The fungal community changes over time in developing wheat heads Morten Hertz a , Iben Ravnborg Jensen a , Laura Østergaard Jensen a , Søren Nøhr Thomsen a , Jacob Winde a , Morten Simonsen Dueholm a , Lydia Henriette Sørensen b , Rasmus Dam Wollenberg a , Hans Otto Sørensen c , Teis Esben Sondergaard a , Jens Laurids Sørensen a,d, ⁎ a Department of Chemistry and Bioscience, Aalborg University, Fredrik Bajers Vej 7H, DK-9220 Aalborg Ø, Denmark b Department of Molecular Biology and Genetics, Center for Quantitative Genetics and Genomics, Aarhus University, Blichers Allé 20, DK-8830 Tjele, Denmark c HOS Agronomen ApS, Gårdkrogsvej 10, DK-6780 Skærbæk, Denmark d Department of Chemistry and Bioscience, Aalborg University, Niels Bohrs Vej 8, DK-6700 Esbjerg, Denmark abstract article info Article history: Received 12 November 2015 Received in revised form 14 January 2016 Accepted 24 January 2016 Available online 28 January 2016 Under normal conditions, wheat is colonized by a multitude of fungi that can have beneficial or adverse effects on plant growth and yield. To study the effect of spraying wheat heads with fungicides on the fungal community from emergence to harvest we applied an amplicon sequencing approach on single wheat heads. The climatic data showed that the spring of 2014 was very dry and without precipitation in the two weeks around flowering. An initial quantitative PCR showed that the total amount of fungal DNA increased during the entire period, without significant difference between sprayed and control wheat heads. Amplicon sequencing of the internal transcribed spacer 2 (ITS2) region showed that operational taxonomic units (OTUs) identified as Sporobolomyces roseus dominated in the first weeks, whereas Alternaria infectoria OTUs dominated in the last weeks before har- vest. The only observed significant difference was that the control wheat heads contained more of the powdery mildew causing Blumeria graminis f. sp. tritici OTUs compared with the sprayed wheat heads. The dry conditions around flowering most likely also had an effect on Fusarium head blight infection as Fusarium OTUs were only sporadically encountered. Analyses of secondary metabolites produced by Fusarium and Alternaria in the wheat heads confirmed the obser- vations from the amplicon sequencing. Enniatin B was the most frequent contaminant present in four sprayed (49–538 ng/g) and three control (56–355 ng/g) wheat heads. The A. infectoria secondary metabolites infectopyrone and 4Z-infectopyrone were however consistently observed in all samples collected the last five weeks before harvest. © 2016 Elsevier B.V. All rights reserved. Keywords: Amplicon sequencing Fusarium Alternaria infectoria Mycotoxins Mycobiome Next generation sequencing 1. Introduction Common wheat (Triticum aestivum L.) is one of the most important crops worldwide, only surpassed by maize and rice (FAOSTAT, 2015). Wheat plants are however under continuous attack from pathogenic fungi, bacteria and insects; these have a huge impact on yield and quality. The most important fungal pathogens include the biotrophs Blumeria graminis (powdery mildew) and Puccinia spp. (rusts), as well as the necrotrophs Pyrenophora tritici-repentis (tan spot), Mycosphaerella graminicola (Septoria blotch), Parastagonospora nodorum (Stagonospora blotch) and Fusarium spp. (Fusarium head blight) (Bockus et al., 2010; King et al., 1983; Parry et al., 1995). Less pathogenic fungi are also very frequently found on wheat, including the black head causing Alternaria, Cladosporium and Epicoccum (Larran et al., 2002). Pink (Sporobolomyces spp.) and white (Cryptococcus spp.) yeasts are also common saprophytes found on wheat leaves and grains (Fokkema, 1971). Of these fungi Fusarium and Alternaria are of concern due to the production of bioactive secondary metabolites of which some are classified as mycotoxins (Andersen et al., 2015; Pereira et al., 2014). The role of the most important wheat pathogens is well studied at single species level, but little is known about the role of the fungal com- munity in disease development. Understanding the complex interac- tions between host plants and their microbial inhabitants is important for development of sustainable disease management and is therefore a high priority. The mycobiota have traditionally been examined through examining visual symptoms of the plants or by isolating and cultivating fungi using different media and growth conditions (Abildgren et al., 1987; Hocking and Pitt, 1980; Sørensen et al., 2009). Biotrophic fungi and slow growing fungi can however be overlooked in the culture dependent methods. The technical advances in next generation sequencing techniques have enabled culture-independent analyses of the mycobiome (fungi and their genes) (Guttman et al., 2014). This International Journal of Food Microbiology 222 (2016) 30–39 ⁎ Corresponding author at: Department of Chemistry and Bioscience, Aalborg University, Fredrik Bajers Vej 7H, DK-9220 Aalborg Ø, Denmark. E-mail address: jls@bio.aau.dk (J.L. Sørensen). http://dx.doi.org/10.1016/j.ijfoodmicro.2016.01.018 0168-1605/© 2016 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro