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Detection of the Bacteria Leifsonia Xyli Subsp. Xyli (Davis)

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Detection of the Bacteria Leifsonia Xyli Subsp. Xyli (Davis) Detection of the Bacteria Leifsonia xyli subsp. xyli (Davis) Using Lateral Flow Immunoassay Test and Modified Culture Media for Improving the diagnosis of Sugarcane Ratoon Stunt Disease Maali Mohamed Elamin Abdalla B.Sc. (Honors) in Agricultural Science (Plant Protection) University of Gezira (2012) A Dissertation Submitted to the University of Gezira in Partial Fulfillment of the Requirements for the Award of the Degree of Master of Science in Plant Pathology Plant Pathology Center Faculty of Agricultural Sciences March / 2020 i Detection of the Bacteria Leifsonia xyli subsp. xyli (Davis) Using Lateral Flow Immunoassay Test and Modified Culture Media for Improving the diagnosis of Sugarcane Ratoon Stunt Disease Maali Mohamed Elamin Abdalla Supervision Committee: Name Position Signature Dr. Adam Mohamed Ahmed Gadelseed Main Supervisor ………................ Dr. Elshafie Elhassan ElshafieElhag Co-supervisor …………………. Date:…………. i Detection of the Bacteria Leifsonia xyli subsp. xyli (Davis) Using Lateral Flow Immunoassay Test and Modified Culture Media for Improving the diagnosis of Sugarcane Ratoon Stunt Disease Maali Mohamed Elamin Abdalla Examination Committee: Name Position Signature Dr. Adam Mohamed Ahmed Gadelseed Chair person ………................ Dr. Omer Osman A. Elbasher Internal Examiner …………………. Dr. OmymaElmahi M. Alkhalifa External Examiner …….................... Date:…………. ii DEDICATION This work is dedicated to: My father, my mother, Myhusband, My sisters, my brother, And my friends Maali iii ACKNOWLEDGMENT First of all my praise and thanks are due to Allah who gave me the strength and patience through the course of this study. I am very grateful tomy supervisor Dr. Adam Mohamed Ahmed Gadelseedfor interest, guidance, patience, valuable advices and encouragement through the period of this study. I thank a lot my co. supervisor Dr. Elshafie Elhassan Elshafie Elhag for his guidance and final correction of this study. Words of thank and gratitude are extended to Dr. Stephan Winter (DSMZ, plant virus division, Germany) for offering the lateral flow immunoassay test which represented the core for this study. I pray may Allah accept the great help I received from my late teacher Ustaz Hamraii Mohamed Ahmed Alimay Allah reward him the paradise (ameen), without him, this work might have not seen the light. My thanks are also to all academic and technical staff of plant pathology center. Special thanks to Uncle Elgaily Mohamed Yousif who took a good care of the work at the greenhouse of plant pathology center. Also I would like to send my deepest thanks to my colleagues at Gunied sugarcane research center and Guneid sugar factory for their encouragement and support through this study stages. Finally, the sincere thanks are to my dad Mohamed Elamin, my mam Mona, my husband Waleed Eltigani, my sisters lina,Tirhab, Soudaa and Waad, my brother Abdalla, my friends Asma, Esraa, Umkalthom, Shadia, Alaa and Amna for supporting me. iv Detection of the Bacteria Leifsonia xyli subsp. xyli (Davis) Using Lateral Flow Immunoassay Test and Modified Culture Media for Improving the diagnosis of Sugarcane Ratoon Stunt Disease By Maali Mohamed ElaminAbdalla Abstract Sugarcane (Saccharum officinarum L.) is an important crop grown in the tropical and subtropical regions. It accounts for 70% of the sugar produced globally. In Sudan, sugarcane is an economically important crop widely cultivated in the country. Sugarcane is affected by ratoon stunt disease (RSD) caused by the bacteria Leifsonia xyli subsp. xyli which results in severe losses in cane yield and quality attributes. The diagnosis of RSD which is an important tool for disease control measures is difficult for some constrains. These constrains include; the RSD has no unique symptoms, the L. xyli subsp. Xyli is extremely fastidious in its nutritional requirements and needs relatively long time for visible pure growth to occur (up to 28 days) and the bacteria concentration in plant tissue is low. This complicates the isolation and microscopic examination and the serological techniques face the problem of sensitivity. The molecular techniques are dependable tools for diagnosis of plant diseases, but they need sophisticated well equipped laboratory. The aims of this study were improving the diagnosis of RSD through detection and identification of L. xyli subsp. xyli using lateral flow immunoassay test, extraction buffer and synthetic culture media for the bacteria. Samples of RSD infected cane stalks were collected from small plots of chewing cane grown by individuals around Wadmedani city. Lateral flow immunoassay which is a simple technique based on immune-chromatography of specific antibodies, was used for detection and identification of the bacteria. The low sensitivity of the technique was compensated for by bacteria multiplication in a synthetic media. A modified media supplemented with amino acid methionine was used to shorten the time for visible bacterial growth. Obvious stunting symptoms were observed on sugarcane grown by individuals as chewing cane around Wad medani city with incidence reaching 100%, and the cane stalk length was reduced to one third. The reddish discoloration was observed on 80% of the diseased cane. The growth of L. xyli subsp. Xyli was noticed on media supplemented with amino acid methionine 5 days after inoculation. Lateral flow immunoassay detected and identified L. xyli subsp. xyli in the cane extract and from the culture and the sensitivity of the test was dependent on the type of extraction buffer. Not all the stunting symptoms were found associated with L. xyli subsp. Xyli on basis of synthetic culture and lateral flow immunoassay test. The lateral flow immunoassay test and the improved synthetic culture are recommended as accurate and dependable tools in the future for detection and identification of L. xyli subsp. Xyli to find out RSD occurrence and incidence on symptomatic and symptomless cane stalks. v كشف تكتيريا)ديفيس( Leifsonia xyli subsp.xyliتإستخذام إختثار اﻷنسياب الجانثي المصلي ووسائط التزريع المعذلة لتحسين تشخيص مرض تقزم الخلف في قصة السكر. معالي محمذ اﻷمين عثذهللا ملخص الذراسة لظت انسكز (.Saccharum officinarum L) يحظٕل يٓى ٚزرع فٙ انًُبؽك انًذارٚخ ٔشجّ انًذارٚخ. ًٚضم 07 ٪ يٍ انسكز انًُزظ ػهٗ يسزٕٖ انؼبنى. فٙ انسٕداٌ، ؼٚزجز لظت انسكز يٍ انًحبطٛم انًًٓخ الزظبدًٚب ٔانًزرػٔخ ػهٗ َطبق ٔاسغ فٙ انجﻻد.ٚزأصز لظت انسكز ثًزع رمزو انخهف )RSD( انذ٘ رسججّ ثكزٛزٚب (Leifsonia xyli subsp. xyli(Davis ٔانذ٘ ٚؤد٘ إنٗ خسبئز فبدحخ فٙ كًٛخ انمظت ٔطفبد انغٕدح. انزشخٛض ؼٚذ أداح يًٓخ نزذاثٛز يكبفحخ اﻷيزاع نكُّ طؼت نًزع )RSD( نجؼغ انٛؼًمبد . ْذِ انٛؼًمبد رشًم:نٛس نـ RSD أػزاع خبرعٛخ ٔداخهٛخ يزفزدح ، فجكززٚب Leifsoniaxyli subsp.xyli شذٚذح انحسبسٛخ فٙ يزطهجبرٓب انغذائٛخ ٔرحزبط إنٗ ٔلذ ٕٚؽم َسجًٛب نحذٔس ًَٕ َمٙ يزئٚ( ٙظم إنٗ ٚ 82ٕ ًيب(. أؼٚب انجكزٛزٚب رٕعذ فٙ أَسغخ انُجبد ثززكٛز يُخفغ. ْذا ٚغؼم انؼزل ٔ انفحض انًغٓز٘ طؼجب ً ٔانزمُٛبد انًظهٛخ رٕاعّ يشكهخ انحسبسٛخ. انزمُٛبد انغزٚئٛخ ْٙ أدٔاد ًٚكٍ اﻻػزًبد ػهٛٓب نزشخٛض اﻷيزاع انُجبرٛخ ، نكُٓب رحزبط إنٗ يخزجز يزطٕر يغٓز عٛ ًذا.ْذفذ ْذِ انذراسخ إنٗ رحسٍٛ رشخٛض يزع رمزو انخهف فٙ لظت انسكز )RSD( يٍ خﻻل كشف ٔرؼزٚف ثكزٛزٚبL. xylisubsp.xyil ثبسزخذاو اخزجبر اﻻَسٛبة انغبَجٙ انًظهٙ، يحبنٛم اﻻسزخﻻص ٔثٛئبد انززرٚغ انغذائٛخ انظُبٛػخ نهجكززٚب.رى عًغ ٛػُبد يٍ لظت انسكز انًظبثخ ثًزع رمزو انخهف (RSD( يٍ انًسبحبد انظغٛزح انًزرػٔخ ثٕاسطخ اﻷفزاد حٕل يذُٚخ ٔد يذَٙ. اﻻَسٛبة انغبَجٙ انًظهٙ ْٕ رمُٛخ ثسٛطخ رؼزًذ ػهٗ انزحهٛم انكزٔيبرٕعزافٙ انًظهٗ ﻷعسبو يؼبدح يٛؼُخ رى اسزخذايٓب نكشف ٔ رؼزٚف ثكزٛزٚب lxx رى رٕٚؼغ انحسبسٛخ انًُخفؼخ نٓذِ انزمُٛخ ؽ ٍػزٚك إكضبر انجكزٛزٚب فٙ ٔسؾ اططُبٙػ. رى اسزخذاو ٔسبئؾ يؼذنخ ثئػبفخ انحًغ اﻷيُٛٗ يٛضٍَٕٛٛ نزمظٛز انفززح انًطهٕثخ نحذٔس ًَٕ ثكزٛز٘ يزئٙ. نٕحظذ أػزاع رمزو ٔاػحخ ػهٗ لظت انسكز انًزرٔع ثٕاسطخ اﻷفزاد فٙ أَحبء يذُٚخ ٔد يذَٙ يغ َسجخ إطبثخ رظم إنٗ %100 ٔخفغ انزمزو ؽٕل سبق انمظت نهضهش. ٔلذ نٕحظ رغٛز نٌٕ أَسغخ انحزيخ انػٕبئٛخ إنٗ انًحًز فٙ %80 يٍ انمظت انًزمزو. حذس ًَٕ يزئٙ نـ ػL. xyli subsp. xyliهٗ انٕسبئؾ انًكًهخ ثحًغ أﻷيُٙٛ يٛضٍَٕٛٛ ثؼذ 5 أٚبو يٍ انؼزل. رى كشف ٔ رؼزٚف ثكزٛزٚبL. xylisubsp. xyli فٙ اخزجبر اﻻَسٛبة انغبَجٙ انًظهٙ فٙ يسزخهض انمظت ٔيٍ انززرٚغ ٔحسبسٛخ اﻻخزجبر رؼزًذ ػهٗ َٕع انًحهٕل انًزؼبدل ﻻسزخﻻص انجكزٛزٚب. حست اﻷٔسبؽ انغذائٛخ اﻻططُبٛػخ ٔاخزجبر اﻻَسٛبة انغبَجٙ انًظهٙ نٛسذ كم أػزاع انزمزو يزرجطخ ثجكٛزٚب .L. xyli subsp xyli.إخزجبر اﻹَسٛبة انغبَجٙ انًظهٙ ٔانززرٚغ اﻻططُبٙػ انًحسٍ أدٔاد دلٛمخ ًٚكٍ اﻻػزًبد ػهٛٓب ي س ز م جﻻ ً نهكشف ػٍ ٔرؼزٚفL. xyli subsp. xyliنؼًزفخ حذٔس َٔسجخ اﻹطبثخ ثًزعػ RSDهٗ سٛمبٌ لظت انسكز انزٙ رظٓز ػهٛٓب أػزاػ ب ً أثذَٔٓب. vi LIST OF CONTENTS DEDICATION ......................................................................................... iii ACKNOWLEDGMENT .......................................................................... iv Abstract .................................................................................................... v vi ................................................................................................يهخض انذاسخ LIST OF CONTENTS ............................................................................. vii LIST OF TABLES .................................................................................... x LIST OF FIGURES .................................................................................. xi CHAPTER ONE......................................................................................1 INTRODUCTION..................................................................................... 1 CHAPTER TWO ....................................................................................4 LITREATURE REVIEW .........................................................................
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