Occurrence and significance of Fusarium and Trichoderma ear rot in maize Dissertation to obtain the Ph.D. degree in the Faculty of Agricultural Sciences, Georg-August-Universität Göttingen, Germany by Annette Pfordt born in Heilbad Heiligenstadt (Thuringia) Göttingen, May 2020 1. Name of referee: Prof. Dr. Andreas von Tiedemann 2. Name of co-referee: Prof. Dr. Petr Karlovsky Date of examination: 15.07.2020 Chapter Ⅰ: Introduction 1. Maize cultivation 01 2. Ear rot diseases in maize 02 2.1. Fusarium ear rot 03 2.2. Trichoderma ear rot 08 3. Aims of the study 11 References 12 Chapter Ⅱ: Impact of environmental conditions and agronomic practices on the prevalence of Fusarium species associated with ear- and stalk rot in maize 1. Introduction 20 2. Results 21 2.1 Fusarium species involved in ear and stalk infections 21 2.2 Effect of previous crop 22 2.3 Effect of tillage 22 2.4 Effect of environmental conditions 23 2.5 Relative impact of main effects 27 3. Discussion 28 4. Material and Methods 31 4.1 Sampling and isolation 31 4.2 Species identification 32 4.3 Meteorological and agronomical data 32 4.4 Statistical analyses 32 Supplementary Materials 33 References 35 Chapter Ⅲ: Occurrence, pathogenicity and mycotoxin production of Fusarium temperatum in relation to other Fusarium species on maize in Germany 1. Introduction 40 2. Results 40 2.1 Natural disease severity 40 2.2 Pathogenicity test on maize cob under field conditions 41 2.3 Pathogenicity test on maize stalk under greenhouse conditions 42 2.4 Effect of temperature on ear infection 43 2.5 Pathogenicity test on wheat under greenhouse conditions 44 2.6 Species identification 44 2.7 Mycotoxin analysis 46 3. Discussion 47 4. Material and Methods 49 4.1 Fungal isolation and cultivation 49 4.2 Inoculum preparation 49 4.3 Pathogenicity test on maize cob under field conditions 49 4.4 Pathogenicity test on maize stalk under greenhouse conditions 50 4.5 Effect of temperature on ear infection 50 4.6 Pathogenicity test on wheat under greenhouse conditions 51 4.7 DNA Extraction, PCR and analysis of tef-1α 51 4.8 Mycotoxin extraction and HPLC-MS/MS 52 4.9 Statistical analyses 53 Supplementary Materials 53 References 56 Chapter Ⅳ: Fusarium ear rot infection in maize in relation to inoculation method, maize variety and Fusarium species 1. Introduction 62 2. Material and Methods 64 2.1 Field site location, experimental design and plant material 64 2.2 Inoculum preparation 64 2.3 Inoculation and disease assessment 65 2.4 Statistical analyses 65 3. Results 65 3.1 Field site management and weather conditions 65 3.2 Disease severity 66 3.2.1 Responses of maize hybrids to different Fusarium species 69 3.2.2 Effect of inoculation method on disease severity 70 4. Discussion 71 4.1 Inoculation method 71 4.2 Location and environmental conditions 72 4.3 Maize genotypes 73 4.4 Fusarium species 74 References 75 Chapter Ⅴ: Trichoderma afroharzianum ear rot– a new disease on maize in Europe 1. Introduction 80 2. Material and Methods 81 2.1 Fungal isolation and cultivation 81 2.2 Inoculation procedure 81 2.3 Plant cultivation and pathogenicity assessment on maize ears 81 2.4 DNA extraction and phylogenetic analysis 82 2.5 Statistical analyses 83 3. Results 83 3.1 Geographic origin of samples 83 3.2 Species identification 84 3.3 Disease symptoms and severity on maize ears 85 4. Discussion 86 References 88 Chapter Ⅵ: Co-authored publication as part of the present investigations High-Resolution Melting (HRM) curve assay for the identification of eight Fusarium species causing ear rot in maize 1. Introduction 91 2. Results 92 2.1 HRM analysis of the sRPB2 and sTEF-1α for the identification of Fusarium species 92 2.2 Identification of Fusarium species in naturally infected maize ears 94 2.3 Fungal colony PCR 94 3. Discussion 95 4. Material and Methods 96 4.1 Reference strains, sample collection and DNA extraction 96 4.2 Fungal colony PCR 97 4.3 Primer design and maximum likelihood tree analysis 97 4.4 HRM analysis 98 4.5 Fluorescence data processing and taxonomic assignment 98 5. Conclusion 98 References 99 Chapter Ⅶ : General discussion 103 References 116 Summary 124 Acknowledgements 126 Curriculum vitae 127 Introduction Chapter Ⅰ: Introduction 1.1 Maize cultivation Maize (Zea mays L.), along with wheat (Triticum aestivum L.) and rice (Oryza sativa L.), belongs to the most important crops worldwide grown on approx. 194 M hectares [1]. Maize is not only one of the most important food crop, but also one of the world's most important animal feeds [2]. In Germany, maize is the second most important crop grown on 2.6 M hectares with a total harvest in 2019 of around 90.4 M tons, which is only exceeded by wheat [3]. The introduction of modern silage preparation techniques in the first half of the 20th century, can be considered as a milestone, which led to the initial cultivation increase in the late 1960s. Further increase in the economic importance of maize is primarily caused by breeding success, i.e. better adapted varieties, which allowed cultivation in cooler climatic regions in Germany, improved sowing and harvest techniques as well as advances in chemical control of weeds [4]. The main types of use in Germany are silage maize and grain maize, as well as corn-cob mix (CCM). In 2019, approximately 2.2 M hectares were harvested as silage maize and 416,000 hectares as grain maize and corn-cob mix [3]. Grain maize and CCM-maize is mainly produced in the southern regions of Germany, especially Bavaria and Baden-Wuerttemberg as well as in the Rhine valley for animal feed production (swine and poultry). In contrast, silage maize is mainly produced in central to northern regions of Germany used as feed for ruminants and as substrate for biogas production. In the last decades, silage maize cultivation increased in particular due to the Renewable Energy Law (EEG) in Germany to promote the use of renewable resources for energy and biogas production [5]. Especially after the adoption of the guideline in 2004, the production area increased by about 70% between 2000 and 2014 due to federal subsidies. In grain maize production, only the grains are harvested, while corn-cob-mix (CCM) contains the grounded grains and spindles. Grain maize is harvested at a dry matter content of 60-65%. To produce silage maize, the whole plant is chopped and harvested and serves as basis for maize silage to be fed to ruminants. In contrast to grain maize, the optimal silage 1 Introduction maize ripeness is around 30-35% DM. A good silage maize variety is also characterized by a high biomass yield and a high energy density in the dry matter content [4]. Due to the increasing maize cultivation, the subsequent increase of this crop within the crop rotation and the spread of insects that promote infestation such as the European corn borer (Ostrinia nubilalis) [6], the infestation pressure and the importance of Fusarium infections on maize have increased in Germany [7,8]. 1.2 Ear rot diseases in maize Ear rots, caused by fungi, are among the most important maize diseases worldwide with high potential yield losses and a reduction of grain quality [9]. The largest damage is caused by their ability to produce toxicogenic secondary metabolites, so called mycotoxins [10]. The most important diseases in maize due to their mycotoxin production are Aspergillus ear rot mainly caused by Aspergillus flavus, Penicillium ear rot and Fusarium ear rot caused by various species of the genus Penicillium and Fusarium [9,11]. Several other fungi are known to cause ear rots in maize, however, usually less harmful with minor incidence and severity, such as Cladosporium ear rot (Cladosporium spp.), Diplodia ear rot (Stenocarpella maydis and S. macrospora), Nigrospora ear rot (Nigrosspora oryzae), Trichoderma ear rot (Trichoderma spp.) and corn smut (Ustilago maydis) [8,12]. Fungal toxins of most concern are produced by species within the genera of Aspergillus, Fusarium and Penicillium. Among these mycotoxins, aflatoxin B1 (AFB1), fumonisin B1 (FB1), and ochratoxin A (OTA) (Figure 1) are the most toxic to mammals, causing a variety of toxic effects including hepatotoxicity, teratogenicity, and mutagenicity, resulting in diseases such as edema, immunosuppression, hepatic carcinoma, esophageal cancer, and kidney failure [10,13,14]. Aflatoxin B1 has been classified as a class I human carcinogen, while fumonisin B1 and ochratoxin A have been classified as class 2B carcinogens by the international agency for research on cancer (IARC) (2002) [15]. 1.2.1 Fusarium ear rot The fungi of the genus Fusarium are widespread pathogens causing economically important diseases, ranging from root and stem rot to ear rot on maize in temperate and semi-tropical areas [16]. Several toxigenic Fusarium species are known to cause yield losses and reduction of grain quality, thus endangering the safety of both animal feed and human food products 2 Introduction [15,17]. Among the most important Fusarium species in pre- and post-harvest ear rots of maize are F. graminearum and F. verticillioides [4–6], but also other species, such as F. poae [7,8], F. proliferatum [1], F. subglutinans [9] and F. temperatum [10], are frequently reported. Infections are typically characterized by the growth of fungal white or reddish mycelium with rotting symptoms on the cob and on stored grains. They are associated with the production of numerous, chemically diverse mycotoxins such as deoxynivalenol, nivalenol, zearalenon and fumonisin [18,19]. Fusarium infected ears develop a white, salmon to cinnamon-colored or pink-purple colored mycelium, which covers the cob and the husk leaves [20].