Occurrence and Importance of Foliar Diseases on Maize (Zea mays L.) in Central Europe Dissertation to obtain the Ph. D. Degree in the Faculty of Agricultural Sciences, Georg-August-University Göttingen, Germany presented by Lucia Ramos Romero Born in Granada, Andalusia, Spain Göttingen, May 2016 D 7 1st Reviewer: Prof. Dr. Andreas von Tiedemann 2nd Reviewer: Prof. Dr. Petr. Karlovsky Date of submission: 26.5.2016 Dedicated to Hubert and Christa -Estudia, que un día te alegrarás- José Luis Ramos Contents Contents Abbreviations 1. Introduction ...................................................................................................................1 1.1. Zea mays (L.): Origin, domestication and actual cultivation in Central Europe ..........1 1.2. Distribution and spread of maize pathogens across continents ................................3 1.3. Main maize leaf diseases in Central Europe .............................................................5 1.3.1. Turcicum leaf blight ...........................................................................................5 1.3.2. Kabatiella eyespot .............................................................................................6 1.4. Maize leaf diseases of secondary importance in Central Europe ..............................9 1.4.1. Common rust.....................................................................................................9 1.4.2. Northern corn leaf spot ....................................................................................10 1.4.3. Maize anthracnose ..........................................................................................10 1.4.4. Phoma spp. complex .......................................................................................12 1.5. Epidemics and potential yield losses ......................................................................14 1.6. Fungicides as control measure ...............................................................................17 1.7. Aim of the thesis .....................................................................................................19 2. Materials and Methods ................................................................................................20 2.1. Materials ................................................................................................................20 2.1.1. Media ..............................................................................................................21 2.1.2. Maize seeds ....................................................................................................22 2.2. Inventory and validation of fungal pathogens on maize leaves ...............................23 2.2.1. Sampling locations ..........................................................................................23 2.2.2. Isolation of fungal organisms ...........................................................................25 2.2.3. Preparation of single spore cultures ................................................................26 2.2.4. In vitro cultivation ............................................................................................27 2.2.5. Morphological identification of causal agents...................................................27 2.2.6. Molecular identification of Phoma spp. ............................................................29 2.2.6.1. Obtaining of DNA from pure cultures ........................................................29 2.2.6.2. Assessment of the obtained DNA yield and quality from cultures .............30 2.2.6.3. Conditions for PCR assay ........................................................................30 2.2.6.4. DNA sequencing and analysis ..................................................................31 2.2.6.5. Identification by the Fungal Biodiversity Center (CBS-KNAW) ..................31 2.2.7. Preservation techniques for fungal organisms .................................................32 I Contents 2.2.8. Production of inoculum ....................................................................................33 2.2.9. Evaluation of pathogenicity in the greenhouse ................................................34 2.2.10. Re-isolation of the pathogen from the infected tissue ......................................38 2.3. Field locations for spore trapping and fungicide application studies ........................38 2.3.1. Locations for fungicide trials ............................................................................38 2.3.2. Locations for spore trapping ............................................................................41 2.4. Epidemiological studies based on spore trapping in the field ..................................42 2.4.1. Trapping season .............................................................................................42 2.4.2. Air sampling and analysis via microscopy .......................................................42 2.4.3. Spore release, development of the disease and weather conditions ...............43 2.5. Coupling spore trapping with PCR and qPCR assays ............................................43 2.5.1. DNA extractions from the spore trap tape ........................................................43 2.5.2. Specific primer sets for amplification ...............................................................44 2.5.3. Conditions for PCR assay ...............................................................................45 2.5.4. Assessment of the obtained DNA yield from tapes via PCR ............................45 2.5.5. Conditions for qPCR assay .............................................................................46 2.5.6. Primer specificity evaluation ............................................................................46 2.5.7. Primer sensitivity evaluation ............................................................................48 2.5.8. Dilutions of DNA yield as template ..................................................................48 2.6. Control of the main diseases through fungicides in the field ...................................49 2.6.1. Treatment design and fungicide application ....................................................49 2.6.2. Disease assessment .......................................................................................53 2.6.3. Biomass and grain yield ..................................................................................55 2.6.4. Correlation of disease development with weather factors ................................55 2.7. Data management and statistical analysis ..............................................................55 2.7.1. Epidemiological studies based on spore trapping in the field ...........................55 2.7.2. Control of the main diseases through fungicides in the field ............................56 3. Results .........................................................................................................................58 3.1. Inventory and validation of fungal pathogens on maize leaves in Central Europe ...58 3.1.1. Symptoms and morphological characterisation of E. turcicum and P. sorghi ...58 3.1.2. K. zeae, B. zeicola and C. graminicola ............................................................60 3.1.3. Symptoms and pathogenicity tests for Phoma spp. .........................................69 3.1.4. Characterisation of Phoma spp. ......................................................................73 3.1.5. Characterisation of Fusarium spp. and pathogenicity tests ..............................86 3.1.6. Testing of further organisms ............................................................................89 3.1.7. Summary of the inventory................................................................................89 II Contents 3.1.8. Distribution and prevalence of fungal pathogens occurring on maize leaves ...93 3.2. Meteorological conditions .......................................................................................96 3.2.1. Mittich and Inzing ............................................................................................96 3.2.2. Ostenfeld .........................................................................................................99 3.2.3. Göttingen ...................................................................................................... 101 3.3. Epidemiological studies based on spore trapping in the field ................................ 102 3.3.1. Seasonal incidence of airborne conidia of E. turcicum ................................... 102 3.3.1.1. Inzing 2014 ............................................................................................ 102 3.3.1.2. Göttingen 2015....................................................................................... 105 3.3.1.3. Comparisons based on climatic conditions, development of Turcicum leaf blight and spore release between Inzing 2014 and Göttingen 2015 ......................... 109 3.3.2. Correlation of microscope counts and DNA yield for E. turcicum ................... 110 3.3.2.1. Inzing ..................................................................................................... 110 3.3.2.2. Göttingen ............................................................................................... 112 3.3.3. Seasonal incidence of inoculum of K. zeae analysed via qPCR .................... 114 3.3.3.1. Ostenfeld...............................................................................................