008, Food additives & contaminants. Part A, Chemistry, analysis, control, exposure & risk assessment.

[40]  T. Karaköy,et al.  The Relationship between Black Point and Fungi Species and Effects of Black Point on Seed Germination Properties in Bread Wheat , 2008 .

[41]  C. Wolf-Hall.  Mold and mycotoxin problems encountered during malting and brewing. , 2007, International journal of food microbiology.

[42]  M. Carmona,et al.  Modelling of wheat black point incidence based on meteorological variables in the southern Argentinean Pampas region , 2006 .

[43]  F. Mateo,et al.  Survey of the mycobiota of Spanish malting barley and evaluation of the mycotoxin producing potential of species of Alternaria, Aspergillus and Fusarium. , 2006, International journal of food microbiology.

[44]  Xiao‐Ru Wang,et al.  Detection and quantification of Cladosporium in aerosols by real-time PCR. , 2006, Journal of environmental monitoring : JEM.

[45]  V. Krstanović,et al.  Contamination of malt barley and wheat by Fusarium graminearum and Fusarium culmorum from the crop years 2001-2003 in eastern Croatia. , 2005, Microbiological research.

[46]  C. Vázquez,et al.  PCR detection assays for the trichothecene-producing species Fusarium graminearum, Fusarium culmorum, Fusarium poae, Fusarium equiseti and Fusarium sporotrichioides. , 2005, Systematic and applied microbiology.

[47]  A. Haikara,et al.  Effects of Three Fusarium Species on the Quality of Barley and Malt , 2005 .

[48]  E. Skjerve,et al.  Alternaria and Fusarium in Norwegian grains of reduced quality--a matched pair sample study. , 2004, International journal of food microbiology.

[49]  T. Guillemette,et al.  Conventional and Real-Time PCR-Based Assay for Detecting Pathogenic Alternaria brassicae in Cruciferous Seed. , 2004, Plant disease.

[50]  J. Clarke,et al.  Effects of foliar fungicides on kernel black point of wheat in southern Saskatchewan , 2002 .

[51]  J. Clarke,et al.  Black Point Reaction of Durum and Common Wheat Cultivars Grown Under Irrigation in Southern Saskatchewan. , 2000, Plant disease.

[52]  A. Ackermann.  Mycoflora of South African barley and malt , 1998 .

[53]  P. Schwarz,et al.  Fate and development of naturally occurring Fusarium mycotoxins during malting and brewing , 1995 .

[54]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[55]  B. Flannigan,et al.  A MICROBIOLOGICAL EVALUATION OF BARLEY MALT PRODUCTION , 1988 .

[56]  C. Christensen,et al.  Microflora of Barley Kernels , 1962, Applied microbiology.

[57]  D. Orčić,et al.  The Fate of Alternaria Toxins in the Wheat-Processing Chain , 2019, Flour and Breads and their Fortification in Health and Disease Prevention.

[58]  S. Cristea,et al.  IDENTIFICATION AND QUANTIFICATION OF FUNGI ASSOCIATED WITH SEEDS OF BARLEY, IN TERMS OF 2014 , 2015 .

[59]  J. Čáslavský,et al.  Determination of seventeen mycotoxins in barley and malt in the Czech Republic , 2015 .

[60]  Anuradha Vegi,et al.  Multiplex real-time PCR method for detection and quantification of mycotoxigenic fungi belonging to three different genera. , 2013, Journal of food science.

[61]  M. Damszel,et al.  Fungi colonizing the grain of spring wheat grown in the conventional and organic systems , 2012 .

[62]  B. Lievens,et al.  Microflora during malting of Barley: Overview and Impact on malt quality , 2011 .

[63]  T. Kulik.  Detection ofFusarium tricinctum from cereal grain using PCR assay , 2010, Journal of Applied Genetics.

[64]  I. Ujčič-Vrhovnik,et al.  The natural occurrence of toxigenic moulds and mycotoxins in Slovenian primary grain production. , 2010 .

[65]  S. Sarandón,et al.  Wheat Grain Discoloration in Argentina: Current Status , 2010 .

[66]  J. Pitt,et al.  Methods for isolation, enumeration and identification , 2009 .

[67]  A. Wilhelmson,et al.  Indigenous Microbial Community of Barley Greatly Influences Grain Germination and Malt Quality , 2007 .

[68]  D. Pejin,et al.  FUNGAL GROWTH DURING MALTING OF BARLEY , 2005 .

[69]  R. Kauppila,et al.  Importance of drying and sound storage conditions on the microbiological safety and viability of malting barley , 2003 .

[70]  P. Ho,et al.  Isolation of genomic DNA from Pichia pastoris without hydrolases , 2002 .

[71]  J. Delcour,et al.  From field barley to malt: detection and specification of microbial activity for quality aspects. , 1999, Critical reviews in microbiology.

[72]  T. White.  Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics , 1990 .

[73]  I. Šafránková,et al.  Seed mycoflora of malting varieties and lines of spring barley in localities Kroměříž and , 2022 .