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

Plant Pathology

Plant Pathology Center

Faculty of Agricultural Sciences

March / 2020

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:………….

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:………….

DEDICATION

This work is dedicated to: My father, my mother, Myhusband, My sisters, my brother, And my friends

Maali

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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.

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.

كشف تكتيريا)ديفيس( Leifsonia xyli subsp.xyliتإستخذام إختثار األنسياب الجانثي المصلي ووسائط التزريع المعذلة لتحسين تشخيص مرض تقزم الخلف في قصة السكر.

معالي محمذ األمين 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 انجالد.ٚ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 األيزاع 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 االػزًبد ػهٛٓب نزشخٛض األيزاع انُجبرٛخ ، نكُٓب رحزبط إنٗ يخزجز يزطٕر يغٓز عٛ ًذا.ْذفذ ْذِ انذراسخ إنٗ رحسٍٛ رشخٛض يزع رمزو انخهف فٙ لظت انسكز )RSD( يٍ خالل كشف ٔرؼزٚف ثكزٛزٚبL. xylisubsp.xyil ثبسزخذاو اخزجبر االَسٛبة انغبَجٙ انًظهٙ، يحبنٛم االسزخالص ٔثٛئبد انززرٚغ انغذائٛخ انظُبٛػخ نهجكززٚب.رى عًغ ٛػُبد يٍ لظت انسكز انًظبثخ ثًزع رمزو انخهف (RSD( يٍ انًسبحبد انظغٛزح انًزرػٔخ ثٕاسطخ األفزاد حٕل يذُٚخ ٔد يذَٙ. االَسٛبة انغبَجٙ انًظهٙ ْٕ رمُٛخ ثسٛطخ رؼزًذ ػهٗ انزحهٛم انكزٔيبرٕعزافٙ انًظهٗ ألعسبو 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 األيُٛٗ يٛضٍَٕٛٛ نزمظٛز انفززح انًطهٕثخ نحذٔس ًَٕ ثكزٛز٘ يزئٙ. نٕحظذ أػزاع رمزو ٔاػحخ ػهٗ لظت انسكز انًزرٔع ثٕاسطخ األفزاد 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 أأليُٙٛ يٛضٍَٕٛٛ ثؼذ 5 أٚبو يٍ انؼزل. رى كشف ٔ رؼزٚف ثكزٛزٚبL. xylisubsp. xyli فٙ اخزجبر االَسٛبة انغبَجٙ انًظهٙ فٙ يسزخهض انمظت ٔيٍ انززرٚغ ٔحسبسٛخ االخزجبر رؼزًذ ػهٗ َٕع انًحهٕل انًزؼبدل السزخالص انجكزٛزٚب. حست األٔسبؽ انغذائٛخ االططُبٛػخ ٔاخزجبر االَسٛبة انغبَجٙ انًظهٙ نٛسذ كم أػزاع انزمزو يزرجطخ ثجكٛزٚب .L. xyli subsp xyli.إخزجبر اإلَسٛبة انغبَجٙ انًظهٙ ٔانززرٚغ االططُبٙػ انًحسٍ أدٔاد دلٛمخ ًٚكٍ االػزًبد ػهٛٓب ي س ز م جال ً نهكشف ػٍ ٔرؼزٚفL. xyli subsp. xyliنؼًزفخ حذٔس َٔسجخ اإلطبثخ ثًزعػ RSDهٗ سٛمبٌ لظت انسكز انزٙ رظٓز ػهٛٓب أػزاػ ب ً أثذَٔٓب.

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 ...... 4

2.1 Sugarcane (Saccharum Officinarum) ...... 4 2.1.1 History ...... 4 2.1.2 Description of the crop ...... 4 2.1.3 Climatic Requirements ...... 4 2.1.4 Cultivation ...... 4 2.1.5 production ...... 5 2.1.6 Sugarcane production in Sudan: ...... 5 2.2 Sugarcane Insect pests: ...... 6 2.3 Sugarcane diseases: ...... 6 2.3.1 Smut diseases...... 7 2.3.2 Sugar cane ratoon stunting disease ...... 7 2.3.2.2 Causal agent ( Leifsonia xyli subsp xyli)...... 7 Taxonomy and Nomenclature...... 7 2.3.2.2.1 Description of bacteria ...... 8 2.3.2.2.2 Distribution of RSD ...... 8 2.3.2.2.3 Biology and Ecology...... 8 2.3.2.2.4 Hosts plant ...... 9 2.3.2.2.5 Symptoms ...... 9 2.3.2.2.6 Isolation...... 10 2.3.2.2.7 Diagnosis...... 10 2.3.2.2.7.1 Microscopic detection of L. xyli subsp. xyli: ...... 10

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2.3.2.2.7.2 Serological Techniques: ...... 11 2.3.2.2.7.2.1 Immuno-microscopy:...... 11 2.3.2.2.7.2.2 Enzyme-linkage immunosorbent assay (ELISA) ...... 11 2.3.2.2.7.2.3 Lateral flow immunoassay test (LFIA): ...... 12 2.3.2.2.8 Pathogenicity Test...... 13 2.3.2.2.9 DNA Techniques ...... 13 2.3.2.2.10 Similarities to other causal of stunting...... 13 2.3.2.2.11 Managements ...... 14 2.3.2.2.11.1 Cultural Control and Sanitary Methods...... 14 2.3.2.2.11.2 Physical control (heat treatment):...... 14 2.3.2.2.11.3 Sanitation: ...... 15 2.3.2.2.11.4 Host-Plant Resistance...... 15 2.3.2.2.11.5 Chemical Control...... 16

CHAPTER THREE...... 17

MATERIAL AND METHODS ...... 17

3.1 Collection of diseased materials: ...... 17 3.2 Observation of external and internal symptoms: ...... 17 3.3. Detection of Lxx bacteria: ...... 17 3.3.1 Lateral flow immunoassay test (serology): ...... 17 3.3.1.1. Extraction of the xylem sap from stunted cane stalks...... 18 3.3.1.2 Extraction buffer...... 18 3.3.1.2.1 Buffer1 (Prepared)...... 18 3.3.1.2.2 Buffer 2 (Ready, provided with the lateral flow test kit)...... 18 3.3.1.3 Loading of Xylem sap on Lateral flow immunoassay test: ...... 19 3.3.2Isolation of L. xyli subsp. xyli ...... 20 3.3.2.1. Preparation of culture media (0.5 L)...... 20 3.3.2.2 Inoculation of the media ...... 20 3.3.2.3 Sub-culture media: ...... 21 3.3.3 Gram staining: ...... 21 Staining mechanism...... 21

CHAPTER FOUR ...... 23

RESULTS...... 23 4.1 Ratoon stunting disease (RSD) incidence and diagnosis based on symptoms:…....23 4.2 Detection of (L. xyli susp. xyli): ...... 24 4.2.1 Lateral flow immunoassay (serology)...... 24 4.2.2 Effect of the culture media: ...... 26

CHAPTER FIVE...... 29

DISCUSSION ...... 29

CHAPTER SIX ...... 32

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CONCLUSION AND RECOMMENDATION ...... 32

1.6CONCLUSION ...... 32

1.6RECOMMENDATIONS ...... 32

REFERENCES...... 33

LIST OF TABLES

Table number Table title Page

Table 1 The average internode length of healthy and stunted cane 24 stalks. Table 2 The effect of extraction buffer on detection sensitivity of 25 lateral flow immunoassay test forLxx.. Table 3 Detection of L .xyli subsp. xyli by symptoms (external and 26 internal) and serological test (lateral flow immunoassay). Table 4 Detection and identification of L. xyli subsp. xyli in cane 27 extracts and culture using lateral flow immunoassay test.

LIST OF FIGURES

Figure No. Figure Title Page Figure 1 Lateral flow test 18 Figure 2 Extraction of stunted cane stalk xylem sap and transfer to Lateral 19 flow test Figure 3 Laminar flow, the hygienic sterilized clean working place 21 Figure 4 The external symptom of Sugarcane ratoon stunt disease (RSD). 23 Figure 5 Internal symptoms of Sugarcane ratoon stunt disease (RSD) 24 Figure 6 The effect of extraction buffer on the detection sensitivity of lateral 25 flow immunoassay test forLxx.. Figure 7 Detection and identification of L. xyli subsp. xyli in cane extracts and 27 culture, using lateral flow immunoassay test.

CHAPTER ONE INTRODUCTION Sugarcane (Saccharumofficinarum) is an economically important crop that is predominantly grown in the tropical and subtropical regions. Historically, sugarcane is indigenous to tropical South America and Southeast Asia with S. barberi in India and S. officinarum in New Guinea. Approximately 70% of the sugar produced globally comes from open pollinated and hybrid ofS. officinarum. Its production faces some agronomical challenges include soil problems, pests and diseases. which are the most serious problems that affected sugarcane. The pests include termite, sugarcane borer (stem borers), and the plant hopper insect Eumetopinaflavipes which acts as virus vector. Sugarcane is also threatened by several fungal, viral and bacterial diseases. Diseases caused by fungi (sugarcane smut), bacteria (ratoon stunt disease), viruses and phytoplasmas (sugarcane grassy shoot) pose a serious threat to sugarcane cultivation and adversely affect the yield and sugar recovery in different sugarcane growing regions. Among the bacterial diseases ratoon stunting disease (RSD) results in severe losses in cane yield and quality attributes. RSD has been identified from different regions of the world using serological and molecular tools. Management of sugarcane ratoon stunt disease depends on proper identification and detection of the causal agent (Leifsonia xyli subsp.xyli).Therefore, diagnosis is an important tool for disease control measures. The diagnosis of ratoon stunting disease (RSD) in sugarcane is difficult due to the little specificity of its symptoms. The RSD has no unique external and internal symptoms which are often inadequate for diagnosis in the field. Therefore the Laboratory tests are almost always required for accurate diagnosis of RSD which are also complicated. The phytobacteria (Leifsonia xyli subsp. xyli (Davis) needs particular nutritional requirements for isolation in the synthetic media besides, the relatively long time for visible pure culture. The bacteria have a low concentration in the diseased plants to be examined under the microscope. Molecular techniques are dependable tools for diagnosis of plant disease, although they might need sophisticated well equipped laboratory. Lateral flow immunoassay is a serological technique with high specificity, does not need specialized devices or equipment, simple and so easy to perform. However, it has relatively low sensitivity compared with enzyme-linked immunosorbent assay (ELISA), this demerit is increased by the low concentration of the L. xyli subsp.xyliin

diseased sugarcane plants. The successful visual examination of sugarcane plants to determine symptomatically diseased plants, isolation and synthetic culture of the bacteria L. xyli subsp. xyli and the testing with lateral flow immunoassay technique could specifically identify and detect the incidence and severity of the RSD.

The general objective of this study: To devolop simple and easy diagnosticmethod/s fordetectionand identification of ratoon stunt disease bacteria (L. xyli subsp.xyli(Davis)) using multiple techniques; symptomatology, serology, microscopic examinationand synthetic culture. The specific objectives of this study are: o To find out suitable synthetic culture media that accelerates the growth rate of bacteria. o To enhance detection and identification sensitivity of lateral flow immunoassay through extraction and isolation of the bacteria L. xyli subsp xyli (Davis).

CHAPTER TWO LITREATURE REVIEW 2.1 Sugarcane (Saccharum officinarum) Sugarcane or sugar cane belongs to the grass family Poaceae, an economically important seed plant family that includes maize, wheat, rice, and sorghum, and many forage crops. Sugarcane are several species of tall perennialtrue grasses of the genus Saccharum, tribe and ropogoneae, used for sugarproduction. All sugarcane species can interbreed and the major commercial cultivars are complex hybrids,Vilela et al.,(2017). 2.1.1 History Sugarcane was an ancient crop of the Austronesian and Papuan people. It was introduced to Polynesia, Island Melanesia, and Madagascar in prehistoric times via Austronesian sailors. Itwas also introduced tosouthern China and India by Austronesian traders at around 1200 to 1000 BC. The Persians, followed by the Greeks, encountered the famous "reeds that produce honey without bees" in India between the 6th and 4th centuries BC. They adopted and then spread sugarcane agriculture, (FAO 2009). 2.1.2 Description of the crop Sugarcane plant is tall perennial true grass that forms lateral shoots at the base to produce multiple stems, typically three to four metre (10 to 13 ft) high and about 5 cm (2 in) in diameter. The stems grow into cane stalk which is stout, jointed, and fibrous and rich in the sugarsucrose accumulates in the internodes. A mature stalk is typically composed of 11–16% fiber, 12–16% soluble sugars, 2–3% non-sugars, and 63–73% water,Schueneman, (2002). 2.1.3 Climatic Requirements Sugarcane is cultivated in the tropics and subtropics in areas with a plentiful supply of water for a continuous period of more than six to seven months each year, either from natural rainfall or through irrigation. It needs a minimum of 60 cm (24 in) of annual moisture. 2.1.4 Cultivation Although some sugarcane produces seeds, modern stem cutting has become the most common reproduction method. Each cutting must contain at least one bud, and the cuttings are sometimes hand-planted. In more technologically advanced countries like

the United States and Australia, billet planting is common. Once planted, a stand can be harvested several times; after each harvest, the cane sends up new stalks, called ratoons. Successive harvests give decreasing yields, eventually justifying replanting. Two to 10 harvests are usually made depending on the type of culture. In a country with a mechanical agriculture looking for a high production of large fields, like in North America, sugar canes are replanted after two or three harvests to avoid a lowering in yields. In countries with a more traditional type of agriculture with smaller fields and hand harvesting, like in the French island, sugar cane is often harvested up to 10 years before replanting. 2.1.5 production Sugarcane is the world's largest crop by production quantity, with 1.9 billion tons produced in 2016, and Brazil accounting for 41% of the world total. In 2012, the Food and Agriculture Organization (FAO) estimated that sugarcane was cultivated on about 26 million hectares (64 million acres), in more than 90 countries, with a worldwide harvest of 1.83 billion tons. The average yield of cane stalk is 60–70 tons per hectare (24–28 long ton/acre; 27–31 short ton/acre) per year. However, this figure can vary between 30 and 180 tons per hectare depending on knowledge and crop management approach used in sugarcane cultivation. Sugarcane is a cash crop, but it is also used as livestock fodder,Perez and Rena,(1997).Brazil is the largest producer of sugar in the world. The next five major producers, in decreasing amounts of production, were India, China, Thailand, Pakistan and Mexico,Draycott, (2006). The main product of sugarcane is sugar (sucrose), cane accounts for 79% of sugar produced. Other products derived from sugarcane include falernum, molasses, rum, cachaça (a traditional spirit from Brazil), bagasse, and ethanol Dahliaet al.,(2009). 2.1.6 Sugarcane production in Sudan: Sugarcane is one of the important economical crops of Sudan, and widely cultivated in several parts of the country. Cane sugar production in the Sudan is rather relatively late since the country depended entirely on imported sugar until 1962. Sugarcane was grown commercially in Sudan for the first time at Guneid area and actual sugar production was realized in 1963 with the establishment of Guneid sugar factory. Since then a number of sugar factories came into operation at New Halfa factory (1964/65), West Sennar Sugar factory (1976/77), Assalya Sugar Factory (1979/81), Kenana Sugar Company (1980/81), and finally White Nile Sugar Company

(2011/2012). The total acreage under sugar production is 376.5 thousand acres Obeid, (2005). Sugarcane production in Sudan was supported by establishment of two research stations, Gunied sugarcane research station and Kenana Sugar Company research station. The earliest sugarcane cultivars used were NCO 310, NCO 376, and CO 527 which replaced later on by the present high yielding main cultivars (co 775, Co 797 and co 6806). Plant crop constituted generally 15% of the total cultivated areas leaving the majority for ratoon crop (5-6 ratoons). The growth period of the crop is 14 - 16 month for plant cane and 11-12 month for ratoons. The crop is harvested using both manual and mechanical method, harvesting is normally done at winter months (season). Research finding coupled with management experience resulted in high improvement in cane varieties and cultural practices. These improvement, late by, boosted the average cane yield to reach 116 ton/ha in some sugarcane estate which is comparable to the international yield levels. 2.2 Sugarcane Insect pests: The sugarcane important insect pests include cane beetle known as cane grub (Tomarus subtropicus (Baltchley)) can substantially reduce crop yield by eating roots; it can be controlled with imidacloprid (Confidor) or chlorpyrifos (Dursban) insecticides. Other important pests are the larvae of some butterfly/moth species, including the turnip moth, sugarcane borer (Diatraea saccharalis), African sugarcane borer (Eldanasa ccharina), Mexican rice borer (Eoreumaloftini),African armyworm (Spodopteraexempta), leaf-cutting ants, termites, spittlebugs (especially Mahanarvafimbriolata and Deoisflavopicta), and the beetleMigdolusfryanus,Samuels et al.,(1990). 2.3Sugarcane diseases: Different types of pathogens infect sugarcane; fungal pathogens such as sugarcane smut (whiptail disease) and pokkahboeng caused by Fusarium moniliforme. The bacteria Leifsonia xyli subsp. xyli, Xanthomonasaxonopodis and Colletotrichumfalcatum, cause the sugarcane ratoon stuntdisease (RSD),gumming disease and red rot disease respectively. Sugarcane grassy shoot disease caused by Phytoplasma. Viral diseases affecting sugarcane include sugarcane mosaic virus, maize streak virus, and sugarcane yellow virus,Rao and Ford (2000).

2.3.1 Smut diseases Among sugarcane fungal disease smut is most devastating and threatening disease in all sugarcane growing areas worldwide. 2.3.2 Sugar cane ratoon stunting disease Sugarcane ratoon stunting disease (RSD) caused by the bacterium Leifsonia xyli subsp.xyli (Lxx) is one of the most economically important diseases of sugarcane in the world,Davis et al.,(1980); Evtushenko et al.,(2000). The ratoon stunting disease (RSD) was first suspected as physiologicaldisorder in cultivator Q28 in Queensland, Australia. Steindl (1961) described it as a new disease of sugarcane and suggested that avirus like pathogen was associated with it. The name RSD is inaccurate since the disease deleteriously retards the growth of plant crop as well. To date disease has been reported from 41out of the 102 sugar cane growing countries in the world. RSD causes significant reduction in cane sugar production due to stunted plant growth, reduced numbers of tillers, thin stalks with shortened internodes and yellowing of foliage. Internally the internodes show discoloration which varies between red to yellow. 2.3.2.1 Economic important Ratoon stunting disease (RSD) is wide distribution and most economically significant disease of sugarcane. RSD can result in up to 50 % yield reduction in stalk weight and number depending on the variety and growing conditions and reduce quality,Taylor et al.,(2003); Comstock,(2002). 2.3.2.2 Causal agent ( Leifsonia xyli subsp xyli) Taxonomy and Nomenclature Taxonomic Tree

• Domain: Bacteria

• Phylum: Actinobacteria [phylum] • Class: Actinobacteria • Subclass: Actinobacteridae • Order: Actinomycetales • Suborder: Micrococcineae • Family: Microbacteriaceae • Genus: Leifsonia • Species: xyli subsp. xyli

The pathogen was taxonomically classified in 1984, Davis et al.,(1984) as Clavibacter xyli subsp. xyli. The subspecies designation distinguishes the ratoon stunting disease pathogen from the closely related pathogen, Clavibacter xyli subsp.cynodontis, which causes stunting disease of Bermuda grass (Cynodondactylon). These pathogens were reclassified in the genus Leifsonia by Evtushenko et al.,(2000) and then became Leifsonia xyli subsp. xyli(Lxx). 2.3.2.2.1 Description of bacteria Colonies of Lxx on semi-solid media are circular with entire margins, convex, non-pigmented, and approximately 0.1-0.3 mm in diameter after 2-3 weeks’ growth. Cells are non-motile pleomorphic rods measuring 0.25-0.50 µm in width and 1-4 µm in length, but longer lengths are not uncommon. The cells apparently divide by septation;'V'forms,which are characteristic of coryneform bacteria undergoing bending or snapping division, are frequently observed in wet mounts by phase-contrast or dark- field microscopy. Ultra-structurally, the cells have a gram-positive type cell wall and frequently contain mesosomes,Bradbury.,(1991). 2.3.2.2.2 Distribution of RSD The wide spread distribution of the disease can be largely attributed to international shipment of infected cuttings , Steindl., (1961)and Dean, (1974). To date, the disease has been reported from Australia, USA, India, Brazil, Florida, China, Fiji, Phillipnes and Africa,Mayeux, et al., (1979); Vishwnathan, (2001); Delaet al.,( 2002); Xu et al., (2008); Johnson and Tyagi, (2010). 2.3.2.2.3 Biology and Ecology The L. xyli subsp.xyli in nature has been found only in sugarcane, no reports of insect transmission, Gillaspie and Davis(1992) and the routes of infection are wounds. The pathogen is transmitted from field to field through propagation from infected sugarcane setts and between plants of the same field through contaminated cutting tools. The RSD disease incidence can increase with each successive harvest, because stalks are harvested from sugarcane plants annually for 3 to 5 years,Hoy et al., (1999); Croft (1994); Grisham et al., (2007). No evidence exists for transmission in true seed. The pathogen can remain viable and infectious for several months apparently in either moribund plant debris or the soil itself, contributing to the persistence of ratoonstunting disease in areas where the disease is common ,Bailey and Tough, (1992).

TheLxx. bacteria systemically invade plants through the xylem. It hasbeen detected in most vegetative parts of sugarcane where maturexylem exists. It was recovered readily from mature stalks and the leaf sheaths and lamina of the lower leaves of infected plants, but not from the midrib and lamina of upper leaves,Teakle et al., (1975). Large populations existed in mature stalks, and smallerpopulations were found in the growing point, leaf lamina, leaf midrib and leaf sheath,Bailey, (1977). 2.3.2.2.4 Hosts plant Sugarcane is the only known natural host of the pathogen (Saccharum spp. AndSaccharum interspecific hybrids), but numbers of grasses have appeared to be hosts after experimental inoculation, Gillaspie and Teakle,(1989).The experimental hosts include Zea mays, Sorghum spp., Brachiariamutica, Brachiaria miliiformis, Chloris gayana,Cynodon dactylon, Echinochloacolonum,Imperatacylindrica, Panicum maximum, Pennisetum purpureum andRhynchelytrumrepens. Host Plants/Plants Affected Plant name Family Context

Saccharum spp. Poaceae Main

Saccharumofficinarum(sugarcane) Poaceae Main

2.3.2.2.5 Symptoms The sugarcane ratoon stunt disease caused by L. xyli subsp.xyli has no unique diagnostic symptoms. Stunting is the only shown symptom but can also be caused by a number of other biotic and abiotic factors. The severity of stunting due to RSD disease may vary considerably. The quantity and quality of the yields can be reduced even when stunting cannot be noticed. Disease expression can be enhanced by stress, especially moisture stress. The growth of diseased crops in stubble or ratoon is slower and the stalks are thinner and shorter and sometimes fewer when the disease is severe. Death of individual plants of extremely susceptible cultivars may occur. Some highly susceptible cultivars may show wilting under moisture stress and even necrosis of leaves at the tips and margins. In mature stalks, vascular bundles at the nodes may reddish discoloration. The intensity of discoloration may vary among cultivars from one time to another, ranging from

yellow, orange, pink, and red to reddish-brown. This may be viewed by slicing longitudinally through a node with a knife and examining the fresh cut for discoloured vascular bundles in the shape of dots, commas, or short lines. Discoloration does not extend into the internode. Similar nodal symptoms may be caused by insects or other pathogens, but usually extend throughout nodes, instead of being limited to the lower portion of nodes, or extend into the internodes. Juvenile stalk symptoms may be observed in some cultivars by longitudinally slicing through 1- to 2- month-old stalks. The symptoms appear as pinkish discoloration justbelow the apical meristematic area and may extended downward as much as a centimeter,Comstock et al;(2002). 2.3.2.2.6 Isolation The pathogen is extremely fastidious in its nutritional requirements and can only be grown in axenic culture on special media such as the SC (synthetic copmlete) medium for 2-3 weeks,Davis et al.,(1980); Burmbleyet al., (2002).The bacterium is aerobic, non-motile, Gram-positive, non-spore-forming, non-acid fast, catalase-positive, and oxidase-negative. 2.3.2.2.7 Diagnosis The morphological symptoms of sugarcane ratoon stunt disease (stunting) are not unique to be sufficient for the accurate diagnosis and its difficult, requiring serological and/or molecular methods,Grisham,(2004). Therefore the diagnosis of RSD primarily depends on laboratory techniques. Diagnostic techniques include microscopic examination of L. xyli subsp. xyli bacterial cells (light and electron microscopy), culture of the bacterium on synthetic media, serological tests, host induced responses, and molecular tools (DNA-based methods). 2.3.2.2.7.1Microscopic detection of L. xyli subsp.xyli: The pathogen is a small, xylem-inhabiting, coryneform bacterium that often can be detected in sugarcane extracts from the nodal tissue using phase-contrast or dark-field microscopy (1000x). L. xyli subsp.xyli,(Davis) cell morphology is sufficiently distinct compared to the other natural micro-organisms associated with sugarcane to permit recognition. False-negative results associated with microscopic visualization of Lxx. bacteria are possible and present a problem, especially when pathogen concentrations are low,Davis and Dean,(1984).Concentrations of the bacteria are greatest in the basal portion of mature stalks at the end of growing season, and the chance of detection is better at this time,Davis et al.,(1988).The pathogen populations are lower in resistance

cultivars than the more susceptible ones ,Bailey, (1977) and can, therefore, present more of a problem for diagnosis. 2.3.2.2.7.2 Serological Techniques: 2.3.2.2.7.2.1 Immuno-microscopy: It is widely employed to localize in situ various components of cells and tissues in both normal and pathological situations. The method is based on the extremely sensitive interaction of a specific antibody (immunoglobulin) with its antigen. An antibody binds specifically only to a small site on the antigen, called an epitope. Serological methods have been developed to detect L. xyli subsp. xyli (Davis), (Harrison and Davis,(1990); Wu et al.,(1990). Fluorescent-antibody staining with a considerable sensitivity has been developed to detect bacterial cells with epifluorescence microscopy. Staining of the bacterium in dried extracts on glass slides was at least tenfold more sensitive than phase-contrast microscopy for detection,Harris and Gillaspie, (1978). At least another tenfold increase in sensitivity was obtained when the bacterium was first stained while still suspended in sap extracts and then concentrated on membrane filters by filtration before observation,Davis and Dean, (1984). The latter method can also be used to estimate the pathogen population size and was named the fluorescent- antibody direct- count on filters (FADCF) technique, Davis, (1985). 2.3.2.2.7.2.2 Enzyme-linkage immunosorbent assay (ELISA) Although not as sensitive, more rapid serological assays have been developed. An ELISA was investigated for detection of L. xyli subsp. xyli; however, problems with non- specific reactions were encountered,Gillaspie and Harris, (1979). A modified ELISA procedure, referred to as the evaporative binding enzyme immunoassay (EB- EIA), was developed,Croft et al.,(1994). A tissue-blot enzyme immunoassay (EIA) was developedto enumerate vascular bundles in cross-sections of sugarcane stalks which have been colonized by the pathogen; this technique can also be used for diagnosis,Harrison and Davis,(1988). A dot-blot EIA was also developed to detect the pathogen in sap extracts. Both the dot-blot and tissue- blot EIA involve the deposition of pathogen cells either from extracts or directly from plant tissue, respectively, onto nitrocellulose membrane filters before serological detection with anindirect EIA.

2.3.2.2.7.2.3 Lateral flow immunoassay test (LFIA): It also called Lateral flow immuno-chromatographic assays is the most popular diagnostic tool that meets the required standards for colorimetric assays,Kim et al., (2019). Lateral flow immunoassays are currently usedfor qualitative, semi quantitative and to some extent quantitativemonitoring in poor equipped or non-laboratory environments.LFIA test unitcomposes of pathogen specific antibody/ies is/are immobilized on a nitrocellulose membrane test. These specific antibodies are labelled by binding to nanoparticles that are oftenmade of colloidal gold, latex or silica within the conjugate padto facilitate visual detection,Posthuma-Trumpieet al., (2009).LFIA typically contains a control line to confirm the test is working properly, along with one or more target or test lines. Other labels include magnetic beads or colored polystyrene beads. Lateral flow immunoassay is widely used in the diagnosis of plant pathogens and considered as an appropriate efficient tool used for in-field pathogen detection Boonhamet al., (2008). LFIA has been used for different purposes, including diagnosis of human diseases, detectionof toxic compounds in food, pregnancy tests, and in environmental settings,Posthuma-Trumpieet al., (2009). Theiruse in phytopathology was focused mostly on fungi,Laneet al.,(2007) Thorntonet al.,(2004) and viruses,Safenkova et al.,(2012) and Safenkova et al.,(2016), although ithas been extended to the specific detection of bacteria such as Erwiniaamylovora,Braun- Kiewnicket al.,(2011), Clavibactermichiganensissubsp. SepedonicusSafenkova et al.,(2014), and Xanthomonascampestrispv. Musacearum,Hodgettset al.,(2015 ). LFIA offers several advantages over traditional techniques used in routine diagnosticprocedures, such as its low cost, simplicity of use, and long shelf life; moreover, results areobtained within 10 minutes and tests can be performed on site by minimally trained staff,Boonhamet al., (2008). It has become a reliable tool for the rapid screening of suspicious samples. Lo´pez-Sorianoet al. (2017) studied the specificity of the LFIA by testing against 87 X. Arboricolapv. Prunistrainsfrom different countries worldwide, 47 strains of other Xanthomonasspecies and 14 strainsrepresenting other bacterial genera. They detected all the 87 X. Arboricolapv. Prunistrains andcross-reactions were observed only with four strains of X. Arboricolapv. corylina, a hazelnutpathogen that does not share habitat with X. Arboricolapv. pruni.

The sensitivity of theLFIA for detection was assessed against threeX. Arboricolapv.Prunistrainssuspensions from pure cultures and spiked leaf extracts. The limit of detection observed with both purecultures and spiked samples was 104 CFU ml-1. The accuracy of the LFIA results were reported to in a high correlationwith those obtained by plate isolation and real time PCR techniques , Lo´pez-Sorianoet al., (2017). Therefore, they proposed the LFIA as a screening tool thatallows a rapid and reliable diagnosis of bacterial pathogenin symptomatic plants. 2.3.2.2.8Pathogenicity Test. Crude extracts of juice from infected plants is used as inoculums and found to be more appropriate inoculum than pure cultures of the pathogen. The quality of crude juice inoculums is difficult to control and may spread other pathogens. The use of pathogenicity test in diagnosing RSD is rare because of the long incubation period (usually at least 3-6 months or longer) required for cane growth and symptom development or detection of the pathogen in the host plant. Sugarcane can be inoculated by dipping freshly cut ends of single node cuttings into a suspension of the pathogen. Cuttingsare then planted and the developed plantsare examined for internal symptoms or the presence of the pathogen after an appropriate incubation period. 2.3.2.2.9 DNA Techniques Cloned DNA probes have been developed for the pathogen.The sensitivities of detection reported for the DNA probes were approximately equal to that reported for the FADCF technique but less than those reported for the various EIAs. A tissue –blot DNA hybridization technique and polymerase chain reaction (PCR) procedures have been reported for the sensitive and specific detection of the pathogen in sugarcane,Pan et al., (1998). 2.3.2.2.10 Similarities to other causal of stunting Any malady that results in stunted growth of sugarcane and does not have characteristic symptoms might be confused with ratoon stunting disease. For example, aluminium toxicity, iron deficiency and nematode infestations may have been confused with this disease. If stunting isuniform throughout the crop, it is probablynot caused by this disease. Uneven growth reduction where some plants are affected more than others is more characteristic; however, nematode infestations can also be uneven.

Diagnosis of ratoon stunting disease should thus be confirmed by detection of the pathogen in the laboratory. 2.3.2.2.11Managements 2.3.2.2.11.1 Cultural Control and Sanitary Methods Planting healthy cane can be used to control ratoon stunting disease. Sanitation is important in keeping healthy cane from becoming infected, because L. xyli subsp. xyli is easilytransmitted mechanically. Seed cane can be monitored for freedom from the disease using appropriate diagnostic techniques. In Australia and South Africa, phase-contrast microscopy has been used to screen thousands of samples annually for L. xylisubsp.xyli (Davis), Bailey and Fox, (1984). Continued vigilance in the selection of seed cane over several years has resulted in a reduction in the incidence of ratoon stunting disease in both plantings for seed cane production and commercial crops. 2.3.2.2.11.2 Physical control (heat treatment): Seed cane can be heat treated with hot water to prevent the spread of L. xyli subsp. xyli from one geographic area to another and to control ratoon stunting disease within areas where it occurs, Steindl., (1961). Hot-water, hot-air, moist-air, and aerated steam treatments have been used, Benda and Ricaud., (1978). Hot-water treatment at 50°C for 2-3 hours has been the most commonly used method, Steindl.,(1961); Gul and Hassan., (1995). The application of streptomycin and hot-water treatment at 52°C for 30 minutes suppressed disease by 22.6% and increased yields of cane and white sugar by 35.54 and 4.55 t/ha, respectively, Gul and Hassan., (1995). Different cultivars vary in their tolerance to injury by heat. Seed cane from mature plants is usually less affected and generally germinates better after hot-water treatment than that from immature or over-mature plants. Pre-treatment of young, heat-sensitive seed cane to increase germination has been reported to reduce the deleterious effects of hot- water treatment, Bendaet al;(1978). Seed cane was cut 1-5 days before treatment, pre-treated at 50°C for 10 minutes in hot water, and then treated the following day at 50°C for 2-3 hours. Hot-air treatment, with inlet air at 58°C for 8 hours or at 50°C for 24 hours, may be less harmful to immature seed cane than hot-water treatment and is used in some areas, Steindl., (1961). Dipping seed cane in water at ambient temperatures following hot-air treatment may protect germination. Moist-air treatment at 54°C for 7 hours in a sealed chamber was developed to overcome the moisture loss

associated with hot-air treatment. Aerated-steam treatment at 53°C for 4 hours uses steam-moistened hot air to reach the necessary internal stalk temperatures more quickly, approaching the treatment time used with hot water. The two major problems limiting the effectiveness of heat treatment, other than expense, are reductions in germination and lack of complete control. Procedures that have been developed to protect germination in addition to those already discussed include treating seed cane with fungicides or other chemicals during and after heat treatment, careful selection of seed cane for treatment, and leaving leaf sheaths over buds during treatment. Proper functioning of the heating unit and temperature control system and proper loading of the heat chamber can favour both germination and control of ratoon stunting disease. Under practical conditions, however, heat treatment is often not completely curative for ratoon stunting disease , Damann and Benda, (1983), and germination rates of treated seed cane may be reduced. Moreover, the expense involved in heat treatment is prohibitive. Consequently, heat treatment is often used to establish pathogen-free nurseries which are then used to supply planting material for commercial fields. Even so, the quantity of seed cane produced in such nurseries is usually inadequate, and additional sources of relatively pathogen-free seed cane are required. Seed cane from sources with a recent history of heat treatment can be used to fill the void , especially when adequate sanitary precautions have been taken to prevent spread of the pathogen and ensure that the level of the disease is minimal. 2.3.2.2.11.3 Sanitation: Because L. xyli subsp. xyli is readily transmitted by mechanical means, sanitation is important in preventing its spread to healthy plants, Comstock et al.,(1996). Precautions can be taken to avoidtransmission of the bacterium from one field to another on contaminated agricultural equipment. Sugarcane fields that are believed to be free from, or with a lower incidence of, ratoon stunting disease can be harvested first each day. Implements which have been used in diseased sugarcane fields can be cleaned and sterilized before entering another field. Hot water, steam, flaming, or chemicals can be used to disinfect implements, Gillaspie and Davis, (1992). 2.3.2.2.11.4 Host-Plant Resistance. Although immunity to infection by L. xyli subsp.xyli is not known to occur in sugarcane, substantial resistance to injury has been found in some cultivars and

appears to have a degree of genetic determination, Roach and Jackson(1992); Milleret al; (1995). Breeding for resistance to the disease was not a part of any breeding program until recently, because no practical methods of screening large numbers of clones for resistance to the disease were available. Selection for disease resistance on the scale required in a sugarcane breeding program is usually made on the basis of a visually assessed parameter of severity or incidence. No such visual parameters exist for ratoon stunting disease. Yield losses have been consistently detectable statistically only in replicated yield-loss trials combined over locations and years. Thus, yield-loss trials are grossly inadequate as a screening procedure on the scale required inabreedingprogram. The tissue-blot EIA and the EB-EIA techniques have been incorporated into sugarcane breeding programs in Florida, USA , Davis etal., (1994) and in Australia,Croft et al.,(1994) , respectively , to permit large-scale screening for resistance to ratoon stunting disease. The number of colonized vascular bundles in stalks is measured by the tissue-blot EIA, and population size of the pathogen in sap extracts is measured by the EB-EIA. Both parameters are measures of the extent of colonization of sugarcane by the pathogen and were found to be highly correlated ,Harrison and Davis, (1988). In addition to the extent of colonization, the frequency of infection of sugarcane clones can be determined by either method. These parameters are then used as estimates of disease incidence and severity as might be encountered in commercial sugarcane production. The validity of using the estimates of pathogen colonization as a measure of disease severity is based on their correlations with yield reduction due to ratoon stunting disease. The possible negative influence on this correlation of clonal tolerance to the disease has been discussed, Davis et al., (1988);Roach andJackson,(1992). It was hypothesized that disease control might best be obtained by disregarding the effects of the disease on yields and concentrating on lowering the potential for epidemics by selection of clones that are less susceptible to infection and produce less inoculum when infected,Davis et al.,(1994).

2.3.2.2.11.5Chemical Control The application of ammonium sulfate to sugarcane crops resulted in 22.89% reduction in disease caused by L. xyli subsp. xyli and yield increases of 29.09 and 2.91 t/ha, in cane and white sugar, respectively,Gul and Hassan, (1995).

CHAPTER THREE MATERIAL AND METHODS 3.1 Collection of diseased materials: Stunted cane stalks were collected from chewing cane planted in shendi foug- WadMedani in season 2018-2019.The sample collection was based on the visual observation of the typical stunt symptoms: shortened internodes and the internal reddish discoloration of the xylem tissue at the nodes of the cane stalks. The stunted cane stalks were removed using knifes that each time treated with ethanol for disinfection. Then the stalks were placed into plastic bags and transferred to plant pathology center laboratory for further examination. A minimum often plants showing RSD symptoms were collected and designated as (A, B, C, D, E, F, G, H, I and J). 3.2 Observation of external and internal symptoms:  External symptoms:Measurements of Stunting: AverageLength of the internode = Total measurements (cm) of internodes length/number of internodes  Internal symptoms (internal reddish discoloration at the stem nodes): 1. The stunted cane stalks were cut into longitudinal and cross sections by blade and knife. 2. The reddish discoloration of the xylem tissue (symptoms ofLxx.) at the lower part of the diseased plant nodes were visually observed.

3.3 . Detection ofLxxbacteria: 3.3.1 Lateral flow immunoassay test (serology): The tests of lateral flow immunoassay test (PHYTO- QUICK) were generously provided by Dr. Stephan Winter, German Institute for microorganism and cell culture, Germany. The test is a simple technique based on immune-chromatography of specific antibodies that was used for detection and identification ofLxx. The lateral flow has three sensitive spots (Fig 1): 1. The sample spot (S) contains antibody with a colored dye. 2. The test lane (T) contain antibodies specific for the pathogen (Lxx).

3. The control lane (C) contains an IgG specific for IgG species.

Fig 6. Lateral flow test; S, T, and C spots in order from bottom to top

3.3.1.1. Extraction of the xylem sap from stunted cane stalks. The basal nodes of plants that showing the stunting symptoms of RSD were disinfected with distilled water and ethanol. The xylem saps of the stunted cane were extracted as the following steps (Fig. 2): o The bottom nodes were separately cut. o The nodes outer layer was removed by knife blade that was disinfected with ethanol and heat for each node. o The peeled node was sectioned longitudinally and separately placed into special sample homogenization plastic bags with built in filter for clarification of the extracts. 3.3.1.2 Extraction buffer. 3.3.1.2.1 Buffer1 (Prepared).  (4g) Peptone.  (0.50g) K2HPO4 (Dibasic potassium phosphate).  (0.50g) KH2 PO4 (Monobasic potassium phosphate).  (10g) MgSO4 (Magnesium sulphate).  (5%) Tween 20 . The above components were added to (500 ml) distilled water and stirred until completely dissolved. The pH was set to (6.6) by addition of KOH solution. Then the buffer was autoclaved for 30 minutes in 15º bar, 121 Cº. 3.3.1.2.2 Buffer 2 (Ready, provided with the lateral flow test kit).  (8g) NaCl (Sodium chloride).  (6.446g) Na2HPO4 (Dibasic sodium phosphate).

 (1.768g) KH2PO4 (Monobasic potassium phosphate).  (0.05%) Triton X-100.  (2%) PVP (Polyvinylpyrrolidone). Dissolved in 1L distilled water, pH was adjusted to 7.4. 3.3.1.3 Loading of Xylem sap on Lateral flow immunoassay test:

o Then the extraction buffer 1 or 2 was added, the ratio of cane weight to buffer volume is 1gram to 10 ml respectively and the cane was ground by pestle.The required volume of extraction buffer was added step by step not at once. o Two to three drops of the extracted xylem sap of the stunted cane node were added by micro plastic pippette to the sample spot (S) on the lateral flow immunoassay test. Two to three drops of the extraction buffer were used as a negative control.The reaction results were recorded in less than 60 minutes after the diffusion started.

A B C

D E

Fig 6. Extraction of stunted cane stalk xylem sap and transfer to Lateral flow test. A: Stunted cane stalks. B: Peeling and sectioning of node. C: Grinding of sections in plastic bag. D: Cane extract. E: Lateral flow test with added extract.

3.3.2 Isolation of L. xyli subsp. xyli 3.3.2.1. Preparation of culture media (0.5 L). The Synthetic Complete (SC) media was prepared according to the recipe of the German institute for microorganisms and cell cultures collection (DSMZ) with a substitution hemin and corn meal agar by methionine and agar agar respectively. The following ingredients were dissolved in 450 ml distilled water.  ( 4g) peptone.  (0.50g) K2HPO4.  (0.50g) KH2PO4.  (0.10g) MgSO4.  (5%) tween 20.  (4.25g) agar.  The above ingredients were dissolved in 450 ml distilled water and the pH set to 6.6 by addition of KOH solution. Then the media mixture was autoclaved for 30 minutes at 15º bar and 121 Cº.  The autoclaved media was cooled but not solidified and the following ingredients were dissolved in buffer1, filter sterilized andadded slowly while stirring intensively using stirrer. o (1g) albumin in (10ml/buffer). o (0.25g) glucose in (10 ml/buffer). o (0.5g) cysteine in (10 ml/buffer). o (2.5 g) methionine in (20 ml/buffer).  The prepared media was then poured into circular Petri dishes and allowed to completely solidify. 3.3.2.2 Inoculation of the media The cane sap that was extracted from the sugarcane xylem which suspected to be infected with L. xyli subsp. xyli was inoculated onto the solidified media and streaked with the help of the loop. The inoculated media were kept in a laminar flow at room temperature (28±Cº) and monitored on daily bacterial growth. The grown bacteria were suspended in extraction buffer1 and 2-3 drops were added to sample spot (S) on the lateral flow test and the reaction results were recorded in less than 60 minutes after the diffusion started. Also 2 - 3 drops of the extraction buffer were used as a negative control. All the processes of bacteria isolation such as extraction from plant tissue, media preparation and inoculation were done under hygienic sterilized clean conditions of laminar flow (Fig 3).

Fig. 3: Laminar flow, the hygienic sterilized clean working place. 3.3.2.3 Sub-culture media: The growth colonies those gave positive results for the presence ofLxx. in the lateral flow test were re-inoculated into the media as in section (3.3.2.2) and the bacterial growth was monitored. The grown bacterial cells were suspended in extraction buffer 1 and 2-3 drops were added to sample spot on the lateral flow test and the reaction results were recorded in less than 60 minutes after the diffusion started. Also 2 - 3 drops of the extraction buffer were used as a negative control. 3.3.3 Gram staining:  The growth colonies those gave positive results for presence of Lxx in culture and sub-culture were examined under microscope after proceeding gram- stain test more times to conformation from gram positive and the rod shape of Lxx.bacteria.  Staining mechanism Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50– 90% of cell envelope), and as a result are stained purple by crystal violet, whereas Gram- negative bacteria have a thinner layer (10% of cell envelope), so do not retain the purple

stain and are counter-stained pink by safranin. There are four basic steps of the Gram stain:

 Applied a primary stain (crystal violet) to a heat-fixed smear of a bacterial culture. Heat fixation kills some bacteria but is mostly used to affix the bacteria to the slide so that they don't rinse out during the staining procedure.

 The iodide was added, which binds to crystal violet and traps it in the cell  Rapid decolorization with ethanol or acetone

 The safranin was added. Carbolfuchsin is sometimes substituted for safranin since it more intensely stains anaerobic bacteria, but it is less commonly used as a counterstain . All the methods were a according to Beveridge, (2001 ) andLeboffe and michael(2014).

CHAPTER FOUR RESULTS 4.1 Ratoon stunting disease (RSD) incidence and diagnosis based on symptoms: The apparent symptoms of sugarcane ratoon stunt disease (RSD), the stunting and the reddish discoloration of the xylem tissue at the nodes of cane stalk were not reported on cane stalks at the farms of sugar factory companies. On the other hand the cane stalks grown by individual in shendi Foug- Wadmedani city for the purpose of selling as a chewing cane at the local market showed obvious stunting symptoms and reddish discoloration of the xylem with the incidence reaching up to 100%. The internode lengthof the stunted cane stalks were found to be reduced to one third (7/20 cm stunted/healthy) (Table1, Fig.4). The reddish discoloration of the vascular bundles (xylem tissues) at the basal nodes was reported on (80%) of the stunted cane stalk ( Fig 5).

B A

Fig. 4. The external symptom of Sugarcane ratoon stunt disease (RSD). A. Sugarcane plants from which the stunted cane stalk was collected. B. Healthy sugarcane stalk left and the diseased (stunted) right.

Table (1). The average internode length of healthy and stunted cane stalks. Average length of Cane stalks internode (cm) Healthy 20 Diseased (stunted) 7

A B

Fig. 5. Internal symptoms of Sugarcane ratoon stunt disease (RSD) A. Diagonal section of stalk: Heathy cane (left) and diseased showing reddish discoloration of vascular bundles at the lower portion of the nodes (right). B. Cross section of cane stalk: Reddish discoloration of vascular bundles at the nodes.

4.2 Detection of (L. xyli susp. xyli): 4.2.1 Lateral flow immunoassay (serology). The specific lateral flow immunoassay test detected and identified L. xyli subsp. xyli (Davis) in the xylem sap extracted from stunted cane stalks and from the culture, the sensitivity of the test was dependent on the type of extraction buffer (Fig. 6 and Table2). The home made buffer1 was more effective and appropriate for extracting the RSD causing bacteria than the ready buffer2. By the use of the extraction buffer1 theLxx. bacteria was detected in 40% of stunted cane stalks compared with 10% for the extraction buffer2.

control band Test band (Lxx) )band

A B Fig. 6: The effect of extraction buffer on the detection sensitivity of lateral flow immunoassay test forLxx. A: Buffer 1 (prepared). B: Buffer 2 (Ready). Two bands mean positive reaction forLxx. bacteria presence.

Table (2): The effect of extraction buffer on detection sensitivity of lateral flow immunoassay test forLxx. .

No. Samples Buffer 2 Buffer 1 1 A1 - + 2 A2 - + 3 B1 - - 4 B2 - - 5 C1 + + 6 D1 - - 7 E1 - - 8 E3 - - 9 F1 - - 10 F3 - + % 10 40

Table (3): Detection of L .xyli subsp. xyli by symptoms (external and internal) and serological test (lateral flow immunoassay). Symptoms Buffer 1 No. Samples Stunting Reddish discoloration 1 A1 + + + 2 A2 + + + 3 B1 + + - 4 B2 + + - 5 C1 + + + 6 D1 + + - 7 E1 + + + 8 E3 + + - 9 F1 + - - 10 F3 + - + % 100 80 40

Not all the stunting symptoms of cane stalk and the reddish discoloration of the nodal xylem tissue were found associated with each other or with the presence of L. xyli subsp. xyli (table 3). The reddish discoloration of the nodal xylem tissues were observed on 80% of the stunted cane stalks and the RSD causal agent (L. xyli subsp. xyli) was reported on 40% (table 3). 4.2.2 Effect of the culture media: Small growth colonies of bacteria were noticed on the media supplemented with amino acid methionine 5 days after inoculation. The bacteria were identified to be L. xyli subsp. xyli when subjected to lateral flow immunoassay test and microscopic examination (Fig 7 and 8, table 4). The culture increased theLxx. serological detection sensitivity up to 70% which was almost two times the direct detection in the cane stalk xylem sap (40%). Under microscopic examination, the bacteria were observed to be rod in shape and had a violet color which means that they were gram-positive, both characters possessed by Lxx bacteria.

Test control band

Lxx band

Fig. 7: Detection and identification of L. xyli subsp. xyli in cane extracts and culture, using lateral flow immunoassay test.A: Cane extract. B: Culture. Two bands mean positive reaction forLxx. bacteria presence.

Table (4): Detection of L. xyli subsp. xyli in cane extracts and culture using lateral flow immunoassay test. No. Samples Cane sap Culture

1 A1 + + 2 A2 + + 3 B1 - + 4 B2 - + 5 C1 + + 6 D1 - + 7 E1 - - 8 E2 - - 9 F1 - + 10 F3 + - % Infected (+) 40 70

Fig. 8. A: Mainculture of Lxx from stunted cane extract. B: Sub-culture

CHAPTER FIVE DISCUSSION Plant diseases diagnosis is one of the most important aspects of plant diseases control measures and depend on accurate causal agent’s detection and identification,Comstock (2002). Some diseases can be diagnosed by visual observation of apparent symptoms that often appear at late disease stage after considerable damage to the crop has been done and it is too late for effective control measures. Some diseases need laboratory examination for diagnosis which may take long time to be done. Nowadays plant diseases diagnostic techniques of quick detection and specific to each pathogen based on proteins or nucleic acids, with a reasonable time requirements and accuracy are under practices, Kazeem and Ikotun,( 2015); Fang and Ramasamy, (2015). Some of these techniques involved procedures require sophisticated laboratory equipment and training, while other procedures can be performed on-site by a person with no special training. The sugarcane ratoon stunt disease (RSD) caused by the bacteria L. xyli subsp.xyli (Davis) is responsible for economical yield loss to the cane stalks and its products,Young and Brumbley, (2004). The RSD disease has no unique morphological symptoms sufficient for its accurate diagnosis,Tiwari et al., (2012), Kazeem and Ikotun, (2015). Therefore the diagnosis of RSD primarily depends on laboratory techniques,Kazeem and Ikotun, (2015). These techniques include microscopic examination of L. xyli subsp. xyli (Davis) bacterial cells (light and electron microscopy), culture of the bacterium on synthetic media, serological tests, host induced responses, and molecular tools (DNA-based methods). Accordingly, in this study simple and easy diagnostic methods were used for detection, identification and isolation of ratoon stunt disease bacteria (L. xyli subsp xyli) that started with observation of symptoms, the use of the serological technique (lateral flow immunoassay test) and modified synthetic media were developed. In contrast to molecular techniques the serological methods in general are widely available and relatively inexpensive, making them applicable without a need for sophisticated equipment and training. In agreement with Tiwari et al. (2012) and Kazeem and Ikotun, (2015) that RSD disease has no specific symptoms, not all the sugarcane stalks showing stunting symptoms were found infected with L. xyli subsp. xyli (Davis). The reddish

discoloration of the nodal xylem tissues were observed on 80% of the stunted cane stalks and the RSD causal agent (L. xyli subsp. xyli (Davis)) was reported on only 40% (table 4). Sometimes stunting of sugarcane may be due to many factors other than L. xyli subsp. xyli (Davis), such as aluminum toxicity, iron deficiency and nematode infestations. The lateral flow immunoassay test which is specific for L. xyli subsp. xyli (Davis) was successfully detected and identified it in the xylem sap extracted from stunted cane stalks and from the culture. The sensitivity of the test was dependent on the type of extraction buffer (fig. 6 and Table 3). This variation of sensitivity might be due to the ability of the buffer to lysis the cane stalk xylem tissues without denaturing the configuration of the bacterial cell that is recognized by the specific antibody in the lateral flow nitrocellulose membrane. The extraction buffer1 that resulted in better sensitivity in lateral flow test was composed of peptone, dibasic potassium phosphate

(K2HPO4), magnesium sulphate (MgSo4)and tween 20 instead of dibasic sodium phosphate (Na2HPO4), triton X-100 and PVP that present in the ready provided buffer2 (see material and methods sections (3.3.1.2.1) and (3.3.1.2.2). TheLxx. bacteria is highly fastidious in its nutritional requirements and can only be grown on special media such as the SC medium (see 3.3.2.1), the incubation period can be up to 2-3 weeks Brumbleyet al., (2002) and Davis et al.,(1980). The growth of bacteria was enhance and the long incubation period reduced by addition of hemin, amino acid cysteine and sometimes methionine. In this study SC media was prepared according to the recipe of the German institute for microorganisms and cell cultures collection (DSMZ) with a substitution of hemin and corn meal agar by methionine and agar agar respectively Monteiro-Vitorello et al., (2004). In contrast to Brumbleyet al., (2002) and Davis et al., (1980) but in accordance with Monteiro-Vitorello et al., (2004), obvious growth colonies of L. xyli subsp. xyli were observed on the media supplemented with amino acid methionine 5 days after inoculation which is far less than the reported 2- 3 weeks (Fig 7, table 5). The culture increased the detection sensitivity ofLxx.and the incidence of sugarcane ratoon stunt by the use the serological lateral flow test. The presence of lxx obtained by culture and the serological test was 70% compared with 40% detected in the cane stalk extracted xylem sap. The lateral flow immunoassay test was found to be simple did not need for specialized equipment and hygienic condition; it was also easy and could be performed by a basic know how laboratory practices.

To our knowledge this is the first time for the use of Lxx. culture and the lateral flow immunoassay test to diagnose the RSD disease in Sudan.

CHAPTER SIX CONCLUSION AND RECOMMENDATION

6.1 CONCLUSION

o Most but not all of the sugarcane stunting symptom and red colour are due to the infection with RSD. o Modification of the SC medium by addition of amino acid methionine resulted in a suitable synthetic growth medium for L. xyli subsp. xyli (Davis) that reduced the incubation period to one week instead of two to three. o Sugarcane ratoon stunting disease (RSD) has no unique external and internal symptoms for effective and accurate diagnosis. o The lateral flow immunoassay test does not need specialised laboratory equipment, was found to be dependable disease diagnosis tool that specifically detect and identify of plant pathogen. 6.2 RECOMMENDATIONS o The lateral flow immunoassay test and the improved synthetic culture are recommended as accurate and dependable tools for detection and identification of L. xyli subsp. xyli (Davis) to find out RSD occurrence and incidence on symptomatic and symptomless cane stalks. o Further studies are needed to identify the possible weed hosts and the carryover of L. xyli subsp. xyli (Davis) and assesses the effectiveness of control measure treatments.

REFERENCES

Bailey, R. A and Fox, P. H. (1984).A large-scale diagnostic service for ratoon stunting disease of sugarcane.Proceedings of the Annual Congress - South African Sugar Technologists' Association58:204-210.

Bailey, R. A. (1977).The systemic distribution and relative occurrence of bacteria in sugarcane varieties affected by ratoon stunting disease.Proceedings of the Annual Congress - South African Sugar Technologists' Association51:55-56.

Bailey, R. A. and Tough, S. A. (1992).Ratoon stunting disease: Survival of Clavibacterxyli subsp. xyli, in field soil and its spread to newly planted sugarcane. Proceedings of the Annual Congress - South African Sugar Technologists ’Association66:75–77. Benda, G. T. A. and Ricaud, C. (1978).The use of hot-water treatment for sugarcane disease control.Proceedings of the International Society of Sugar Cane Technologists.16:483-496.

Beveridge,T.J.(2001). Use of the Gram stain inmicrobiology. Biotechnic & Histochemistry .76(3):111 – 118. Boonham, N. Glover R. Tomlinson, J. and Mumford, R. (2008).Exploiting generic platform technologies for the detection and identification of plant pathogens.European Journal of Plant Pathology. 121: 355±363.

Bradbury, J. F. (1991).Clavibacter xyli subsp. xyli.I M I Descriptions of Fungi and Bacteria No. 105, sheet 1044.CAB International,Wallingford, UK.

Braun-Kiewnick, A. Altenbach, D. Oberha, È. Nsli, T. Bitterlin, W. Duffy, B. A. (2011).Rapidlateral-flow immunoassay for phytosanitary detection of Erwinia amylovora and on-site fire blight diagnosis. J Microbiol Meth.;87: 1-9.

Brumbley, S.M. Petrasovits, L. A. Birch, R. G. and Taylor, P. W. (2002).Transformationandtransposon mutagenesisofLeifsonia xylisubsp.xyli, causalorganism of ratoon stunting disease of sugarcane.Mol.plant- Microbe interact.15:262-268.

Comstock, J. C. (2002). Ratoon stunting disease.Sugar Tech 4(1–2):1 - 6.

Comstock, J. C. Shine, J. M. J. Davis, M. J. and Dean, J. L. (1996).Relationship between resistance to Clavibacter xyli subsp. xyli colonization in sugarcane and spread of ratoon stunting disease in the field.Plant Disease80(6):704-708;23.

Croft, B. J. Greet, A. D. Leaman, T. M. and Teakle, D. S. (1994).RSD diagnosis and varietal resistance screening in sugarcane using the EB-EIA technique. Proceedings of the 1994 Conference of the Australian Society of Sugar Cane Technologists held at Townsville, Queensland, 26th April to 29th April 1994 [edited by Egan, B. T.] Brisbane, Australia; Watson Ferguson & Company 143- 151.

Dahlia, La. Kurniawan, I. Anggakusuma, D. and Roshetko, J. M. (2009).Consumer Preference for Indigenous Vegetables.World Agroforestry centre.

Damann, K. E. J. and Benda, G.T. A. (1983).Evaluation of commercial heat-treatment methods for control of ratoon stunting disease of sugarcane.Plant Disease67(9):966-967.

Davis, M. J. and Dean, J. L. (1984).Comparison of diagnostic techniques for determining incidence of ratoon stunting disease of sugarcane in Florida.Plant Disease, 68(10):896-899.

Davis, M. J. Dean, J. L. Miller, J. D. andShine, J. M. J. (1994).A method to screen for resistance to ratoon stunting disease of sugarcane.Sugar Cane6:9-16.

Davis, M. J. Gillaspie, A. G. J. Harris, R. W. and Lawson,R. H. (1980). Ratoon stunting disease of sugarcane: isolation of the causal bacterium. Science USA210:1365- 1367.

Davis, M. J. (1985). Direct-count techniques for enumerating Clavibacterxyli subsp.xyli which causes ratoon stunting disease of sugarcane. Phytopathology75(11):1226- 1231.

Davis, M. J., and R. A. Bailey. 2000. “Ratoon stunting.” In A Guide to Sugarcane Diseases, edited by Philippe Rott, Roger A. Bailey, Jack C. Comstock, Barry J. Croft, and A. Salem Saumtally, 49–54.

Davis, M. J. Dean, J. L. and Harrison, N. A. (1988). Distribution of Clavibacter xyli subsp. xyli in stalks of sugarcane cultivars differing in resistance to ratoon stunting disease. Plant Disease72 (5):443-448.

Davis, M. J. Dean, J. L. and Harrison, N. A. (1988). Quantitative variability of Clavibacter xyli subsp. xyli populations in sugarcane cultivars differing in resistance to ratoon stunting disease. Phytopathology78 (4):462-468.

Davis, M. J. Gillaspie, A. G. J. Harris, R. W. and Lawson, R. H. (1980). Ratoon stunting disease of sugarcane: Isolation of the causal bacterium. Science, USA, 210:1365-1367.

Davis, M. J. Gillaspie, A. G. J. Vidaver, A. K. and Harris R. W. (1984). Clavibacter: a new genus containing some phytopathogenic coryneform bacteria, including Clavibacter xyli subsp. xyli sp. nov., subsp. nov. and Clavibacter xyli subsp. cynodontis subsp. nov., pathogens that cause ratoon stunting disease of sugarcane and Bermudagrass stunting disease. International Journal of Systematic Bacteriology, 34 (2):107-117; [3 fig., 4 tab.]; 51 ref.

Dean, J. L. (1974). Ratoon stunting disease in relation to shipment of cuttings from Canal Point, Florida.Sugarcane Pathologists' Newsletter, 11:12-7.

DelaCueva, F.M. Natural, M. P. Bayot, R. G. Mendoza, E. M. T and Ilag, L.L.(2002) Geographic distribution of ratoon stunting disease of sugarcane in the Philippines.Journal of Tropical Plant Pathol 38: 11-18.

Draycott, A.P., 2006. Sugar beet.Blackwell Publishing,514 p

Egan, B.T. (1970). RSD in North Queensland.Proceedings of the Queensland Society of Sugar Cane Technologists37:221-224.

Evtushenko, L.I. Dorofeeva, L.V. Subbotin, S.A. Cole, J. and Tiedje, J.M.( 2000).Leifsoniapoae gen. nov., sp. nov., isolated from nematode galls on Poaannua, and reclassification of 'Corynebacterium aquaticum' Leifson 1962 as Leifsoniaaquatica (ex Leifson 1962) gen. nov., nom. rev., comb. nov.andClavibacter xyli Davis et al. 1984 with two subspecies as Leifsonia xyli (Davis et al. 1984) gen. nov., comb. nov.International Journal of Systematic and Evolutionary Microbiology, 50(1):371-380.

Fang, Y. and R.P. Ramasamy (2015).Current and prospective methods for plant disease detection.Biosensors (Basel), 5 pp. 537-561.

FAO STAT. (2009). Food and Agriculture Organization Statistics Division [Online].Available by Food and Agriculture Organization of the Unit Nations http://www.fao.org/statistics/ (verified February 21)..

Gillaspie, A. G. J. and Davis, M.J. (1992). Ratoon stunting of sugarcane. In: Mukhopadhyay AN, Kamar J, Chaube HS, Singh US, eds. Plant Diseases of International Importance. Diseases of Sugar, Forest, and Plantation Crops, Vol. 4. New Jersey, USA: Prentice Hall, 41-61.

Gillaspie, A. G. J. and Harris, R.W. (1979). Limitations of ELISA for detection of the RSD-associated bacterium in sugarcane and sudan grass. SugarcanePathologists' Newsletter, No. 22:25-28.

Gillaspie, A. G. J. and Teakle, D.S. (1989). Ratoon stunting disease. In: Ricaud C, Egan BT, Gillaspie AG Jr, Hughes CG, eds. Diseases of Sugarcane - Major Diseases. Amsterdam, Netherlands: Elsevier, 59-80.

Grisham, M. P. (2004).Ratoon stunting disease.in: Rao GP, saumatally AS, RottP, eds ,sugarcane pathology vol .111 : Bacteria and Nematode disease. Enfield, NH USA :Science Publishers,77-96.

Grisham, M. P. Pan, Y.B., and Richard, E. P. J. (2007).Early detection of Leifsonia xyli subsp. xyli in sugarcane leaves by real-time polymerase chain reaction. Plant Disease91:430-434.

Gul, F. and Hassan, S. (1995). Further studies on the control of ratoon stunting disease of sugarcane in the N.W.F.P. Pakistan Journal of Phytopathology, 7(2):163-165; 8 ref.

Harris, R.W. and Gillaspie, A.G. J. (1978).Immunofluorescent diagnosis of ratoon stunting disease.Plant Disease Reporter, 62(3):193-196.

Harrison, N.A.and Davis, M.J. (1990).Comparison of serological techniques for diagnosis of ratoon stunting disease. Sugar Cane, Spring Supplement:5-9.

Harrison, N.A. Davis, M.J. (1988).Colonization of vascular tissues by Clavibacter xyli subsp. xyli in stalks of sugarcane cultivars differing in susceptibility to ratoon stunting disease.Phytopathology, 78(6):722-727.

Harrison, N. and Davis, M. J. (1990). Comparison of serological techniques for diagnosis of ratoon stunting disease. Sugar Cane, Spring Supplement:5-9.

Hodgetts, J. Karamura, G. Johnson, G. Hall, J. Perkins, K. Beed, F. (2015).Development of a lateral flow device for in-field detection and evaluation of PCR-based diagnostic methods for Xanthomonas campestris pv. musacearum, the causal agent of banana xanthomonas wilt. Plant Pathol. 64: 559-567

Hoy, J. W. Grisham, M. P.and Damann, K.E. (1999).Spread and increase of ratoon stunting disease of sugarcane and comparison of disease detection methods. Plant Disease83:1170-117.

Johnson,S. S. and Tyagi, A. P.(2010) Effect of Ratoon Stunting Disease of Sugarcane yieldin Fiji.The South P acific Journal of Natural and Applied Sciences. 28: 69-73.

Kazeem, S.A. and Ikotun, B. (2015).Evaluation of different detection assays for use in Leifsonia xyli subsp. xyli assessment programs. Journal of Tropical Agriculture, Food, Environment and Extension,Volume14 pp. 27-34.

Kim, H. Chung, D. R. and Kang, M. (2019).A new point-of-care test for the diagnosis of infectious diseases based on multiplex lateral flow immunoassays.Analyst 144(8):2460-2466. Lane, C. R. Hobden, E. Walker, L. Barton, V.C. Inman, A. J. Hughes, K. J. D. (2007).Evaluation of a rapid diagnostic field test kit for identification of Phytophthora species, including P. ramorum and P. kernoviae at the point of inspection. Plant Pathol.56: 828-835.

Leboffe, and Michael. (2014). Microbiology Laboratory Theory and Application (3rd ed.). Englewood, CO: Morton Publishing Company. p. 105.ISBN 978-1617312809.

Lo´pez-Soriano, P. Noguera, P. Gorris, M. T. Puchades, R. Maquieira, A. Marco- Noales E.and LoÂpez, M.M.(2017) Lateral flow immunoassay for on-site detection of Xanthomonasarboricolapv. pruni in symptomatic field samples.PLoS ONE 12(4):e0176201.

Maramorosch, K. Plavsic-Banjac, B. Bird, J. and Liu, L. J. (1973). Electron microscopy of ratoon stunted sugar cane: microorganisms in xylem. Phytopatholog ische Zeitschrift, 77(3):270-273.

Mayeux, M. M. Cochran, B. J. and Steib. J. ( 1979).An aerated steam system for controlling ratoon stunting disease.Transactions of the American Society of Agricultural Engineers, 22(3):653-656.

Miller, J. D. Davis, M. J. and Dean, J. L. Shine, J. M. J. (1995). Heritability of resistance to ratoon stunting disease in sugarcane. Sugar Cane, No. 1:3-8; 24 ref.

Monteiro-Vitorello, C.B., Camargo, L.E.A., Van Sluys, M.A., Kitajima, J.P., Truffi, D.,do Amaral. A.M., Harakava, R., de Oliveria J.C., Wood, D., deOliveria, M.C., Miyaki. (2004). The genome sequence of the Gram- positive sugarcane pathogen leifsonia xyli subsp. xyli. Mol. Plant Microbe Interact. 17,827-836.

Obeid, A.(2005) Sudanese sugar company – History, present and future prospect. Annual conference of the international sugar organization.Friendship Hall-Sudan.

Pan, Y.B. Grisham, M.P. Burner, D.M. Damann, K. E. J.and Wei, Q. (1998). A polymerase chain reaction protocol for the detection of Clavibacter xyli subsp. xyli, the causal bacterium of sugarcane ratoon stunting disease. Plant Disease, 82(3):285-290; 39 ref.

Perez and Rena (1997)."Chapter3: Sugarcane".Feeding pigs in the tropics.Food and Agriculture Organization of the United Nations.Archived from the original on 21 February 2018.Retrieved 2 September 2018.

Posthuma-Trumpie, G. A., Korf, J. and van Amerongen, A. (2009). Lateral flow immunoassay, its strengths, weaknesses,opportunities and threats. A literature survey.Analytical and Bioanalytical Chemistry. 393: 569 - 582.

Roach, B. T. and Jackson, P. A. ( 1992). Screening sugar cane clones for resistance to ratoon stunting disease. Sugar Cane, No. 2:2-12; 35 ref.

Rao, G. P. and Ford, R. E. (2000) Vectors of virus and Phytoplasma diseases of Sugarcane: An Overview. In: Sugarcane Pathology, Vol. III. Virus and Phytoplasma diseases, USA, Pg: 265-314

Safenkova, I. V. Pankratova, G. K. Zaitsev, I. A. Varitsev, Y. A. Vengerov, Y. Y. Zherdev, A. V. (2016). Multiarray on a test trip (MATS): rapid multiple. immunodetection of priority potato pathogens. Anal Bioanal Chem.408: 6009-6017

Safenkova, I. V. Zherdev, A. V. Dzantiev, B. B. (2012).Factors influencing the detection limit of the lateral-flow sandwich immunoassay, a case study with potato virus X. Anal Bioanal Chem. 403: 1595-1605.

Safenkova, I. V. Zaitsev, I. A. Pankratova, G. K. Varitsev, Y. A. Zherdev, A. V. Dzantiev, B. B. (2014). Lateral flow immunoassay for rapid detection of potato ring rot caused by Clavibacter michiganensis subsp. sepedonicus.Appl BiochemMicro.50 (6): 675-682.

Samuels, K. D. Z. D. E. Pinnock, R. M. and Bull. (1990). Scarabeid larvae control in sugarcane using Metarhiziumanisopliae. J. Invertebr. Pathology. 55: 135-137

Schueneman, T.J. (2002).Backyard sugarcane 1- Growth and development of sugarcane.SS- AGR-253.Agronomy Department.Florida Cooperative Extension services.Institute of foot and Agric. Science.University of florida

Steindl, D.R.L. (1961). Ratoon stunting disease. In: Martin JP, Abbott EV, Hughes CG, eds. Sugarcane Diseases of the World, Vol. I. Amsterdam, Netherlands: Elsevier, 433-459.

Taylor, P. W. J.Petrasovits,L.A. Van der Velde, R. Birch, R. G. Croft, B. J. Fegan, M. Smith, G. R. Brumbley, S. M. (2003).Development of PCR-based markers for detection of leifsonia xylisubsp.xyli in fibrovascular fluid of infected sugarcane plants.Australas plant pathol 32:367-375.

Thornton, C. R.Groenhof, A .C. Forrest, R. Lamotte, R. A.(2004).immunochromatographic lateral flow device specific to Rhizoctonia solani and certain related species, and its use to detect and quantify R. solani in soil. Phytopathology. 94: 280

Tiwari, A. K., Vishwakarma, S. K., Kumar, P. P., Pandey, N. and Lal, M. (2012).Ratoon stunting disease (Leifsonia xyli) of sugarcane(Mini Review). Plant Knowledge Journal (1) 20-24.

Vilela, M. M. Del Bem, L. E. Van Sluys, M. A. de Setta, N. Kitajima, J. P. Cruz, G. M. Sforça, D. A. de Souza, A. P. Ferreira, P. C. Grativol, C. Cardoso- Silva, C. B. Vicentini, R. and Vincentz, M. (2017).Analysis of Three Sugarcane Homo/Homeologous Regions Suggests IndependentPolyploidization Events of Saccharum officinarum and Saccharum spontaneum.Genome Biology and Evolution. 9(2): pp. 266-278.

Vishwnathan, V. (2001).Growing severity of ratoon stunting disease of sugarcane in India.Sugar Tech. 3: 154-159.

Wu, M.F. Lin, C.P. and Chen, C.T. (1990). Isolation, cultivation and monoclonal antibody production of Clavibacterxyli subsp. xyli, the causal agent of sugarcane ratoon stunting disease. Plant Protection Bulletin (Taipei), 32(2):91-99.

XU ing-Sheng, XU Li-Ping, QUE You-Xiong, GAO San-Ji.and Chen Ru-Kai (2008).Advances in the Ratoon Stunting Disease of Sugarcane. DOI CNKI:SUN: RYZB.0.2008-02-01.

Young, A. Brumbley, S.M.(2004). Ratoon stuning disease of sugarcane. History, Management and new research. Sugarcane pathology. Vol. lll: Bacterial and nematode disease. Edited by Rao, G. P., Saumtally, Salem , and Rott, Philippe. Enfield, U.S. Science Publishres.97-124.