VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF AGRONOMY -*** - UNDERGRADUATE THESIS TITLE: STUDY ON BACTERIAL LEAF BLIGHT (Xanthomonas oryzae) OF RICE Student name : BUI HUU QUANG Class : K61KHCTT Student code : 614114 Department : PLANT PATHOLOGY Supervisor : Assoc.Prof HA VIET CUONG HANOI – 2021 DECLAIRE I hereby declare that this graduation thesis is my own original work and has not been submitted before to any institution for assessment purposes Further, I have acknowledged all sources used and have cited them in the reference section Hanoi, January 2021 Signature Bui Huu Quang i ACKNOWLEDGEMENTS First of all, I would like to express my deep gratitude to Assoc.Prof.Dr Ha Viet Cuong, Lecturer of Plant Disease Department; Dr Nguyen Van Muoi, Plant Research Institute, Faculty of Agriculture, Vietnam National Academy of Agriculture The two instructors were always by my side, caring, helping, giving suggestions, commenting and conveying to me valuable experiences during the graduation thesis With the most sincere feelings, I would like to thank all staff of the Center for Tropical Plant Disease, Center for Conservation and Development of Plant Genetic Resources, and Plant Research Institute for their enthusiastic guidance and assistance has taught enthusiastically about the expertise and skills of the laboratory along with the support of experimental materials during the practice of the thesis And finally, with all my infinite respect and gratitude, I would like to extend my parent; is a solid rear for me on the road of life Thank you very much! Bui Huu Quang ii TABLE OF CONTENTS DECLAIRE .i ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iii LIST OF ABBREVIATIONS v LIST OF TABLES vi LIST OF FIG vii SUMMARY ix PART I: INTRODUCTION 1.1 Introduction 1.2.Objectives and requirements 1.2.1 Objectives 1.2.2 Requirements PART II: LITERATURE REVIEW 2.1 Status of research on Bacterial leaf blight of rice in the world 2.2 Status of research on Bacterial leaf blight of rice in Vietnam 2.3 Discovery and classFIcation of Xanthomonas oryzae 2.4 Distribution and economic impact 2.5 Pathogen modes of infection, symptoms, and signs 2.6 Sources of primary inoculum, dissemination and survival 2.7 Control 10 2.8 Xanthomonas oryzae pv oryzae (Xoo) 14 2.8.1 Morphology and structural 14 2.8.2 Genomic 16 2.8.3.Pathogenicity (related genes) genes 18 PART III MATERIALS AND METHODS 21 3.1 Location and time for research 21 3.1.1 Research location 21 3.1.2 Time for research 21 3.1.3 Subjects 21 3.2 Materials 21 iii 3.2.1 Media and preparation 21 3.2.2 Chemicals and buffers 23 3.2.3 Commercial enzymes and markers 23 3.2.4 Antibiotics and bactericide 23 3.2.5 Primers 24 3.2.6 Key facilities 24 3.3 methods 24 3.3.1 Collection of leaf samples 24 3.3.2 Bacterial isolation 25 3.3.3 PCR test 25 3.3.4 Pathogenicity test 27 3.3.5 Isolation of Xanthomonas oryzae from rice leaves 28 3.3.6 Inoculation of Xoo 28 3.3.7 Agarose Electrophoresis 28 PART IV RESULTS AND DISCUSSION 30 4.1 ISOLATION OF XANTHOMONAS ORYZAE PV ORYZAE 30 4.1.1 Bacterial leaf blight (BLB) sampling 30 4.1.2 Isolation of Xanthomonas oryzae from collected samples 31 4.1.3 Identification of Xanthomonas oryzae pv oryzae by multiplex PCR 33 4.2 MORPHOLOGICAL and biological characteristics of XOO isolates 35 4.2.1 Identification of Gram group of Xoo isolates by KOH test 35 4.2.2 Colony characteristics of Xoo isolates on different media 37 4.2.3 Starch hydrolysis 40 4.2.4 Cellulose hydrolysis 42 4.2.5 Catalase test 44 4.2.6 Hypersensitive reaction (HR) on tobacco leaf 46 4.2.7 Reaction of Xoo isolates with antibiotics and bactericide 47 4.2.8 Pathogenicity test 50 PART V CONCLUSION AND SUGGESTION 54 5.1 Conclusion 54 5.2 Proposal for further work 54 REFERENCES: 55 iv LIST OF ABBREVIATIONS ABBREVIATION TERM Ab Antibody Ag Antigen BLB Bacterial leaf blight BLS Bacterial leaf streak Bp Base pair DNA Deoxyribonucleic acid dNTP Deoxynucleoside triphosphate EDTA Ethylene diamine tetra acetic acid ELISA Enzyme linked immunosorbent assay EPS Capsular extracellular polysaccharide Ig Immunoglobulins OD Optical Density ODD Ouchterlony Double Diffusion PCR Plolymerase Chain Reaction PEG Polyethylen PSA Peptone Sucrose Agar TAE Tris – acetate – EDTA UV Utra Violet Xo Xanthomonas oryzae Xoc Xanthomonas oryzae pv oryzicola Xoo Xanthomonas oryzae pv oryzae v LIST OF TABLES Table 3.1 PCR master mix (25 µL) 26 Table 3.2 PCR profile 26 Table 4.1 Rice bacterial leaf blight samples collected in 2020 30 Table 4.2 Isolation of Xanthomonas oryzae from diseased rice leaf samples on mXOS medium 32 Table 4.3 Detection of Xanthomonas oryzae pv oryzae by multiplex PCR 34 Table 4.4 Gram differentiation of Xoo isolates by KOH test 36 Table 4.5 Colony characteristic of Xoo isolates on different media after days at 28 oC 37 Table 4.6 Starch hydrolysis test of Xoo isolates 41 Table 4.7 Cellulose hydrolysis test of Xoo isolates 43 Table 4.8 Catalase test of Xoo isolates 45 Table 4.9 Hypersensitive reaction (HR) of selected Xoo isolates on tobacco leaf (Nicotiana tabacum cv K326) 46 Table 4.10 Growth inhibition of antibiotics and bactericide against Xoo isolates 48 Table 4.11: Inoculation result of Xoo isolates on rice seedling 51 vi LIST OF FIG Fig 2.1 Morphology of Xanthomonas oryzae (a) Strands and condensed droplets of ooze consisting of X oryzae cells coated in extracellular polysaccharide exuded on to the surface of an infected rice leaf (b) Colonies of X oryzae pv oryzae on glucose yeast extract agar (c) Scanning electron micrograph of a single X oryzae pv oryzae cell (bar, 1.0 16 Figure 2.2 Genomic map of Xoo strain MAFF311018 17 Figure 4.1 Symptoms of collected rice bacterial leaf blight samples 31 Figure 4.2 Isolation of Xoo on mXOS medium by 3-ways streaking (A) and dilution (B) methods The plates are from sample The plate B is at 102 dilution The arrow indicates the Xoo-like colonies 33 Figure 4.3 Multiplex PCR of Xoo isolates selected from positive colonies M1 is 100 bp DNA ladder with denoted reference bands M2 is kb DNA ladder (GeneRuler kb, Thermo Scientific) Two bands, 331 and 162 bp, specific to Xoo pathovar are indicated by arrow 35 Figure 4.4 KOH reaction of Xoo isolates depictured from the Xoo-01 isolate 36 Figure 4.5 Culture of Xoo isolates on different media after incubation at 28 oC for days 40 Figure 4.6 Starch hydrolysis test of Xoo isolates show all of them are positive 42 Figure 4.7 Cellulose hydrolysis test of Xoo isolates showed all of them were positive 44 Figure 4.8 Catalase test of Xoo isolates The Xoo isolates are indicated 45 Figure 4.9 Hypersensitive reaction (HR) of selected Xoo isolates on tobacco leaf (Nicotiana tabacum cv K326) 47 Figure 4.10a Evaluation of growth inhibition of antibiotics and bactericide at recommended 10X concentration against Xoo isolates 49 vii Figure 4.10b Illustration of position and growth inhibition of antibiotics and bactericide at recommended 10X concentration against Xoo isolates (Xoo-1) 49 Figure 4.11 XOO-1 isolate infection in rice varieties 52 Figure 4.12 XOO-2 isolate infection in rice varieties 52 Figure 4.13 XOO-3 isolate infection in rice varieties 52 Figure 4.14 XOO-4 isolate infection in rice varieties 53 Figure 4.15 XOO-6 isolate infection in rice varieties 53 viii SUMMARY Title: “Study on Bacterial leaf blight of rice Purpose -This study aimed to detect and evalution of biological, physiological and pathogenic characteristics of Xanthomonas oryzae pv Oryzae Research method -To collect the infected leaves samples from different locations -To isolate and identify Xanthomonas oryzae pv oryzae (Xoo) from collected samples -To evaluate the biological characteristics of isolated Xoo isolates -To evaluate the physiological characteristics of isolated Xoo isolates -To evaluate the pathogenicity of isolated Xoo isolates Newness, creativity Leaf blight is caused by bacteria Xanthomonas oryzae pv oryzae (Xoo) causes severe damage to rice growing regions across the country, reducing rice yield by up to 50% Although some chemicals have been used in recent years, they cannot control the disease under severe conditions They also cause environmental problems, especially water pollution Therefore, my research contributes to the evaluation of the pathogenicity of current blight bacteria, while also preventing the risk of outbreaks Research results - Using semi-selective medium, mXOS, several anthomonas oryzae – like isolates collected from provinced in the Red River Delta were isolated Based on Multiplex PCR, Xoo isolates were identified and selected for futher studies - The Xoo isolates have several identical characteristics including: Gram negative (KOH test), grow slowly on nutrient media, colony being convex, mucoid, shiny, pale to straw yellow colour in PSA, mWakimoto and YDC ix Figure 4.7 Cellulose hydrolysis test of Xoo isolates showed all of them were positive 4.2.5 Catalase test Catalase mediates the breakdown of hydrogen peroxide H2O2 into oxygen and water To find out if Xoo isolates are able to produce catalase enzyme, a small inoculum of each isolate grown on PSA medium plate was mixed into hydrogen peroxide solution (3%) and is observed for the rapid elaboration of oxygen bubbles The lack of catalase is evident by a lack of or weak bubble production Catalase-positive bacteria include strict aerobes as well as facultative anaerobes They all have the ability to respire using oxygen as a terminal electron acceptor 44 For this test, all our Xoo isolates were catalase-positive (Table 4.8 and Figure 4.8) Table 4.8 Catalase test of Xoo isolates No Isolates Formation of oxygen bubbles Reaction conclusion Xoo-01 Yes Positive Xoo-02 Yes Positive Xoo-03 Yes Positive Xoo-04 Yes Positive Xoo-05 Yes Positive Xoo-06 Yes Positive Xoo-08 Yes Positive Xoo-09 Yes Positive Figure 4.8 Catalase test of Xoo isolates The Xoo isolates are indicated 45 4.2.6 Hypersensitive reaction (HR) on tobacco leaf All phytopathogenic bacteria produce a hypersensitive reaction (HR) in leaf mesophyll tissue Saprophytes allegedly not induce this reaction, making HR a quick and useful determinative test to differentiate saprophytes from plant pathogens The reaction is invaluable for the characterization of phytopathogenic bacteria recovered from outside their host or recovered from a host with latent infections Most Gram negative phytopathogenic bacteria produce hypersensitivity in tobacco leaf (Nicotiana tobaccum) For this test, all Xoo isolates were suspended in sterile water and the bacterial suspensions were infilled in leaves of tobacco plant (Nicotian tabacum cv K326) After 48 hours, the leaf tissues at the infiltration positions of all isolates were collapsed, indicating they induced the HR reaction in the tobacco leaf (Table 4.9, Figure 4.9) Table 4.9 Hypersensitive reaction (HR) of selected Xoo isolates on tobacco leaf (Nicotiana tabacum cv K326) Formation of death tissue at 48 No Isolates Xoo-01 Yes Positive Xoo-02 Yes Positive Xoo-03 Yes Positive Xoo-04 Yes Positive Xoo-05 Yes Positive Xoo-06 Yes Positive Xoo-08 Yes Positive Xoo-09 Yes Positive hours after infiltration Reaction conclusion 46 Figure 4.9 Hypersensitive reaction (HR) of selected Xoo isolates on tobacco leaf (Nicotiana tabacum cv K326) 4.2.7 Reaction of Xoo isolates with antibiotics and bactericide In this experiment, two isolates Xoo-1 and Xoo-9 were selected to evaluate the response of Xoo to antibiotics (Ampicillin, Chloramphenicol, Streptomycin, Rifampicin and Tetracycline) and one bactericide for numerous plant bacteria including Xoo (Kasugamycin under trademark Kasumin 2SL from Arista company) When tested with the filter paper disks supplemented with antibiotics and bactericide at the recommended concentration (ie Ampicillin 100 ppm, Chloramphenicol 34 ppm, Kasugamycin (Kasumin 2SL, Arista), 10 ppm, Streptomycin 100 ppm, Rifampicin 34 ppm, Tetracycline 12.5 ppm), both two isolates still grow well on medium, indicating that they were not inhibited by these antibiotics and bactericide at the recommended concentrations When the concentrations of the antibiotics and bactericide were raised 10X, only Streptomycin (1000 ppm) showed inhibition to both Xoo isolates 47 with the average diameter of inhibition zone being approximately 30 mm after days of incubation (Table 4.10 and Figure 4.10a and 4.10b) It is worthy to note that Streptomycin have been being the most popular antibiotic used in plant agriculture for bacterial disease control including Xoo One benefit outcome from this experiment is streptomycin can be used as a selective component supplemented in media for isolation of Xoo from rice plants or seeds Table 4.10 Growth inhibition of antibiotics and bactericide against Xoo isolates Antibiotic/Bac Concentration Recommended tericide (µg/mL = ppm) concentration 100 Average diameter of inhibition zone (mm) Xoo-1 Xoo-9 1X 0 1000 10 X 0 Chloramphenic 34 1X 0 ol 340 10 X 0 Kasugamycin 10 1X 0 100 10 X 0 100 1X 0 1000 10 X 29.8 ± 3.01 30.3 ± 0.35 34 1X 0 340 10 X 0 12.5 1X 0 125 10 X 0 0 Ampicillin (Kasumin 2SL, Arista) Streptomycin Rifampicin Tetracycline Control (H2O) 48 Figure 4.10a Evaluation of growth inhibition of antibiotics and bactericide at recommended 10X concentration against Xoo isolates Figure 4.10b Illustration of position and growth inhibition of antibiotics and bactericide at recommended 10X concentration against Xoo isolates (Xoo-1) 49 4.2.8 Pathogenicity test To identify the pathogenicity of Xoo isolates, we conducted inoculation of these isolates on rice seedlings of 10 diffrentiate cultivars (IRBB2, -3, -4, -5, -7, -10, -11, -14, -21, -24) and other rice cultivars which are popularly grown in the Northern Vietnam (Bac Thom 7, Thanh Em, and Nep) or being used in rice breeding program (IR64, R6, HC6, R99n, R353 and R26) by a leaf cutting method The plants of all cultivar were were planted in pots until 7-10 leaf stage Five isolates Xoo, representative for provinces, Xoo-1, Xoo-2, Xoo-3, Xoo-4 and Xoo-66, were selected for inoculation The isolates were cultivated on m Wakimoto medium After days of incubation, the isolates were harvested by resuspending in steril water (OD 600 approximatelly 2) and used for inoculation on tip of leaves by cutting method At least leaves of each plant were inoculated per each isolate After 18 days of inoculation, the length of lesions was measured, each isolate measured typical leaves and then averaged The inoculation results are shown in Table 4.11 and Figure 4.11-4.15 Inoculation result showed that all Xoo isolates could produce the lesion of BLB on inoculated leaves of all 19 rice cultivars However, the lesions were not severe and not typical This less severity of the disease on inoculated leaves could be resulted from some factors such as low temprature during inoculation, which is known to be unfourable for development of BLB disaese, or the resistance of rice plants at the seedling stage or the resistance induced by stress due to growth condition when plant were grown in pots 50 Table 4.11: Inoculation result of Xoo isolates on rice seedling Average length of lesion (mm) after 18 days of No inoculation Cultivar Xoo-1 Xoo-2 Xoo-3 Xoo-4 Xoo-6 control (H2O) IRBB 2.0 3.3 5.3 4.7 3.7 IRBB 4.7 8.0 9.3 7.0 14.3 IRBB 2.8 6.8 3.2 5.8 2.0 IRBB 2.2 8.7 2.0 4.5 6.3 IRBB 2.5 7.33 4.0 9.0 5.0 IRBB 10 10.3 8.0 7.0 6.8 6.3 IRBB 11 7.0 9.0 5.7 2.7 5.3 IRBB 14 5.7 8.0 3.0 6.0 6.0 IRBB 21 4.3 9.7 2.5 3.2 5.2 10 IRBB 24 3.5 4.0 0.8 1.0 1.0 11 IR64 3.3 3.5 3.0 3.0 1.8 12 R6 1.3 2.8 2.7 1.3 1.8 13 HC6 3.0 4.7 3.0 4.0 2.7 14 R99n 3.8 2.7 3.7 2.3 2.3 15 Bắc Thơm 1.5 3.3 2.3 2.7 2.5 16 R353 3.3 2.5 1.7 3.0 1.3 17 R26 1.5 2.2 3.0 1.77 2.2 18 Thành em 4.7 4.7 3.7 3.3 4.0 19 Nếp 3.0 2.3 1.7 2.2 3.0 51 Figure 4.11 XOO-1 isolate infection in rice varieties Figure 4.12 XOO-2 isolate infection in rice varieties Figure 4.13 XOO-3 isolate infection in rice varieties 52 Figure 4.14 XOO-4 isolate infection in rice varieties Figure 4.15 XOO-6 isolate infection in rice varieties 53 PART V CONCLUSION AND SUGGESTION 5.1 CONCLUSION Using semi-selective medium, mXOS, several Xanthomonas oryzae – like isolates collected from provinces in the Red River Delta were isolated Based on Multiplex PCR, Xoo isolates were identified and selected for further studies The Xoo isolates have several identical characteristics including: Gram negative (KOH test), grow slowly on nutrient media, colony being convex, mucoid, shiny, pale to straw yellow colour in PSA, mWakimoto and YDC medium, rose pink colour in mXOS, starch hydrolysis positive, cellulose hydrolysis positive and catalase positive All of them induced the Hypersensitive Reaction (HR) on tobacco leaves Invitro test on two Xoo isolates, Xoo-1 and X00-9, indicated that Streptomycin could inhibit growth of Xoo at 1000 ppm In contrast, other antibiotics including chloramphenicol, rifampicin, tetracycline and ampicillin and on bactericide, kasugamycin from Kasumin SL (Arysta), could not inhibit Xoo, even at concentrations that are 10 times higher than recommended ones Inoculation of Xoo isolates representative for provinces on rice seedlings by leaf cutting method showed all of them produced the BLB lesions on inoculated leaves of all 19 rice cultivars However, all the lesions were not severe and typical 5.2 PROPOSAL FOR FURTHER WORK - To collect more Xoo isolates in other province - To characterize further physiological and particularly pathogenic characteristics of the collected isolates - To apply the antibiotics that are not inhibit Xoo for selection of this 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