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Ph D THESIS agrobiologie
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2011 by Hammad Ketta All rights reserved, The great aim of education is not knowledge but action. Herbert Spencer,Declaration, I declare that I have elaborated my dissertation work aimed at Cereal viruses. transmitted by Polymyxa graminis I have used only the sources quoted in References. This PhD thesis is submitted in partial fulfillment of the requirements for the degree of. Doctor of Philosophy in Plant Protection Sciences Czech University of Life Sciences. Prague 2011, All rights reserved No part of this publication may be reproduced without written. permission of the copyright holder,Prague 2011,In Prague date 13 12 2011 Signature. ACKNOWLEDGMENTS,ACKNOWLEDGMENTS, I would like to express my deepest thanks to my mentor the wise fairly minded and.
honest Prof Ing Pavel Ry nek CSc head of the Department of Plant Protection Faculty. of Food and Natural Resources Czech University of Life Sciences Prague in the Czech. Republic for his guidance helpful encouragement and continuous support during the. practical stages of the experiments and dissertation writing. Similarly I wish to express my special gratitude and appreciation to Ing Miloslav. Zouhar Ph D for his sincere help diligent effect valuable ideas and remarks during the. research work, I am grateful to my parents and my wife for their help and supporting me during all. stages of the research work, I thank also all the colleagues and technicians of the Department of Plant Protection for. their assistance and friendship that made me feel home. Barley mild mosaic virus BaMMV and Barley yellow mosaic virus BaYMV that. belong to Bymovirus genus familiy Potyvirideae are causal agents of soil borne mosaic. disease in winter barley in the UK China Japan and several European countries but they are. not yet reported in the Czech Republic This disease is economically important and in years. particularly favourable for disease development is known to cause crop yield losses of up to. 50 70 Mechanical transmission of BaMMV into barley seedlings is generally inefficient. and is the major constraint for testing cultivar resistance to the virus Both viruses are. transmitted to plants exclusively by a vector Polymyxa graminis which is a relatively poor. characterised eukaryotic fungal like micro organism It survives in the soil as resting spores. cystosori containing the viruses within their protective shells When the host plants are. present and the environmental conditions are favourable these spores release swimming. zoospores which invade and develop in the roots of barley and some other cereal crops. During root invasion the viruses are transmitted to healthy root cells from where they move. upwards systemically into the shoots and leaves where they cause mosaic symptoms general. stunting of the crop and poor seed set, There are no known chemical control agents against P graminis and control of. BaYMV and BaMMV is only possible by deploying virus resistant cultivars Thus the. cultivation of resistant varieties is the only way to prevent the considerable yield losses. caused by the pathogen Czech isolates of P graminis may be able to transmit BaYMV and. To explore mechanical transmission BaMMV infected barley plants were grown at. glasshouse conditions and used as inoculum sources to inoculate seedlings of susceptible. winter barley cv Florian Extracts were prepared from BaMMV infected Florian plant leaves. at 30 and 60 days after symptom appearance DASA and grown at 12 to 14 C High. infection rates were attained in addition of some compounds in inoculation buffer potassium. phosphate buffer 0 04 M Especially addition of sodium diethyldithiocarbamate Na DIECA. was found to increase infectivity in mechanical inoculation of BaMMV as polyphenoloxidase. inhibitor and chelator of copper Inoculated plants showed visible symptoms after four weeks. from inoculation symptomatic plants were tested by ELISA DAS to confirm the results. From the results of first experiment we observed that the chemical additives Na. DIECA 0 01 M and Na DIECA 0 001 M were more effective during the mechanical. inoculation than other chemical additives For the previous reason we designed the second. experiment of mechanical inoculation with potassium phosphate buffers 0 04 M and. potassium phosphate buffers 0 04 M with additives Na DIECA 0 001 M and Na DIECA 0 01. M in different pH values 6 5 7 7 5 and 8 The obtained results confirmed that the. chemical additive Na DIECA 0 001 M with pH value 8 was the most effective chemical. substance for mechanical inoculation of BaMMV to susceptible barley plants cv Florian. For the monitoring of BaMMV and BaYMV in some localities in the Czech Republic. seventeen soil samples which collected separately from different localities of cereal. production barley during April 2008 using a digging fork were placed separately and planted. to barley in pots in a greenhouse at 12 14 C and fifty six tested barley plant samples planted. into soil samples collected from different localities of sugar beet production during 1989 and. 1999 were infected neither by BaMMV nor BaYMV, From the obtained results the preservation method with CaCl2 was the best way for. BaMMV infected tissues preservation for a long term 13 and 14 months. Homogenization method of plant materials by the machine RETSCH Mixer Mills MM. 400 was much easier and quicker than other used methods of homogenization for the reason. of BaMMV detection by ELISA Detection sensitivity using the classical ELISA method. reported that BaMMV was detected using ELISA in the three greatest concentrations A. sample was considered positive for BaMMV when absorbance at 405 nm was more than three. times the mean of the negative control, Although the using of mortar and pestles with liquid nitrogen for homogenization of.
plant materials for RNA extraction is a labor intensive and it takes much time it was the best. method for homogenization of plant material All tested RNA extraction methods the. isolation of RNA using phenol chloroform isolation isolation with GeneJET RNA kit and. magnetic isolation were used for the detection of BaMMV in the infected leaves of barley. were on the bases of RT PCR analysis to be reliable The presence of BaMMV was confirmed. in all samples analyzed, Purity of RNA expressed as an A260 A280 coefficient ranged between 2 02 2 06 with the. isolation method GeneJET RNA kit and isolation by magnetic method while the method of. phenol chloroform isolation values ranged between 1 59 1 65 In terms of the time. consumption and total steps during RNA isolation was evaluated as the optimal method for. the isolation of total RNA using the magnetic method The separated fragments of cDNA on. the gel were so sharp and clear with the extraction method by magnetic isolation in. comparison with other methods of extraction, For the monitoring of P graminis in some localities in the Czech Republic seventeen. soil samples from different localities of cereal fields barley with prediction of P graminis. occurrence were collected during April 2008 and fifty six soil samples collected from. different localities of sugar beet production during 1989 and 1999 All collected soil samples. were planted to barley The protist was identified as P graminis on the basis of morphology. of resting spores cystosori and sporangia and the size of individual cystosori 4 to 5 m in. diameter Cystosori of P graminis were observed in the roots of barley plants grown in 2 of. the 17 soil samples and 20 of the 56 soil samples especially the samples from esk Mezi i. The presence of P graminis in the roots of plants grown in the soil samples and the positive. control sample versus the absence of the vector in roots of plants in the negative control soil. was verified by PCR assay with DNA extracts The method of extraction and homogenization. of barley roots material by using RETSCH Mixer Mills MM 400 was more suitable for. obtaining the DNA of P graminis cystosri and the time consumption was shorter in. comparison with the first method of extraction and homogenization. To characterize the P graminis isolates the amplified PCR product a DNA fragment. of 472 bp was sequenced and blasted for each of the samples that tested positive These. sequences were aligned with a known sequence GenBank Accession No AM259276 for P. graminis The sequences from P graminis on barley were 100 homologous to the. published sequence of P graminis f sp temperata To our knowledge this is the first report. of P graminis f sp temperata in the Czech Republic. The obtained results from the experiments of the sand culture indicated that all tested. barley cv Florian roots were parasitized by P graminis but were not infected by BaMMV. after one and half month of growing The chosen plants were tested by ELISA and RT PCR. Later after more than three and half months we obtained clear symptoms of BaMMV with the. seedlings of barley cv Florian The results confirmed that the Czech isolates of P graminis. are able to transmit the BaMMV,TABLE OF CONTENTS,TABLE OF CONTENTS. ACKNOWLEDGMENTS I,ABSTRACT II,TABLE OF CONTENTS V,LIST OF TABLES VIII. LIST OF FIGURES IX,LIST OF USED ABBREVIATIONS X,1 INTRODUCTION 1.
2 REVIEW OF LITERATURE 3,2 1 FAMILY POTYVIRIDEAE 4. 2 2 BYMOVIRUSES 4,2 3 DISTRIBUTION OF SOIL BORNE BARLEY VIRUSES 5. 2 4 BIOLOGICAL AND SEROLOGICAL PROPERTIES OF BAMMV AND BAYMV 6. 2 5 CONTROLLING OF SOIL BORNE BARLEY VIRUSES 8,2 6 GENOME AND COAT PROTEIN OF BAYMV 8. 2 7 GENOME ORGANIZATION AND COAT PROTEIN OF BAMMV 10. 2 8 PHYLOGENETIC ANALYSIS 11, 2 9 MECHANICAL INOCULATION AND TESTS FOR RESISTANCE 12. 2 9 1 Modified inoculation technique 14, 2 9 2 Reducing agents and substances protecting against phenolic compounds 14.
2 10 LONG TERM PRESERVATION OF VIRUS INFECTED TISSUES 15. 2 11 PLASMODIOPHORID TAXONOMY 16,2 12 PLASMODIOPHORID DISTINCTIVE FEATURES 17. 2 13 POLYMYXA GRAMINIS A VECTOR OF SOIL BORNE BARLEY VIRUSES 17. 2 14 LIFE CYCLE OF POLYMYXA GRAMINIS 19,2 15 VIRUS ACQUISITION BY POLYMYXA GRAMINIS 21. 2 16 RISK FACTORS 22, 2 17 MOLECULAR DIVERSITY AND PHYLOGENETIC RELATIONSHIPS OF. PLASMODIOPHOROMYCETES 22, 2 18 ENZYME LINKED IMMUNOSORBENT ASSAY ELISA PROCEDURES 26. TABLE OF CONTENTS,2 18 1 Direct Double Antibody Sandwich Method 26.
2 18 2 Indirect Double Antibody Sandwich Methods 26. 2 19 MOLECULAR METHODS 28, 2 19 1 RNA extraction methods for viral detection 28. 2 19 2 Concentration of total RNA 28,2 19 3 Reverse transcription RT 28. 2 19 4 Polymerase Chain Reaction PCR 29,2 19 4 1 PCR Modifications 30. 2 19 5 Separation of nucleic acids by electrophoresis 31. 2 19 5 1 Agarose gel electrophoresis 31,2 19 5 2 Polyacrylamide gel electrophoresis 31. 3 THESIS OBJECTIVES 33,4 MATERIALS AND METHODS 34,4 1 INOCULUM SOURCE 34.
4 2 MECHANICAL INOCULATION 34, 4 3 MEANS OF EFFECTIVE MECHANICAL TRANSMISSION OF BAMMV 35. 4 4 MONITORING OF BAMMV AND BAYMV IN BARLEY IN THE CZECH REPUBLIC 36. 4 5 LONG TERM PRESERVATION OF BAMMV INFECTED TISSUES 36. 4 6 DAS ELISA PROTOCOL PAB 38,4 7 RNA EXTRACTION OF BAMMV 40. 4 8 REVERSE TRANSCRIPTION RT 45,4 9 POLYMERASE CHAIN REACTION PCR 45. 4 10 AGAROSE GEL ELECTROPHORESIS 46,4 11 MONITORING OF P GRAMINIS 47. 4 12 TRANSMISSIBILITY OF THE BAMMV BY CZECH ISOLATES OF P GRAMINIS 53. 5 RESULTS AND DISCUSSION 54,5 1 INOCULUM SOURCE 54.
5 2 MECHANICAL INOCULATION METHODS 54, 5 3 MEANS OF EFFECTIVE MECHANICAL TRANSMISSION OF BAMMV 56. 5 4 MONITORING OF BAMMV AND BAYMV IN BARLEY IN THE CZECH REPUBLIC 60. 5 5 LONG TERM PRESERVATION OF BAMMV INFECTED TISSUES 60. 5 6 DAS ELISA PROTOCOL PAB 61,5 7 RNA EXTRACTION AND RT PCR METHOD 62. 5 8 MONITORING OF P GRAMINIS 63,TABLE OF CONTENTS, 5 9 TRANSMISSIBILITY OF THE BAMMV BY CZECH ISOLATES OF P GRAMINIS 71. 6 CONCLUSION 72,7 REFERENCES 75,APPENDIX 94,LIST OF TABLES. LIST OF TABLES,Table 1 Important soil borne viruses of cereals 3.
Table 2 Cereal viruses transmitted by Polymyxa graminis 19. Table 3 Subgroups of Polymyxa graminis 23, Table 4 Chemical additives to the inoculation buffer 35. Table 5 Chemical additives to the inoculation buffer in different pH values 36. Table 6 The percentage of infected barley plants cv Florian after mechanical inoculation 60. Table 7 Purity and concentration of total RNA extracted from BaMMV infected barley 62. Table 8 Presence of P graminis in soil samples collected from different localities of cereal. production barley in April 2008 66, Table 9 Presence of P graminis in soil samples collected from different localities of sugar. beet production in 1999 67, Table 10 Presence of P graminis in soil samples collected from different localities of sugar. beet production in 1989 67,LIST OF FIGURES,LIST OF FIGURES. Fig 1 Occurrence of Barley yellow mosaic virus and Barley mild mosaic virus in Germany 6. Fig 2 Map of BaYMV genome and putative protein products 9. Fig 3 Genome organization of Barley mild mosaic virus BaMMV 11. Fig 4 Phylogenetic trees of the coat protein top and the P2 fragment bottom 12. Fig 5 Polymyxa graminis 21, Fig 6 Phylogenetic analysis of Plasmodiophoromycetes on the basis of the rDNA 25.
Fig 7 Preliminary steps of the InnuPure C12 44, Fig 8 Percent of BaMMV infected plants by two methods of mechanical inoculation 55. Fig 9 Inoculation method stick with gauze SWG 56, Fig 10 Symptoms of BaMMV infected winter barley cv Florian 56. Fig 11 Final frequencies of infected plants of cv Florian six weeks after inoculation 57. Fig 12 Progression of the frequency of BaMMV infected cv Florian plants 58. Fig 13 Percentage of infected barley plants cv Florian four weeks after inoculation 59. Fig 14 Sensitivity the ELISA assays for BaMMV detection 61. Fig 15 Comparison of sharpness and clearness of BaMMV DNA 63. Fig 16 Photomicrographs of sporosori of P graminis in roots of barley cv Florian 65. Fig 17 Sporosori of Ligniera spp in the roots of barley cv Florian 65. Fig 18 A PCR products of DNA fragments of 472 bp obtained from P graminis 68. Fig 19 Sand culture for growing of barley cv Florian 71. LIST OF USED ABBREVIATIONS,LIST OF USED ABBREVIATIONS. A260 UV in wave length 260 nm Maximum absorption of nucleic. AMV RT AMV Reverse Transcriptase,bp Base pair,BSA Bovine serum albumin. C Cytosine,cDNA Complementary DNA,Chl Iaa Chloroform isoamylalcohol.
CP Capsid Coat Protein,CTAB Cetyltrimethylammonium bromide. cv Cultivar,ddH2O Double distilled water,DNA Deoxyribonucleic acid. dNTP Dinucleotid tri phosphate,dsDNA Double strand DNA. dsRNA Double strand RNA,EDTA Ethylendiaminetetraacetic acid. ELISA Enzyme Linked Immunosorbent Assay,Fig Figure.
g Times gravity, ICTV International Committee on Taxonomy of Viruses. kDa Kilo Dalton molecular weight of proteins,kbp Kilo base pair. ME Mercaptoethanol 2 Mercaptoethanol,min Minute,MP Movement protein. mRNA Messenger ribonucleic acid,PAGE Poly Acrylamide Gel Electrophoresis. PBS Phosphate buffer saline,PCR Polymerase Chain Reaction.
PVP Polyvinylpyrrolidone,LIST OF USED ABBREVIATIONS. RdRp RNA dependent RNA polymerase,Ribolock Ribonuclease inhibitor. RNA Ribonucleic acid,RNAse Ribonuclease,rRNA Ribosomal ribonucleic acid. RT PCR Reverse Transcriptase Polymerase Chain Reaction. SDS Sodium dodecyl sulfate,sec Second,ssDNA Single strand DNA. ssRNA Single strand RNA,TBE Tris borate EDTA,TGB Triple gene block.
Tris Tris hydroxymethyl aminomethane,tRNA Transfer ribonucleic acid.

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