Inducing systemic resistance against some tomato virus ... Title: INDUCING SYSTEMIC RESISTANCE...

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  • Inducing systemic resistance

    against some tomato virus diseases

    By

    E m an Shahw an M oheb E lE m an Shahw an M oheb E lE m an Shahw an M oheb E lE m an Shahw an M oheb E l----D in Shahw anD in Shahw anD in Shahw anD in Shahw an

    B.Sc. Agric. Sci., (Plant Pathology) 2002 M.Sc. Agric. Sci. (Plant Pathology) 2007

    Fac. Agric., Moshtohor, Banha Univ.

    DISSERTATION

    Submitted in Partial Fulfillment of the Requirements for

    The Degree of

    DOCTOR OF PHILOSOPHY

    in PLANT PATHOLOGY

    (V iral D iseasesV iral D iseasesV iral D iseasesV iral D iseases)

    Agricultural Botany Department (Plant Pathology)

    Faculty of Agriculture, Moshtohor Banha University

    2010

  • Title: INDUCING SYSTEMIC RESISTANCE AGAINST SOME TOMATO VIRUS DISEASES

    Name: E m an Shahw an M oheb E lE m an Shahw an M oheb E lE m an Shahw an M oheb E lE m an Shahw an M oheb E l----D in Shahw anD in Shahw anD in Shahw anD in Shahw an

    Degree: DOCTOR OF PHILOSOPHY

    Department: Agricultural Botany (Plant Pathology)

    ABSTRACT

    The objectives of this study were isolation and identification of the most

    frequently and economically viruses causing serious losses in tomato crop in

    the different location of Qalyoubia Governorate, evaluating some medicinal

    plant extracts and kombucha filtrate as biotic inducers to induction systemic

    acquired resistance in the tomato plants against CMV and using more effective

    bioinducers as bioelicitors for control viruses infection via induction

    'pathogenesis-related' (PR-1a) genes.

    Target virus was chosen according to its more frequently and severity

    among the isolated viruses in these locations at the winter season from the

    study year. Isolated virus was confirmed biologically and serologically assays.

    Extracts of two medicinal plants (Clerodendrum inerme L. Gaertn and

    Mirabilis jalapa L.) and were individually or in mixture in addition to

    kombucha filtrate were evaluated as bioinducers. All the four inducers were

    successfully in the induction of systemic acquire resistance (SAR) in the

    uninoculated tomato plants and sprayed with (50% v/v) of inducers.

    Tested bioinducers were used as biocontrol to inhibiting the virus

    infection of tomato plants as spraying every 15 days under greenhouse

    conditions. Pathogenesis-related (PR-1a) gene was molecularly isolated and

    identified via sequencer which compared with those recorded in the Gen-Bank.

    In conclusion, using medicinal extracts and other natural inducers were promise with good systemic acquired resistance against the great numbers of plant pathogens. In future, induction of resistance can be done cheaply and easily using natural substances

  • CONTENTS Page

    LIST OF FIGURES ...................................................................................... i LIST OF TABLES ......................................................................................iv LIST OF PLATES..................................................................................... vii LIST OF ABBREVIATIONS .................................................................ix INTRODUCTION ........................................................................................1 REVIEW OF LITERATURE..................................................................4 MATERIALS AND METHODS ..........................................................44 EXPERIMENTAL RESULTS ..............................................................73

    Part I 1- Disease incidence and frequency of virus ....................................73 2- Confirmation of Cucumber mosaic virus (CMV) ......................75

    2.1. Host range .........................................................................................75 2.2. Transmission of CMV ...................................................................78 2.3. In vitro properties............................................................................78 2.4. Inclusion bodies...............................................................................80 2.5. Dot blot immunoassay (DBIA) ...................................................80

    Part II Evaluation of biotic inducers of systemic acquired resistance and biocontrol of CMV ................................................ 82 A. Induction of systemic acquired resistance (SAR)

    by biotic inducers before virus inoculation ....................82 1. Histopathology changes ...........................................................82 2. Biochemical changes .................................................................86

    2.1. Antiviral Proteins......................................................................86 a. Determination the elicited antiviral protein as response to

    induction SAR (pre-inoculation) after 7-days...........................86 b. Determination the elicited antiviral protein as response to

    induction SAR (post-inoculation) after 25-days.......................89 2.2- Oxidative enzymes .......................................................................92 a. Peroxidase isozyme in tomato plants sprayed with biotic

    inducers to induce SAR (pre-inoculation) after 7-days ............92 b. Peroxidase isozyme in tomato plants sprayed with biotic

    inducers to induce SAR (post-inoculation) after 25-days........95 c. Polyphenol oxidase isozyme in tomato plants sprayed

    with biotic to induce SAR (pre-inoculation) after 7-days........ 98

  • d. Polyphenol oxidase isozyme in tomato plants sprayed with biotic to induce SAR (pre-inoculation) after 25-day.... 101

    2.3. Quantification of total SA in tomato plants treated with biotic pre-virus inoculation .....................104

    2.4. Photosynthetic pigments content..........................................109 2.5. Determination of phenolic compounds ..........................110 2.6. RNA determination in tomato plants treated with

    biotic inducers pre-virus inoculation.......................... 112 3. Molecular marker for SAR detection ............................113

    Analysis of molecular data by Bioinformatics ...................... 116 1- Nucleotide sequence....................................................................... 116 2- Translation of partial nucleotide sequence of PR-la

    gene for tomato plants treated with biotic inducers ....121 4- Effect of biotic inducers on virus infectivity during

    induction of SAR as follows................................................... 126 B- Using of biotic inducers as bioinducers to control CMV

    infection ................................................................................................ 128 1. Histopathological changes ....................................................... 128 2. Biochemical changes ................................................................... 131

    2.1. Antiviral proteins.................................................................. 131 a. Determination the elicited antiviral protein as

    response to treatment with bioinducers to control infected tomato plants after 7 days of spraying............... 131

    b. Determination the elicited antiviral protein as response to treatment with bioinducers to control infected tomato plants after 25 days of spraying ............ 134

    2.2. Oxidative enzymes................................................................ 137 a. Peroxidase isozyme in infected tomato plants and

    sprayed with bioinducers to control CMV after 7 days of spraying.................................................................................. 137

    b. Peroxidase isozyme in infected tomato plants and sprayed with bioinducers to control CMV after 25- days of spraying........................................................................ 139

    c. Polyphenol oxidase isozyme in infected tomato plants and sprayed with bioinducers to control CMV after 7 days of spraying .......................................................... 143

    d. Polyphenol oxidase isozyme in infected tomato plants and sprayed with bioinducers to control CMV after 25 days of spraying........................................................ 145

  • 2.3- Photosynthetic pigments content ................................... 149 2.4. Determination of total phenols ........................................ 151 2.5. Total free amino acids content in inoculated

    tomato plants and treated with bioinducers ............ 152 2.6. Total carbohydrates content in inoculated tomato

    plants and treated with bioinducers ........................... 154 3. Effect of bioinducers on virus infectivity ........................... 155

    DISCUSSION ............................................................................................ 157 SUMMERY ................................................................................................ 188 REFERENCES ......................................................................................... 198 ARABIC SUMMARY

  • i

    LIST OF FIGURES Page

    Figure 1 Conceptual model for the pathway leading to the establishment of SAR...........................................................29

    Figure 2 Standard curve of the protein concentration using bovine serum albumin as a standard protein. ..................................57

    Figure 3 Sta