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Control of Root Rot of Chickpea Caused by Sclerotium Rolfsii by Different Agents and Gamma Radiation

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Control of Root Rot of Chickpea Caused by Sclerotium Rolfsii by Different Agents and Gamma Radiation Tanta University Faculty of Science Botany Department Control of root rot of chickpea caused by Sclerotium rolfsii by different agents and gamma radiation. A thesis submitted to Faculty of Science – Tanta University In partial fulfillment of the requirements for the degree of master in Microbiology (Mycology) Submitted by Rasha Mohammed Fathy El- Said B.Sc. Microbiology- 2004- Al- Azhar University 2012 Supervisors Prof. Dr. Abd El Wahab Anter Ismail. Head of Integrated control department, Giza Research Institute. Prof. Dr. Ahmed Ibrahim El- Batal. Professor of Applied Microbiology and Biotechnology, National Center for Radiation Research & Technology (NCRRT). Prof. Dr. Hanan Mahmoud Mubarak. Associate. Prof. of Mycology, Botany Department, Faculty of Science, Tanta University. Supervisors Prof. Dr. Abd El Wahab Anter Ismail Head of Integrated control department, Giza Research Institute. Prof. Dr. Ahmed Ibrahim El- Batal Professor of Applied Microbiology and Biotechnology, National Center for Radiation Research & Technology (NCRRT). Prof. Dr. Hanan Mahmoud Mubarak, Associate. Prof. of Mycology, Botany Department, Faculty of Science, Tanta University. Head of Botany Department Prof. Dr. Hassan Fared El-Kady. This thesis has not been previously submitted for any degree at this or any other University. Rasha Mohammed Fathy TO WHOM IT MAY CONCERN This is to certify that Ms. Rasha Mohamed Fathy has attended and passed successfully the following post-graduate courses (theoretical and practical) as a partial fulfillment of the requirement for the degree of Master of Science (Microbiology) Botany Department, Faculty of Science, Tanta University during the academic year 2004/2005. The courses cover the following topics: 1- General and applied bacteriology, the use of microorganisms in preparation of leathers, methods and instruments used in microbiology. 2- Biochemistry, fermentation chemistry and immunological reactions. 3- Plant pathology, virology and special fungi. 4- Phycology, physiology of algae and physiology of fungi. 5- Biostatistics. 6- German language. 7- Computer science. This certificate is issued at here own request. Head of Botany Department Prof. Dr. Hassan Fared El-Kady CURRICULUM VITAE Name: Rasha Mohamed Fathy El-Said Locality: Tanta - Gharbia Governorate. Nationality: Egyptian. Occupation: Dministrator at National Center for Radiation Research & Technology (NCRRT) - Atomic Energy Authority – Egypt. Qualification: B.Sc. Degree in Microbiology (2004), Excellent, Faculty of Science, Al-Azhar University. Attended and passed successfully the post graduate courses in partial fulfillment of M. Sc. (2005) Head of Botany Department Prof. Dr. Hassan Fared El-Kady ACKNOWLEDGMENT Firstly my unlimited thanks to Allah, the most merciful, the most greatful, real helper and real support for all. I am also deeply thankful to Prof. Dr. Abd El Wahab Anter Ismail Head of Integrated control department, Giza Research Institute for suggestion the topic, supervising the work and revising the manuscript. And also for his great effort throughout the work. I am also indebted to Prof. Dr. Ahmed Ibrahim El- Batal Professor of Applied Microbiology and Biotechnology, National Center for Radiation Research & Technology (NCRRT) for supervising this work and for his kind and valuable helping in radiation experiments. I want to express about my keen thanks and my great gratitude to Prof. Dr. Hanan Mahmoud Mubarak, Associate. Prof. of Mycology, Botany Department, Faculty of Science, Tanta University for supervising this work, encouragement during practical work and for her keen interest and unfailing help. I wish to express my appreciation to Prof. Dr. Yehia A.G. Mahmoud Professor of Mycology, Botany department, Faculty of Science, Tanta University, for his cooperation and help during the progress of this work, I would like to truly thank him for his stimulating, care, support and encouragement through out the course of this thesis. I want also to express about my deepest appreciation and my special thanks to whom working at Mycology lab. at Botany Department, Faculty of Science, Tanta University and National Center for Radiation Research & Technology (NCRRT) lab. for their sincere help. Finally, I must offer my deepest appreciation and very special thanks to all members of my family for their excellent help, and continues encouragement to make this thesis to arise. Rasha Abstract Abstract Sclerotium rolfsii causes root rot disease in several crops including chickpea that result in low yield. Artificial infection of chickpea seedlings by S. rolfsii in vitro demonstrated that different tissues of the plant completely disintegrated by fungal infection. In vitro and green house pot experiments demonstrated that inducers in combination with fungicides, oils and bioagents resulted in about 80 % suppression of root rot disease. Treatments have no phytotoxic effect on chickpea seedlings at low doses. Gliocladium virens and Gliocladium deliquescens were effective as biocontrol agents against Sclerotium rolfsii. The percent of survival plants, fresh weight, dry weight and plant height of chickpea plants increased with different treatments with inducers compared with the control. Chlorophyll a, b, and total chlorophyll amounts increased to the maximum values. The activity of two plant enzymes, peroxidase and polyphenol oxidase increased. In this study, gamma irradiation of chickpea seeds at doses 5, 10, 15, 20, 25 and 30 Gy have negative effect on survival, plant height, fresh weight and dry weight of chickpea. The effect of gamma irradiation at doses 0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 kGy on the antagonistic effect of Gliocladium virens and Gliocladium deliquescens against S. rolfsii were investigated. The results revealed that gamma irradiation increase the antagonistic effect of Gliocladium virens and Gliocladium deliquescens against S. rolfsii . Abstract Effect of gamma irradiation at doses of 0, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 5 kGy on the mycelial growth and pathogenicity of S. rolfsii were investigated. The results revealed that gamma irradiation at doses 0.25 up to 3.0 kGy increase the pathogenicity of S. rolfsii but gamma irradiation at dose 5.0 kGy completely inhibited the growth of S. rolfsii. Extracellular polygalacturonase was characterized and purified by precipitation with 70 % ammonium sulfate, dialysis and gel filtration through Sephadex 75 for irradiated and unirradiated S. rolfsii isolates. The purified enzyme exhibited its maximum activity at gamma irradiation dose 3.0 kGy compared with the control. The molecular weight was determined by SDS- PAGE for irradiated and un-irradiated isolates to be a single band at 72 kDa. Key words: Sclerotium rolfsii, chickpea, root rot, biocontrol, gamma irradiation, polygalacturonase. Contents Page Introduction…………………………………………………………… 1 Preface………………………………………………………... 1 Aim of the work…………………………………………………… 5 Review of literature…………………………………………... 6 Chickpea (Cicer arietinum)…………………………………. 6 Sclerotium rolfsii………………………………………… 8 Effect of the fungicides, oils, inducers and bioagents on the growth of the pathogenic fungus S. rolfsii……………………………… 11 1. Effect of fungicides……………………………………… 11 2. Effect of oils……………………………………………….. 13 3. Effect of inducers…………………………………………... 17 4. Effect of bioagents………………………………………… 20 Phytotoxicity of the tested fungicides, oils and inducers on the length of shoot and root system………………………………… 22 Effect of some combinations of fungicides, biocontrol agents, and oils on fungi……………………………………………………. 25 Effect of fungicides, oils, biocontrol agents on chlorophyll content and plant chemical constituents……………………………………. 26 Polygalacturonase content in Sclerotium rolfsii……………………. 28 Effect of gamma radiation…………………………………………... 30 Types of radiation…………………………………………… 30 1- Ionizing radiation……………………………………… 31 2- Non- ionizing radiation………………………………… 31 Effect of ionizing radiation on microorganisms.................................. 31 Factors affecting the sensitivity of microorganisms to radiation…… 32 Effect of gamma radiation on microorganisms………………………… 33 1- The direct action…………………………………………….. 33 2- The indirect action…………………………………………… 33 Materials and Methods…………………………………………….. 36 Tested fungicides…………………………………………………… 36 1-Vitavax-200……………………………………………….. 36 2-Monceren – T……………………………………………… 37 The tested oils……………………………………………………….. 37 Potato dextrose agar medium……………………………………… 38 Tested inducers………………………………………………………. 38 Tested fungi…………………………………………………………… 38 1- Mode of artificial infection of Cicer arietinum as a host tissue in vitro……………………………………………………………. 39 a) Preparation of inoculum…………………………………… 39 b) Plant materials………………………………………………. 39 c) Examination of the infected tissues ………………………. 40 d) Light microscopy…………………………………………… 40 2- Biological tests ( In vitro studies )………………………………….. 40 2.a- Antagonism between pathogenic fungus and antagonistic fungi (biocontrol agents)………………………………………… 40 2. b- Fungicidal activity of fungicides, oils and inducers……...... 41 2.c- Phytotoxicity test………..………………………… 42 2.d- Joint toxic effect vitavax fungicide combined with inducers and oils against S. rolfsii…………………………………………. .. 43 3- Greenhouse experiments ( In vivo )…………………………………. 44 3.a- Pathogenecity test…………………………………………….. 44 3.b- Preparation of inoculum and soil infestation…………………. 44 3.c- Effect of infection by S. rolfsii on growth parameters of Cicer arietinum…………………………………………. 45 4- Effect of fungicides, oils, inducers, bioagents and their combinations on the infected Cicer arietinum
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