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Identification of Pathogens and Control of Spot Blotch Disease of Barley (Hordeum Vulgare) by Combining Plant Resistance and Biological Control

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Identification of Pathogens and Control of Spot Blotch Disease of Barley (Hordeum Vulgare) by Combining Plant Resistance and Biological Control Identification of pathogens and control of spot blotch disease of barley (Hordeum vulgare) by combining plant resistance and biological control Abdallah Mohamed M Ali Aada A Thesis Submitted for the Degree of Doctor of Philosophy April 2013 School of Biology Faculty of Science, Agriculture and Engineering Newcastle University Newcastle upon Tyne UK Summary Identification of pathogens and control of spot blotch disease of barley (Hordeum vulgare) by combining plant resistance and biological control agents with different methods of application Spot blotch is one of the most important diseases of barley (Hordeum vulgare) in Libya and worldwide. The overall aim of this study was to investigate the potential of biological control in combination with disease resistance to control spot blotch without the potential hazards of chemical application. Fungi were isolated from barley plants with spot blotch symptoms from different areas in Libya. As well as the commonly known spot blotch pathogen Bipolaris sorokiniana (teleomorph Cochliobolus sativus), Bipolaris spicifera (teleomorph Cochliobolus spicifer), Curvularia inaequalis, and Alternaria alternata were identified by their morphology and ribosomal DNA sequences. Bipolaris sorokiniana was the most serious pathogen under the test conditions; the others infected barley but caused less severe symptoms. Spot blotch resistance of barley seedlings was tested under greenhouse conditions with four Libyan cultivars (ACSAD, Nibola, Rehan, and Wadi Utbah) and two UK cultivars (Gaelic and Pastoral). Nibola was the most resistant. The ability of the organisms in three commercial biocontrol products, Trichoderma harzianum T-22 (Trianum), Streptomyces lydicus WYEC 108 (Actinovate) and Bacillus subtilis QST 713 (Serenade), to control spot blotch individually and in combination was investigated. On agar plates, all three inhibited growth of the pathogens completely on the second day of culture, except that for B. spicifera with S. lydicus there was an inhibition zone and the pathogen grew in the opposite direction. Disease severity was lowest when T. harzianum T-22 was applied individually to the most resistant cultivar, Nibola. Foliar application, soil treatment and seed coating all reduced disease severity. With foliar application, T. harzianum T-22 was more effective when applied at the same time as the pathogen than when applied one week before or four days after. In a field experiment with T. harzianum T-22, foliar application combined with seed treatment suppressed spot blotch more effectively than either method individually. i Dedication I would like to dedicate this research To My Parents, My wife and My Children ii Acknowledgements I am grateful and very happy to thank all the people who supported me during the period of my studies. I cannot thank by name all the people who have supported me by physical action, by advice or by encouraging me to achieve my study successfully, especially those who taught me from my childhood till this stage of my study, but nevertheless, thank you for all. I would like to deeply express my feelings, thanks, and sincere appreciation to Dr Ethan Hack, my supervisor, for his guidance, continuous encouragement, support, and advice during my research and especially for his proficient help in the design of the field study and patience in making suggestions during my study and all his valuable comments till the end of writing of my thesis. I would like also thank following people and institutions. Professor Ezarug Edongali, University of Tripoli, who has supported me in many ways, and his continuing encouragement to study in this field and especially study in this country. Dr Saleh Hassan, Sebha University, who supported me and gave me the opportunity to do part of my work in his lab during my field study in Libya, and for continually asking and encouraging me to achieve the goals of my study. Dr Gordon Port, Newcastle University, my second supervisor, for his advice and guidance for planning my work from the beginning and his support during my experiments in Close House and in labs. Dr Robert Shiel, Newcastle University, for advice on statistical analysis and design of the field experiment. The Libyan Authorities, who sponsored me to do my study in the UK, and the Libyan Embassy in London, who supported me during my time in the UK. People working in Tesawa project for production of improved seeds who provided me with barley seeds for my study. iii People in Agricultural Maknusah project, who supported me during my field study in Libya. Matt Kowalski, Natural Industries, Inc., Houston. USA, and Dr Paul Sopp, Managing Director, Fargro Ltd., Littlehampton, UK, who supplied samples of Actinovate® AG and Serenade®, respectively. Ros Brown, technician, and all her colleagues for their support during my work in the labs. Alan Craig and his colleagues who supported me during my experimental work at Close House field station, especially Robert Hodgson (who has retired). All my colleagues in the School of Biology for their help during my study in all ways, advising, discussion and sharing knowledge in school meetings and conferences. All my friends in Libya, especially Omar Alkilani, Agila Mousa, Saad Alafi, and Ali Lamin who supported me during my field study in Libya. Naji M. Adda, my brother, who supported me in many ways during all the period of my study in this country and during my field study in Libya. Mohamed Ali M. Adda, my brother who supported me in many ways during all the period of my study in the UK and in my home country. Omar Mohamed Ali Adda, my nephew who supported me and takes responsibility for looking after my farm, which is helping me financially during my study. Hatow Omar, my mother, for her unlimited support since I was created until she passed away during my study in the UK. I feel I cannot return even part of her unlimited support. My children, Allaedin, Ibrahim, Mohamed, Marwa, Safa, and Omar, who have lived with me during this journey of my study and accepted with happiness delays in my support and service for some of them because of the limited time I could spend with them. iv Finally and not least, I would like to deeply thank my wife Soad Hassan, for her unlimited support in many ways and her continuing encouragement to do my research and take responsibility and care for looking after our children, which helped me to focus on my studies. v Table of Contents Summary…… ................................................................................................................ i Dedication…. ................................................................................................................ ii Acknowledgements ................................................................................................... iii List of Tables ............................................................................................................. xii List of Figures .......................................................................................................... xvii Chapter 1 General Introduction ........................................................................... 1 1.1 Background of the Study ................................................................................. 1 1.2 Spot blotch disease ............................................................................................ 2 1.2.1 Effect of environment on spot blotch disease distribution .......................... 3 1.2.2 Cochliobolus (anamorphs Bipolaris, Curvularia) ...................................... 4 1.2.3 Alternaria alternata .................................................................................... 8 1.2.4 Spot blotch disease management ................................................................ 9 1.3 Biological control ............................................................................................ 10 1.3.1 Trichoderma as biocontrol agent .............................................................. 13 1.3.2 Methods of applying biological control .................................................... 16 1.3.3 The use of Bacillus subtilis to control plant diseases ................................ 18 1.3.4 The use of Streptomyces species as biocontrol agents .............................. 20 1.3.5 Combination of biocontrol agents ............................................................. 22 1.3.6 Combination of biocontrol agents with fungicides ................................... 23 1.3.7 Combining of biocontrol agents with plant disease resistance ................. 24 1.4 Aims of this project ........................................................................................ 25 1.5 Research Questions ........................................................................................ 26 Chapter 2 Materials and Methods .................................................................... 29 2.1 General plant growth ..................................................................................... 29 2.2 Pathogens ........................................................................................................ 29 2.3 Biocontrol agents ............................................................................................ 30 2.4 General microbial culture methods .............................................................. 30 2.5 General methods of plant treatment ............................................................. 31 2.5.1 Foliar inoculation of plants with pathogen spore suspensions
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