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In Vitro Control of Ascosphaera Apis Fungus by Some Plant Extracts

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In Vitro Control of Ascosphaera Apis Fungus by Some Plant Extracts id110707841 pdfMachine by Broadgun Software - a great PDF writer! - a great PDF creator! - http://www.pdfmachine.com http://www.broadgun.com ÜÜϴϣϼÜÜÜ˰γϹ΍ΔόϣΎΠϟ΍ﺔ ﻏΓΰ˰ÜÜÜÜÜÜ – Islamic University Gaza - ﻋﻤÜÜÜÜÜÜÜϴϠόϟ΍ΕΎÜÜÜ˰˰˰˰γ΍έΪϟ΍ΓΩΎÜÜﺎ Deanship of Graduate Studies Faculty of Science ϡϮ˰˰˰˰˰˰ÜÜ˰˰˰˰˰˰Ϡόϟ΍Δ˰˰˰˰˰˰ÜÜÜÜÜÜÜÜ˰˰˰˰˰ϴϠϛ Biological Sciences Master Program ΔϴΗΎϴΤϟ΍ϡϮϠόϟ΍ήϴΘδΟΎϣΞϣΎϧήΑ Botany & Mycology ﻧﺒΕΎ˰˰ϳή˰˰˰˰˰˰˰τϓϭ ΕΎ˰˰˰˰ÜÜÜÜÜÜÜÜÜÜÜÜÜÜ In Vitro Control of Ascosphaera apis Fungus by Some Plant Extracts Prepared By Ashraf A. F. El Shafai Supervisors Dr. Kamal J. El Nabris Dr. Abboud Y. El Kichaoui Ph.D. Marine Biology Ph.D. Botany A thesis Submitted in Partial Fulfillment of The Requirements for the Degree of Master of Biological Sciences April, 2012 Dedication This thesis is dedicated to my parents. My father who is always supporting me, through the years. To the spirit of my mother who paid the price for my rest and my success. To my wife and my children who are the closest to me and who have always given me a warmth love. I Declaration "I hereby declare that this submission is my own work and that, to the best of my knowledge and belief, it contains material neither previously published or written by another person nor material which to a substantial extent has been accepted for the award of any other degree of the university of other institute, except where due a acknowledgment has been made in the text'. Name Signature Date Ashraf A. F. Al-Shafai Ashraf February,2012 ________________________________________________________________________ © 2012. No part of this work can be copied, translated or stored in a All Rights Reserved retrieval system, without prior permission of the author. II Acknowledgment First, I am very grateful to Allah who made all the things possible. There are a number of people without whom this thesis might not have been written, and to whom I am greatly indebted. I would like to express my greatest gratitude to my supervisors, Dr. Kamal El Nabris and Dr Abboud El kichawi for their continuous support, guidance, sage advice, insightful criticisms, and patient encouragement aided the writing of this thesis. My special thanks goes to all the stuff of the department of Biological Sciences, the Islamic University of Gaza (IUG) for their help and support. I would also like to thank D. Tarek Albashiti and Dr. Kamal Alkahloot for providing references in this study and for their support. I owe my thanks to Mr. Khassan Al Ashi (retired chairman of beekeeping unit, Ministry of Agriculture) for providing information and support. A special thank of mine goes to my colleagues, Mr.Yussef Abou Sylmya, Mr. Mohamed Abou Auda, and Mr. Marwan Odwan for their help, continuous support, and motivation. To All the staff members and colleagues of Biology and Biotechnology Department (IUG) for their help and support. I would also like to thank, the Disscuction committee for their views that will enhance this thesis. At last but not the least I want to thank my father, step mother, wife, sons and friends whom appreciated me for my work and motivated me. At the end, I am very grateful to those who participated and helped me to complete this study. III ϡϳΣέϟ΍ϥϣΣέϟ΍ௌϡγΑ ˱ΎΗϭϳΑϝΎΑΟϟ˸ ΍ϥ˴ ϣ˶ ϱΫ˶ Χ˶ Η͉ ΍ϥ˴΃ϝΣϧ͉ ϟ΍ϰϟ˴ ·ϙ˴ ΑέϰΣϭ˴΃ϭ ˵ ˵ ˶ ˴ ˶ ˶ ˶ ˸ ˶ ͊ ˴ ˴ ˸ ˴ } ϝϛ˵ ϥϣ˶ ϲ˶Ϡϛ˵ ϡΛ˵ ˿́ ϥ˴ ϭη˵ έόϳ Ύϣϣ˶ ϭ έΟη͉ ϟ΍ ϥ˴ ϣ˶ ϭ ͋ ͉ ( ) ˶ ˸ ˴ ͉ ˴ ˶ ˴ ˴ ΎϬ˴ ϧ˶ ϭρ˵ Α˵ ϥϣ˶ Ν˵ έ˵ Χ˸ ϳ˴ ˱ϼ˵ϟΫ˵ ϙ˶ Α͋ έ˴ ˴ϝΑ˵ γ˵ ϲϛ˶ ˵Ϡγ˸ Ύϓ˴ Ε˶ ΍έ˴ ϣ˴ Λ͉ ϟ΍ ϙ˴ ˶ϟΫ˴ ϲ˶ϓ ϥ͉ ˶· α˶ Ύϧ͉ Ϡ˶ϟ ˯Ύϔ˴ η˶ Ϫ˶ ϳ˶ϓ Ϫ˵ ϧ˵ ΍ϭ˴ ϟ˸ ˴΃ ϑ˲ ˶ϠΗ˴ Χ˸ ϣ͊ Ώ˲ ΍έ˴ η˴ ϝΣϧϟ΍Γέϭγ ˿̂ ϥ˴ ϭέϛ͉ ϔ˴ Η˴ ϳϡϭϘ˴ ϟ͋ Δ˱ ϳϵ { ( ) ˵ ˴ ˳ ˸ ˴ IV Abstract Chalkbrood is one of the most dangerous disease of the honeybees, Apis mellifera. It causes a significant reduction in brood and honey production thus, leading to an economic loss in apiculture. In present study, the pathogenic fungus was isolated from infected larvae in Northern Governorate- Palestine and identified based on morphological and cultural characters as Ascosphaera apis. Crude aqueous extracts of ten different plant species including Punica granatum, Artemisia monosperma, A. absinthium, A. herba-alba, Cyperus rotundus, Callistemon viminalis, Cinnamomum zeylanicum, Tagetes patula, Annona squamosa and Psidium guajava were evaluated in vitro for their antifungal activities against A. apis. Results showed that extract of P. granatum was the most active one among all. Other extracts vis., P. guajava, C. zeylanicum, and C. viminalis demonstrated a moderate activity with MICs of 1.25, 2.5 and 5% respectively. The remaining extracts of A. monosperma, A. absinthium, A. herba-alba, T. patula, C. rotundus, and A. squamosa were totally inactive against A. apis. For further evaluation of the antifungal properties of the different plant extracts, the radial mycelial growth inhibition test was also performed on agar media supplemented by plant extracts to achieve different concentrations of 5%, 10% and 20%. The results showed that the average radial mycelia growth of A. apis was significantly reduced by P. granatum, P. guajava, C. viminalis and C. zeylanicum (P < 0.05). Contrary to this, The remaining extracts of A. monosperma, A. absinthium, A. herba-alba, C. rotundus, T. patula and, A. squamosa were found to have weak antifungal activities at all concentrations tested. In order to assess the antifungal effect of the P. granatum extract on A. apis, the growth of profile for the fungus was followed at extract free agar media after incubation at different concentrations of P. granatum extract. The results found that the extract of P. granatum flower exhibits a fungistatic effect because it causes changes on the normal growth profile of A. apis at the different concentrations tested (5%, 10% and 20%). Since the extract of P. granatum flower was proved to be the most effective in vitro against A. apis, its toxicity to worker bees was evaluated. The results of the toxicity test showed that, the flower extract of P. granatum was not lethal to adult workers at low concentrations tested since the a cumulative mortality percentages were 4.4%, 1.1 % and 3.6% at 0% 12.5% and 25% concentrations respectively. V In conclusion, results from these findings suggest that the aqueous extract of P. granatum flower may be used as natural antifungal agents to inhibit growth of A. apis. These findings however need to be progressed to field applications to evaluate the efficacy of the most active antifungal plant extract identified in this study against the causative agent of chalkbrood disease in an apiary system. Keywords: MIC, Plant extracts, fungistatic, Ascosphaera apis, Apis mellifera, Punica granatum, Artemisia monosperma, A. absinthium, A. herba-alba, Cyperus rotundus, Callistemon viminalis, Cinnamomum zeylanicum, Tagetes patula, Annona squamosa, Psidium guajava VI Δγ΍έΪϟ΍κΨϠϣ ˬϞδόϟ΍ ϞΤϧ ϲϓ ϱήϴηΎΒτϟ΍ ΔϨπΤϟ΍ νήϤϟ ϲδϴ΋ήϟ΍ ΐΒδϤϟ΍ ήτϓ ήΒΘόϳ Ascosphaera apis Ω΍ήϓ΃ ΩΪϋϞϠϘϳ ΎϤϣ ˬϞΤϨϟ΍ Ε˶ ΎϗήϳϰϠϋϲΒϠδϟ΍ ϩήϴΛ΄Η ΐΒδΑ ΔϳΩΎμΘϗ΍ Γ˳ έΎδΧ ϰϟ· ϱΩΆϳ Ύϣ ΎΒϟΎϏϭ τϠΨϟ΍ ΢ϴϘϠΘϟ΍ ϰϠϋ ΪϤΘόϤϟ΍ ϲΗΎΒϨϟ΍ ΝΎΘϧϹ΍ ϭ Ϟδόϟ΍ ΝΎΘϧ· ϞϠϘϴϓ Δϔ΋Ύτϟ΍ﻲ ϥϵ΍ ϰΘΣϭˬ ﻻ ﺗﻮﺟﺪ . ϦϋΚΤΒϠϟΔγ΍έΪϟ΍ϩάϫϑΪϬΗˬϚϟΫϰϠϋ˯ΎϨΑϭ ˱ΎϴϤϟΎϋΓΪϤΘόϣνήϤϟ΍΍άϫΔΤϓΎϜϤϟΓΩΪΤϣΔϴΠϴΗ΍ήΘγ· . ϟΔϠϳΪΑΔϘϳήρﻤﻜﺎﻓﺤﺔ ϭΎϬΘϔϠϜΗΔϠϘΑϭΔϘτϨϤϟ΍ϲϓΎϫήϓϮΘΑΰϴϤΘΗΔϴΗΎΒϧΕΎμϠΨΘδϣϡ΍ΪΨΘγΎΑήτϔϟ΍΍άϫ ΔΌϴΒϟ΍ϰϠϋϲΒϠδϟ΍ΎϫήϴΛ΄ΗϡΪϋ . ΎϳϼΧϦϣΖόϤΟϲΘϟ΍ϭΔΘϴϤϟ΍Ϟδόϟ΍ϞΤϧΕΎϗήϳϦϣ ήτϔϟ΍ϝΰϋϢΗΔγ΍έΪϟ΍ϩάϫϝϼΧ A. apis ήϴϳΎόϤϟ΍Δτγ΍ϮΑϪϴϠϋϑήόΘϟ΍ϭήτϔϟ΍ΪϳΪΤΗϢΗϭˬϦϴτδϠϓˬΓΰϏωΎτϗˬϝΎϤθϟ΍ΔψϓΎΤϣϲϓΔΑΎμϣ ΔϴΗΎΒϧΕΎμϠΨΘδϣΓήθόϟ ΔϴϟΎόϔϟ΍ϢϴϴϘΗϢΗ ϭ Νϭ΍ΰΘϟ΍έΎΒΘΧ΍Δτγ΍ϮΑϭϪϟΔϳήϬΠϤϟ΍ϭΔϳήϬψϤϟ΍ . ﺗﺸﻤﻞ Δϓ΍ϮΠϟ΍ϭΔτθϘϟ΍ϭΔϔϴτϘϟ΍ϭΔϓήϘϟ΍ϭΓΎηήϔϟ΍ϭΪόδϟ΍ϭ΢ϴθϟ΍ϭ ΔδϴδϣΪϟ΍ϭέΫΎόϟ΍ϭϥΎϣήϟ΍ : ϮϤϨϟ΍έΎτϗ΃ϑΎμϧ΃ΕϻΪόϣέΎΒΘΧ΍ϭ ςϴΒΜΘϠϟϰϧΩϷ΍ΰϴϛήΘϟ΍έΎΒΘΧ΍Δτγ΍ϮΑήτϔϟ΍ϮϤϧϰϠϋ (MIC) γ΍έΩ ΎϤϛ ˬϱήτϔϟ΍ ϝΰϐϠϟﺖ ΖϳήΟ΃ϭ ˬέϮϛάϤϟ΍ ήτϔϟ΍ ϰϠϋ ϥΎϣήϟ΍ ΓήϫίκϠΨΘδϤϟ ήϴΛ΄Θϟ΍ ωϮϧ Ϟδόϟ΍ϞΤϧΔϟΎϐηϰϠϋϥΎϣήϟ΍ΓήϫίκϠΨΘδϤϟΔϴϤδϟ΍Δγ΍έΪϟΔΑήΠΗ . ΔϴϠϋΎϓήΜϛϷ΍ϥΎϛϥΎϣήϟ΍ΓήϫίκϠΨΘδϣϥ΃Ξ΋ΎΘϨϟ΍ΕήϬυ΃ΪϘϟϭ ﺿﺪ ΰϴϛήΘϟ΍ϥΎϛΚϴΣ A. apis ΓΎηήϔϟ΍ϭΔϓήϘϟ΍ϭΔϓ΍ϮΠϟ΍κϠΨΘδϣϦϣϼϛήϴΛ΄Θϟ΍ϲϓϩϼΗ Ϫϟ ςϴΒΜΘϠϟϰϧΩϷ΍ .(0.625% ) (MIC) ήϴΛ΄Η Ύϣ΃ ϲϟ΍ϮΘϟ΍ ϰϠϋ ϭ ˬ Ξ΋ΎΘϨΑ ˱ΎτγϮΘϣ ΍ήϴΛ΄Η ΕήϬυ΃ ΚϴΣ . 5% 2.5% 1.23% (MIC) ϝΎόϓ΍ήΛ΃ϲτόΗϢϟΔτθϘϟ΍ϭΪόδϟ΍ϭΔϔϴτϘϟ΍ϭ΢ϴθϟ΍ϭ ΔδϴδϣΪϟ΍ ϭέΫΎόϠϟϯήΧϷ΍ΕΎμϠΨΘδϤϟ΍ ϱήτϔϟ΍ϮϤϨϠϟέΎτϗϷ΍ϑΎμϧ΃ΕϻΪόϣΔγ΍έΩϰϟ·ΔΒδϨϟΎΑϭ Δγ΍έΪϟ΍ϩάϫϲϓέϮϛάϤϟ΍ήτϔϟ΍ϰϠϋˬﻓﻘﺪ . ΔϓήϘϟ΍ϭΓΎηήϔϟ΍ϭΔϓ΍ϮΠϟ΍ϭϥΎϣήϟ΍ΕΎμϠΨΘδϤϟ Δϴ΋ΎμΣ·ΕϻϻΩΞ΋ΎΘϨϟ΍ΕήϬυ΃ (P < 0.05) ϝΰϐϟ΍ϮϤϧϰϠϋϒϴόοϯήΧϷ΍ ΕΎμϠΨΘδϤϟ΍ήϴΛ΄ΗϥΎϛΎϤϨϴΑϝϭϷ΍έΎΒΘΧϻ΍ϊϣΔϘϔΘϣΞ΋ΎΘϨϟ΍ΖϧΎϛϭ ΕΪΟϭ΃ϭ Δϴ΋ΎμΣ·ΔϟϻΩϱ΃ϲτόΗϢϟϭΔγ΍έΪϟ΍ϲϓΔϣΪΨΘδϤϟ΍ΰϴϛ΍ήΘϟ΍ϊϴϤΠϟϱήτϔϟ΍ .(P < 0.05) βϴϟϭ ήτϔϠϟ΢ΑΎϛήϴΛ΄ΗήΒΘόϳϥΎϣήϟ΍ΓήϫίκϠΨΘδϤϟΔϔϠΘΨϤϟ΍ΰϴϛ΍ήΘϟ΍ήϴΛ΄Ηϥ΃Ξ΋ΎΘϨϟ΍ A. apis ϪϟϦϜϳϢϟϥΎϣήϟ΍ΓήϫίκϠΨΘδϤϟΔϣΪΨΘδϤϟ΍ΰϴϛ΍ήΘϟ΍ϥ΃Δγ΍έΪϟ΍ΕήϬυ΃ΎϤϛϭ Ϫϟ˱ϼΗΎϗ˱΍ήϴΛ΄Η . δόϟ΍ϞΤϧΕϻΎϐηϰϠϋΖϴϤϣήϴΛ΄Ηﻞˬϭ ΕϻΎϐθϠϟΔϴϤϛ΍ήΘϟ΍ΕΎϴϓϮϟ΍ΔΒδϧΖϧΎϛΚϴΣ 3.6% 1.1% ϲϟ΍ϮΘϟ΍ϰϠϋκϠΨΘδϤϠϟ ϭ ϭ ΰϴϛήΘϠϟ ϭ . 0% 25% %12.5 4.4% ήτϓΪοϱϮϗρΎθϧήϬψϳϱάϟ΍ϥΎϣήϟ΍ΓήϫίκϠΨΘδϣϥ΃ϰϟ·ϞλϮΘϟ΍ϢΗˬΞ΋ΎΘϨϟ΍ϰϠϋ˯ΎϨΑϭ A. ˱ ΐϠτΘϳϚϟΫϥ΃ϻ·ˬϞδόϟ΍ϞΤϧϲϓϱήϴηΎΒτϟ΍ΔϨπΤϟ΍νήϤϟϱήτϓΩΎπϤϛ ϩέΎΒΘϋ΍ϦϜϤϳ apis ν΍ήϣ΃ΝϼόϟΎϬϣ΍ΪΨΘγ΍ϦϜϤϳΔϣΪΨΘδϤϟ΍ΕΎμϠΨΘδϤϟ΍ΖϧΎϛ΍Ϋ·ΎϣέΎϬυϹΕΎγ΍έΪϟ΍ϦϣΪϳΰϤϟ΍ ΔϴϠΤϨϟ΍ΕΎΠΘϨϤϟ΍ΓΩϮΟϰϠϋϭϞΤϨϟ΍ϰϠϋΔϴϤδϟ΍έΎΛϵ΍ϞϴϠϘΘϟϞδόϟ΍ϞΤϧ . ϱήτϔϟ΍ ΢ΒϜϟ΍ ΔϴΗΎΒϧ ΕΎμϠΨΘδϣ Δϴδϴ΋ήϟ΍ ΕΎϤϠϜϟ΍ ﻧﺤﻞ - - - MIC - Ascosphaera apis : ΕΎΒϧ –ΓΎηήϔϟ΍ΕΎΒϧ Ϊόδϟ΍ΕΎΒϧ ΢ϴθϟ΍ΕΎΒϧ ΔδϴδϣΪϟ΍ΕΎΒϧ έΫΎόϟ΍ΕΎΒϧ ϥΎϣήϟ΍ΕΎΒϧ Ϟδόϟ΍ - - - - - - Δϓ΍ϮΠϟ΍ΕΎΒϧ ΔτθϘϟ΍ΕΎΒϧ ΔϔϴτϘϟ΍ΕΎΒϧ –ΔϓήϘϟ΍ . - - VII Table of contents Items Pages ……………………………………………………………... Dedication I ……………………………………………………………. Declaration II ……………………………………………………… Acknowledgment III …………………………………………………….. Abstract (English) V ……………………………………………………... Abstract (Arabic) VII ……………………………………………………… Table of contents . VIII …………………………………………………………… List of tables XII …………………………………………………………. List of figures XIII …………………………………………………………. Abbreviations XV Chapter 1: Introduction …………………………………………………………. 1.1. Overview 1 ……………………………………………………….. 1.2. Significance 4 ………………………………………….. 1.3. Objectives of the study 6 …………………………………………. 1.3.1. General objective 7 …………………………………………. 1.3.2. Specific objectives 7 Chapter 2: Literature Review ………………………………. 2.1. Honey bees (Apis mellifera L.)..... 8 …………………………………………... 2.2. Chalkbrood disease... 9 ……………………………………………….. 2.2.1. Description 9 ………………… 2.2.2. Morphology of Ascosphaera apis fungi 10 ……………... 2.2.3. Taxonomy of Ascosphaera apis fungi ..... 10 ………………………….. 2.2.4. Life Cycle of Ascosphaera apis 11 ……………………………………..
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    First Detection of the Larval Chalkbrood Disease Pathogen Ascosphaera apis (Ascomycota: Eurotiomycetes: Ascosphaerales) in Adult Bumble Bees Maxfield-Taylor, S.A., Mujic, A.B., Rao, S. (2015) First Detection of the Larval Chalkbrood Disease Pathogen Ascosphaera apis (Ascomycota: Eurotiomycetes: Ascosphaerales) in Adult Bumble Bees. PLoS ONE 10(4). doi:10.1371/journal.pone.0124868 10.1371/journal.pone.0124868 Public Library of Science Version of Record http://cdss.library.oregonstate.edu/sa-termsofuse RESEARCH ARTICLE First Detection of the Larval Chalkbrood Disease Pathogen Ascosphaera apis (Ascomycota: Eurotiomycetes: Ascosphaerales) in Adult Bumble Bees Sarah A. Maxfield-Taylor1, Alija B. Mujic2, Sujaya Rao1* 1 Department of Crop and Soil Science, Oregon State University, Corvallis, Oregon, United States of America, 2 Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon, United States of America * [email protected] Abstract OPEN ACCESS Fungi in the genus Ascosphaera (Ascomycota: Eurotiomycetes: Ascosphaerales) cause Citation: Maxfield-Taylor SA, Mujic AB, Rao S (2015) chalkbrood disease in larvae of bees. Here, we report the first-ever detection of the fungus First Detection of the Larval Chalkbrood Disease in adult bumble bees that were raised in captivity for studies on colony development. Wild Pathogen Ascosphaera apis (Ascomycota: queens of Bombus griseocollis, B. nevadensis and B. vosnesenskii were collected and Eurotiomycetes: Ascosphaerales) in Adult Bumble maintained for establishment of nests. Queens that died during rearing or that did not lay Bees. PLoS ONE 10(4): e0124868. doi:10.1371/ journal.pone.0124868 eggs within one month of capture were dissected, and tissues were examined microscopi- cally for the presence of pathogens.
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