pathological and molecular studies

PATHOLOGICAL AND MOLECULAR STUDIES ON SPOTTED WILT VIRUS INFECTING TOMATO PLANTS BY

GAMALAT MOHAMED ALLAM B.Sc. Agric. Sc. (Plant Pathology), Ain Shams University 2002

A thesis submitted in partial fulfillment of

the requirements for the degree of

MASTER OF SCIENCE In Agricultural Science (Plant Pathology)

Department of Plant Pathology Faculty of Agriculture Ain Shams University

2006

Approval Sheet PATHOLOGICAL AND MOLECULAR STUDIES ON SPOTTED WILT VIRUS INFECTING TOMATO PLANTS BY GAMALAT MOHAMED ALLAM B.Sc. Agric. Sc. (Plant Pathology), Ain Shams University 2002

This thesis for M.Sc. degree (Plant Pathology) has been approved by: Prof. Dr. Mohamed A. Awad

--------------------------

Prof. of Plant Pathology, Fac, of Agric., El-Menofiya University Prof. Dr. Fawzy M. Abo El-Abbas

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Prof. of Plant Pathology, Fac. of Agric., Ain Shams University Prof. Dr. Mostafa H. El-Hammady

----------------------

Prof. Emeritus of Plant Pathology, Fac. of Agric., Ain Shams University

Date of Examination: 22 / 5 / 2006

PATHOLOGICAL AND MOLECULAR STUDIES ON SPOTTED WILT VIRUS INFECTING TOMATO PLANTS

BY

GAMALAT MOHAMED ALLAM B.Sc. Agric. Sc. (Plant Pathology), Ain Shams University 2002

Under the supervision of: Prof. Dr. Mostafa Helmy El-Hammady Professor Emeritus of Plant Pathology, Department of Plant Pathology, Faculty of Agriculture, Ain Shams University (Principal Supervisor) Dr. Tarek Abd El-Karim Moustafa Assistant Prof. of Plant Pathology, Department of Plant Pathology, Faculty of Agriculture, Ain Shams University Dr. Hayam Samy Abdelkader Researcher of Molecular Virology, Virus and Phytoplasma Research Department, Plant Pathology Research Institute, Agricultural Research Center

ABSTRACT Gamalat Mohamed Allam, Pathological and Molecular Studies on Spotted Wilt Virus Infecting Tomato Plants. Published M.Sc. Thesis, Department of Plant Pathology, Faculty of Agriculture, Ain Shams University, 2006. Tomato spotted wilt virus (TSWV) has become an economically important virus infecting major crops and causing serious losses in Egypt and worldwide. The virus under study was isolated from tomato fruits expressing typical symptoms that induced by TSWV from the experimental farm, Faculty of Agriculture, Ain Shams University. TSWV induced local and systemic infection on successive leaves of tomato upon mechanical inoculation. The results revealed that the virus is sap and insect transmissible, its thermal inactivation point is 45-50 ºC; the dilution endpoint is 10-3 and the virus was completely inactivated after incubation for 5-6 hours at room temperature. The host range studies revealed that the virus is capable to infect 33 plant belongs to 31 Genus and 16 Family including Amaranthacea, Apiaceae, Araceae, Asteracea, Brassicaceae, Chenopodiaceae, Convolvulaceae, Cucurbitaceae, Fabaceae, Geraniaceae, Malvaceae, Meliaceae, Poaceae, Primulaceae, Scrophulariaceae, and Solanaceae. The plant expressing symptoms varied in its reaction whereas two Genus following the Family Cucurbitaceae and Poaceae expressed no symptoms upon mechanical inoculation, while all the rest plant expressedlocal and systemic infection according to the infected host and these results were confirmed serologically by in-direct ELISA. Electron micrograph of the virus showed that it is quasispherical and its diameter ranges from 80-100 nm. Serological studies

were carried out to produce polyclonal antibodies against TSWV by using molecular cloning and expression of the purified fusion nucleoprotein (NP/6xHis) expressed in E.coli and isolated in a pure form and in a large quantity to be used in immunization experiment. The results indicated that the Egyptian polyclonal antibodies (TSWV/IgG-EG) obtained during this study were successfully capable to detect TSWV in infected tomato plants (Native protein) using indirect ELISA and also reacted specifically with the purified nucleoprotein (Denatured protein) which was injected into the experimental rabbit. It was found that the best antibody dilution that can be used in the detection of TSWV in infected plants was 1/1000. When TSWV/IgG-EG was compared with the Indian antiserum by in-direct plate trapping ELISA and DBIA, it was found that the Egyptian antibodies had proved high specificity and detectability in virus detection in all infected samples under test as well as the Indian one. The results of molecular studies showed the ability of detection of TSWV in total nucleic acid extracts sing reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocapture reverse transcriptase-polymerase chain reaction (IC-RT-PCR). The nucleocapsid (NP) protein gene was isolated by RT-PCR from total nucleic acid extracts from TSWV-infected tomato plants. A fragment of the NP gene of Tomato spotted wilt virus (TSWV) Egyptian isolate was amplified by RT-PCR using one set of primer TSW1 ( 5' – ATGTCTAAGGTTAAGCTC-3') and TSW2 (5'- TTAAGCAAGTTCTGTGAG-3'). DNA fragment (780 bp) amplicon was cloned, sequenced and expressed into the pBAD-Topo expression vector in E.coli. Immunocapture RT-PCR detected TSWV in infected tissues at a dilution of (10-3) of cDNA in infected tomato plants. cDNA probe hybridized strongly and specifically to TSWVinfected samples collected from Fayoum region and El-Menia region with a detection limit of 0.1 ng of total RNA.No background

reactions were observed when the DNA probe was tested with extracts from healthy plants. Sequence analysis of the NP gene showed that the TSWV Egyptian isolate is displaying about 81 % amino acid sequence homology to eight published amino acid sequences of TSWV/NP. The fusion nucleoprotein (NP/6xHis) was expressed in E.coli using 0.002 % of the L-arabinose after a time course of induction for 4 hours. The results indicated that the purified native NP/6xHis fusion nucleoprotein purified from E.coli was strongly reacted with Indian TSWV-AS. The results were confirmed by Western blotting technique which detected a major protein band of molecular mass ~ 28 kDa when probed with Indian TSWV-AS. The molecular expression results confirmed that the purified viral nucleoprotein expressed and purified from E.coli is a strong immunogenic that can be used in immunization experiment to produce diagnostic polyclonal antibodies specific to TSWV under Egyptian conditions. Key Words: TSWV; Nucleoprotein gene (NP) ; RT-PCR ; IC - RT - PCR; Western blotting; DBIA; Cloning; Protein Expression; Sequence analysis; Polyclonal antibodies; TSWV/IgG-EG.

Acknowledgment Thanks God I am deeply grateful to Prof. Dr. El-Hammady, Prof. of Plant Pathology, Faculty of Agriculture; Ain Shams University. His support and help have been invaluable. His reviewing of the manuscript has really been crucial. For I warmly thank Dr. Tarek A. Moustafa, Assistant Prof. of Plant Pathology, Faculty of Agriculture; Ain Shams University, for offering valuable suggestions during reviewing the manuscript. I wish to express my gratitude to Dr. Hayam S. Abdelkader, molecular biologist, Plant Pathology Reasearch Institute, (PPRI), ARC., for creating a supportive and encouraging working atmosphere and providing excellent research guidance. It has been a privilege to work with her. I am grateful to Prof. Dr. Ali M. Abdel-Salam, Prof of Plant Pathology, Faculty of Agriculture; Cairo University for his help in the purification of TSWV/IgG. He has always been willing to offer help when needed. His expertise with antiserum production has been invaluable. I thank Maha Al-Kahazendar Lecturer assistant, Faculty of Science, Cairo University, Microbiology Department for her help during the virus experiments. My deepest gratitude to the members of the molecular biology group. I never forget a nice and friendly atmosphere they created inside and outside the lab. I would like to thank all other colleagues in the Department of Virology at the Faculty of Agriculture, Ain Shams University who

have helped me in a way or another. Without the support from mother, sisters and brother I would not have the patience and persistence to accomplish this work.

CONTENTS Page LIST OF TABLES ............................................................ LIST OF FIGURES ..........................................................

iii v

I. INTRODUCTION ......................................................... II. REVIEW OF LITERATURE ..................................... III. MATERIALS AND METHODS ............................. Part I

1 4 18

I. Isolation and Identification of TSWV .............................. A. Isolation ...................................................................... B. Identification ............................................................... B. 1. Transmission .......................................................

18 18 18 19

B. 1.a. Mechanical transmission .................................. B. 1.b .Insect transmission ........................................... B.2. Host range and Symptomatology ......................... B.3. Virus stability ........................................................

19 19 20 20

B.4. Serological studies ................................................ B.4.1.Reactivity of TSWV nucleoprotein (NP) against Indian antiserum ........................................................... B.4.2. Antiserum production ........................................

21 22

B.4.2.a. Determination of Antiserum titer ..................... B.4.2.b. Separation of TSWV/IgG ................................ B.4.2.c. Titration of TSWV / IgG-EG ........................... B.4.3. Determination of dilution end-point of TSWV

23 23 24 24

22

infected sap by indirect ELISA method ......................... B.4.4. Evaluation of Egyptian TSWV-IgG by dot- blot

25

Page Immuno binding assay (DBIA) .................................... B.5 .Electron Microscopy ............................................. Part II II. Molecular studies ........................................................... A. High Pure RNA tissue kit:............................................

26

B. Cetyl trimethyl ammonium bromide method (CTAB): ... II. 2. RT-PCR amplification .............................................. II. 3. Molecular Cloning ...................................................... II.3.A. Preparation of E. coli competent cells ...................

27 28 29 29

II.3.B. Bacterial Transformation ...................................... II 3.C. Rapid screening of the transformed colonies by PCR ................................................................................ II.3.D. Preparation of recombinant plasmids ....................

26 26

30 30 31

II 3.E. Agarose gel electrophoresis .................................. II. 4. Southern hybridization ............................................. II.4.A. TSWV/NP DNA probe preparation ...................... II.4.B. Dot Blot Hybridization Assay ..............................

32 32 33 34

II.4.C. Hybridization technique......................................... II.4.D. Colorimetric detection .......................................... II. 5 .Immunocapture – Reverse Transcription – Polymerase Chain Reaction (IC-RT-PCR) ....................................

34 35 35

II.6. Nucleotide sequencing of TSWV-nucleoprotein gene .. II.7. Production of recombinant TSWV/NP-6xHis fusion protein via gene expression ....................................... II.7.A. Rapid screening of small cultures (Mini-prep) ...

36 37

II.7.B. Large scale production of TSWV/NPs ................. II.7.C. SDS- PAGE Electrophoresis of nucleoprotein ... II.7.D. Purification of 6xHis-tagged TSWV-nucleoprotein under denaturing conditions ............................................

38 38 39

II.7.E. Purification of 6xHis-tagged TSWV-nucleoprotein

39

under native conditions ....................................................

37

IV. RESULTS ................................................................... Part 1 Isolation and Identification of tomato spotted wilt virus(TSWV) ............................................................ A. Isolation ......................................................................

Page 41 41 41

B. Identification ................................................................ B.1. Transmission ............................................................. B.1.a. Mechanical transmission ....................................... B.1.b.Insect transmission (Thrips transmission ) ..............

41 41 41 42

B.2. Host range and symptomatology .............................. B.3. Virus stability ............................................................ B.4. Serological studies ..................................................... B.4.1.Reictivity of TSWVnucleoprotein ( NP ) against

42 49 50 50

Indian antiserum ....................................................... B.4.2 . Antiserum production ............................................ B.4.2 . a . Determination of TSWV antiserum titer ......... B.4.2. b . Separation of TSWV / IgG ................................

52 52 53

B.4.2.c . Titration of TSWV / IgG / EG and its reactivity with different infected tissues by in direct ELISA .. B.4. 3 . Determiation of diluation end – point of TSWV infected sap .............................................................

54

B.4. 4 . Evaluation of the antiserum by dot – blot Immunoassay ( DBIA ) ............................................ B.5. Electron microscopy ................................................. Part II

59

II. Molecular studies of TSWV ............................................ 1. RNA extraction ............................................................. 2. Detection of TSWV by RT-PCR ................................... 2.A. RT-PCR of TSWV/NPs ............................................

61 61 62 62

2. B. Southern Blot Analysis of the PCR products ...........

64

3. Molecular Cloning of partial sequence of TSWV/NP gene

65

55

60

Page in E.coli ................................................................... 4. Validation of Cloning of TSWV/NPs gene by PCR ..... 5. Dot Blot Hybridization Assay ........................................ 6. Immune Capture Reverse Transcription Polymerase Chain Reaction (IC- RT-PCR) ...........................................

68 69 71

7. Nucleotide sequencing analysis ..................................... 8. Expression of TSWV nucleocapsid gene in E.coli ........ 8. A. Rapid screening of small cultures .............................. 8. B. Purification of TSWV 6xHis-tagged rNP under

71 73 74 75

denaturing conditions ............................................... V. DISCUSSION VI. SUMMARY VII REFERENCES

76 88 94

Table No. 1 2 3 4 5 6 7

Host range of Tomato spotted wilt virus ( Tospovirus) tested by mechanical inoculation.

Page 43-44

Determination of thermal inactivation point (TIP) of TSWV in crude sap. Determination of the dilution end point (DEP) of TSWV in crude sap. Determination of the longevity in vitro of TSWV in crude sap. Antiserum titration produced against TSWV

49

Titration of TSWV/IgG/EG and its reactivity with different infected tissues Determination of dilution end-point of TSWV infected sap.

54

List of Tables

49 50 52

55

List of Figures Fig. No. 1A, 1,2

Page Symptoms expression on mechanically infected

45-46

host range. 1B

DBIA of twenty tomato plants exposed to 47 viriluferous Thrips tabaci insects and showed symptoms after 14 - 21 days from inoculation using TSWV Indian Antiserum was used.

2 A,1,2

Symptoms expression on naturally infected host range.

47-48

2B

DBIA showing the reactivities of naturally infected reservoir plants collected from the experimental farm, Fac of Agric, Cairo Univ. nominated (a, b, c, d, e, f, g, h, I, j, k, and l)

48

with Indian TSWV/AS. 3

DBIA showing the reactivity of TSWV/NP expressed in E.coli using Indian antiserum (dil 1/4000)asprimaryantiserum.Nitrocellulose membrane shows the crud preparation from bacteria expressing TSWV/NP (CB). CL: cleared lysates of bacteria expressing the NPs. Flow-through (Fth), Wash 1 (W1), Wash 2 (W2), E1 and E2: purified 6x-His/NPs fusion

51

Fig. No.

Page protein purified under native conditions. Anti rabbit IgG alkaline phosphatase-conjugated (Sigma) was used as second antibody at dilution 1-7500.

Fig. No.

Pag

4

Titration of TSWV antiserum.

53

5

Ultraviolet spectrum of purified TSWV/IgG

53

6

Schematic diagram showing the absorbance values of indirect ELISA measured at 405 nm for determination of approximate working dilution of

55

Egyptian TSWV/NP IgG against different sap extracts from four plants: Pelargonium spp, Tomato, Petunia, and Nicotiana rustica infected with TSWV NP. Positive and negative controls are 7

included. Schematic diagram showing the absorbance values of indirect ELISA measured at 405 nm for determination of approximate dilution end point of the crude sap that can be used to detect TSWV in infected samples. Four plants were used; Pelargonium spp, Tomato, Petunia, Nicotiana rustica infected with TSWV/NP: nucleoprotein as positive control is included. Absorbance values of healthy plant samples were also indicated. Results are presented as O.D. ratios between the infected and healthy samples (I/H).

58

8

DBIA showing the serologic reactivities of Indian (A) TSWV/AS (1-4000) and Egyptian (B) TSWV/NP/IgG (1:1000) with different tested protein sap preparations from 1 to 30 different TSWV infected host plant as indicated in Tabl 1.Healthy sap (control)

59

Figur No.

Pae Showing No colored signal. The nitrocellulose membrane was color developed using NBT/BCIP. Fig. (A) Showing sample could not be detected by Indian TSWV/AS. While in Fig (B) showing the samples that could not be detected by Egyptian TSWV/IgG .

60

9

Electron micrograph of partially purified virus preparation.

61

10

1% agarose gel electrophoresis showing the total RNA extraction from different tissues infected with TSWV.

63

11 A

Nucleotide sequence of the nucleoprotein gene of TSWV infecting Chrysanthemum isolate accession number (AB038341) and the specific primers TSW1 (+) and TSW2 (-) designed for RT-PCR.

64

11 B

1 % agarose gel electrophoresis showing the results of RT-PCR products amplified from Lycopersicon esculentum,Datura metel, Pelargonium spp, and Nicotiana rustica infected with TSWV.

65

12

Southern hybridization of TSWV PCR products amplified from different infected tissues using TSWV-NP DNA-probe labeled with Dig-11 dUTP. Schematic diagram showing the steps used in

66

13 A

cleaning of the TSWV/NP- PCR product from Agarose gel band using QiAquick Gel Extraction Kit (Qiagen).

13 B

Cleaned PCR product (TSWV/ NP gene) after

14

QiAquick purification of the correct size. Genomic organization of TSWV (L, M, and S RNAs); and the partial restriction map of pBADTopo expression vector (Invitrogen).

66

67 68

Fiuger No. 16 A 16 B 17

Page Screening of presumptive pBAD-Topo-NP plasmid clones by PCR . Southern hybridization with digoxigeninlabeled TSWV-NP probe.

69

Dot blot hybridization assay performed on sap extracts from Lycopersicon esculentum infected

70

69

samples collected from Fayoum region (18 samples) and El-Menia region (12 samples). 18

Gel electrophoresis showing the sensitivity of IC-RT-PCR for detection of TSWV in different tomato tisues infected with TSWV.

71

19

Multiple sequence alignment of deduced amino acid sequences encoding the Nucleoprotein (NP) gene of TSWV-EG isolate with that published in the gene bank.

72

20

Phylogentic tree of the deduced amino acid sequences of TSWV-NP-EG Tomato isolate and published sequences of TSWV isolates in gene bank under the accession numbers

73

21 22 A 22 B

AB088385,AB038341,AB038342, AJ242772, AY611529, Z36882, AB175809, and X94550 12% SDS-polyacrylamide gel electrophoresis of TSWV NPs gene expressed in E.coli. 12% SDS-polyacrylamide gel electrophoresis of TSWV NPs gene expressed in E.coli. Western blots analysis showing the reactivity of TSWV polyclonal antibodies with the produced 6x-His/NP fusion protein

74 75 76

List of Abbreviations A Aa

Amino acids

BCIP bp

5-bromo-4-chloro-3-indolyle phosphate Base pair

B

Blocking buffer

1% skimmed dried milk in PBS.

C cv cvs

Cultivar Cultivars

cDNA CMV

Complementary deoxyribonucleic acid Cucumber mosaic virus

DBIA

Dot-blotting immunobinding assay

DEP Dig-11-dUTP DNA dNTP

Dilution end point Digoxiginen 11 deoxyuridine triphosphate Deoxyribonucleic acid Deoxynucleotides triphosphate

ds DNA DTT

Double-stranded deoxyribonucleic Dithiothritol

E.coli

Escherichia coli

E.M. EDTA ELISA EtBr

Electron microscope Ethylenediamine tetracetic acid Enzyme-linked immunosorbent assay Ethidium bromide

Fac Feddan Fig

Faculty Feddan=4200m2 Figure

D

E

F

I

IC-RTPCR

Immunocapture reverse transcriptase

IgG IPM

polymerase chain reaction Immunoglobulin G Integrated Pest Management

IPTG

Isopropylthiogalactoside

KDa

Kilo Dalton

LB LIV L-RNA

Luria Bertani Longevity in vitro Large ribonucleic acid

µg µl M M-RNA

microgram microliter Molar medium ribonucleic acid

MCA mg MgCl2 min

Monoclonal antibodies milligram magnesium chloride minute

mM MW

millimolar Molecular weight

Ni-NTA

Nnickel-nitrilotriacetic acid

nM NSm NSs

nanomole

O.D. ORF

Optical density Open reading frame

K L

M

N

Non structural protein (medium) Non structural protein (small)

O

P PAGE PBS

Polyacrylamide gel electrophoresis Phosphate-buffered saline

PBST PCR

PBS containing 0.05% (v/v) Tween-20. Polymerase Chain Reaction

Pm pNPP:

Picomole Paranitrophenyl phosphate

PVY

Potato Virus Y

RdRp RNA

RNA dependent RNA polymerase Ribonucleic acid

RNase rNP rpm rRNA

Ribonuclease Recomb inant nucleopr otein Revolution per minute Ribosomal RNA

RT

Reverse Transcription

SDS S-RNA

Sodium dodecyl sulphate small ribonucleic acid

SSC

Sodium chloride/Sodium citrate buffer

TAE Taq

Tris: Acetate: EDTA Thermus aquaticus

TBE TBS TE TEMED TIP

Tris: Borate: EDTA 50mM Tris-HCl, 150mM NaCl (pH 7.5). 10 mM Tris- HCl, pH 7.5, and 1 mM EDTA. N,N,N',N'-tetramethyl-ethylenediamine Thermal inactivation point

TMV

Tobacco mosaic virus

R

S

T

TOMV tRNA TSWV TYLCV

Tomato mosaic virus Transfer ribonucleic acid Tomato spotted Wilt Virus Tomato yellow leaf curl virus

U U

unit

Univ UV

University Ultraviolet

v/v

Volume/volume ratio

V W W/V

Weight/Volume ratio

I. INTRODUCTION Tomato is one of the most important vegetable crops in Egypt and worldwide. There are more than 4,000 varieties of tomatoes. In Egypt, tomatoes are grown in three seasons, winter, summer and autumn, on about 3 percent of Egypt's total planted area. In the old land of Egypt-Delta area, 4.0 million acres most of them are cultivated intensively with various crops, to obtain maximal yield. Tomatoes are cultivated on about 180,000 Feddans of total Egyptian planted area. The United States produces more tomatoes than any other nation, 10 million metric tons; Turkey, China, Italy, and Egypt are also major producers of tomatoes (3.72 million metric tons) (Abdel-Monem, 2004). Much of the tomato crop grown in industrial countries is processed for use in making a variety of food products. These products include tomato ketchup, tomato juice, tomato soup, tomato paste, tomato sauce, and canned whole tomatoes. Tomatoes are an important source of vitamins A and C and of certain minerals. Several major virus diseases affect tomato production throughout the world including tobacco and tomato mosaic tobamoviruses (TMV and ToMV), CMV, tomato spotted wilt tospovirus (TSWV), tomato yellow leaf curl geminivirus (TYLCV), and tobacco etch potyvirus (TEV). The genus Tospovirus, Family Bunyaviridae, includes several viruses that cause severe worldwide economic problems (Zitter and Daughtrey, 1989). Tomato spotted wilt virus (TSWV) a key member of Tospovirus, inflicts Tomato spotted wilt virus genus (TSWV) Tospovirus is also widespread but in most cases, its incidence was low to moderate (23.8 % to 30.9 %) in Egypt (EPPO/CABI, 1997), however, very high incidence (64.2 %) was reported in one case in South Africa (OEPP/EPPO, 1999 a, b). Crop losses may be reached

to 100 % (Berling et al. 1990; Rodriguez, 1990) in some other continents. Tomato Spotted Wilt Virus is an emerging plant disease. An emerging disease is a pathogen with increasing incidence in the last 20 years. An average of 3.5 new hosts of TSWV has been identified per year since 1987. The virus was first described in 1915 in Australia (Brittlebank, 1919). TSWV is a spherical virus (80 – 120 ; Martin, 1964; Best and Palk, 1964 and 71nm diameters) (Ie, 19 Kitajima, 1965). It composes of 5 % nucleic acid (RNA), 70 % protein, 5 % carbohydrate and 20 % lipid (De Avila et al., 1991). The immature larvae of Thrips tabaci insects acquire and transmit TSWV (Sakimura, 1963). TSWV has become an increasingly important factor in the production of tomato and other many economic crops such as peanut and different vegetables in many countries worldwide by (Berling et al. 1990, Rodriguez, 1990, EPPO/CABI, 1997, Hull, 2002, Culbreath et al. 2003). TSWV infects many economic plants in both mono and dicots (Cho et al. 1987, Berling, 1990, Sether and De Angles, 1991, and Goldbach and Peters, 1996). The virus was first isolated in Egypt from Physalis peruviana by AlKhazindar, (1999), and from tomato plants by Abd-El Nazeir, (1999). TSWV molecular detection has been developed using cDNA probes (Ronco et al. 1989; Rice et al. 1990) and riboprobes (Huguenot et al. 1990), both of which have proved useful for the sanitary certification of plant material (Saldarelli et al. 1996). Several PCR-based methods have been developed for the specific detection of TSWV. The first PCR-based assay was developed by Mumford et al. (1994). Immunocapture PCR and RT-PCR were developed by Nolasco et al. (1993) and Weekes et al. (1996), respectively. A reliable and rapid detection of TSWV from a single infected thrips by RT-PCR has recently been reported by Mason et al. (2003).

Two main concomitant factors that make TSWV one of the most destructive and widely distributed plant viruses are the highly polyphagous nature of its vectors and the relative lack of host specificity of the virus. Consequently, TSWV has a very large host range and its control remains problematic. Resistant varieties are useful in minimizing losses, but infections by field resistancebreaking (RB). TSWV isolates have been reported in nominally resistant pepper hybrids (Roggero et al. 1999) as well as in resistant tomato hybrids (Aramburu and Marti, 2003). Integrated pest management (IPM) approach is being considered for several crop protections grown in Egypt. Egyptian researchers have bred tomatoes to increase the number of fruit per plant and to improve their quality and disease resistance. In recent years, tomato plantations in Egypt have shown syndrome typical to TSWV infection. However, no detailed studies were reported on this disease in Egypt. This study focused on four main goals; first, is to identify and characterize TSWV by biological means; second, is to molecularly characterized TSWV by using molecular biological tools via cloning, sequencing and expression of nucleoprotein (NP) gene in E.coli, the third aim is to use the purified recombinant N protein expressed in the E.coli system as an immunogen for production of polyclonal antibodies for detection purposes, and fourth aim is the application of the antibodies produced via this study to detect and monitor TSWV infection in tomato plants.

II. REVIEW OF LITERATURE Part I. Isolation and Identification of TSWV Isolation Brittlebank (1919) detected TSWV on tomato crop in Victoria State (Australia). Holmes (1946) stated that, spotted wilt is a viral disease of world-wide distribution. He mentioned that the virus has reached every continent and many oceanic islands, and the infection seems to be much more frequent in some areas than in others, probably in accordance with availability of suitable plant hosts to act as reservoirs and thrips to act as vectors of the causative virus. Best (1968) reported the presence of TSWV on tomatoes in all the Australian states. In India, Bidari and Reddy (1985) isolated Tomato Spotted Virus (TSWV) from capsicum plants showing symptoms of bright yellow chlorotic rings. Honda et al. (1989) isolated TSWV from watermelons in Japan. Pitblado et al. (1990) isolated TSWV from tomato and pepper plants in Canada. De Avila et al. (1991) isolated a tomato spotted wilt virus from diseased Capsicum annuum cv. Gedeon showing heavy mosaic, leaf distortion and stem necrosis. They designated this isolate as TSWV-C. Smith (1932), Baker and Jones (1988), Bellardi and Vicchi (1990), Jorda et al. (1995) and Cuadrado et al. (1991) reported the occurrence of TSWV in Europe. The outbreak of the disease followed the introduction of the thrips Frankliniella occidentalis. Kaminska and Korbin (1994) isolated tomato spotted wilt tospovirus from tomatoes, pepper, gerbera, chrysanthemum, dieffenbachia and some

other plants. Hassan (1995) investigated the incidence, etiology and epidemiology of viruses infecting winter tomatoes in Pakistan. From those viruses, he identified tomato spotted wilt virus (TSWV) on the basis of serology and biology. Kovalenko and Shepelevych (2004) studied the pathogenecity and virulence of tomato spotted wilt virus isolated from tobacco under the field conditions of the Crimea in the tobacco plants of Immunny 580 varieties and Datura stramonium. According to their virulence, the obtained 26 isolates were divided into three groups: strongly virulent (severe), middle-virulent and weakly virulent (mosaic). It has been shown that strongly virulent isolates interfere with weakly virulent ones and induce the development of nonspecific resistance in tobacco plants. efficient to the tobacco mosaic virus.

This resistance was also

Identification Brodsgaard (1989) identified TSWV on the basis of general symptoms, specific vectors (Thrips tabaci and Frankliniella occidentalis), and major host plants (mainly ornamentals). Sherwood et al. (1989) identified tomato spotted wilt virus in greenhouse crops in Oklahoma on the basis of serology. They used the Monoclonal antibodies against the lettuce strain (L) and polyclonal antibody against the impatiens strain (I) of TSWV for virus identification employing ELISA. Pitblado et al (1990) identified TSWV from tomato and pepper plants in Canada by ELISA, EM and host range. Kaminska and Korbin (1994) identified the virus by symptomatology, host range, stability in vitro and serology (ELISA). Rosello et al (1994) stated that tomato is susceptible to more than 200 diseases that cause great economic losses. TSWV is among the important viral diseases that are widely spread in tomato crops all

over the world. Hassan (1995) studied a virus infecting winter tomatoes in Pakistan. He identified the virus on the basis of serology and biology as tomato spotted wilt tospovirus.

Physical and biological properties Best (1968), Honda et al. (1989) and Da Graca (1985) reported that TSWV showed a thermal inactivation point of 40 – 45 ºC for 10 min and longevity in vitro of 2 – 4 h at 30ºC. Roggero and Pennazio (1997) studied the thermal inactivation point of TSWV in tobacco plants used isolate was infective after 10 min at 44°C, but not at 46°C. In vivo studies showed that the virus was infective in plants treated for 1 h at 50°C. AlKhazindar, (1999) and El-Sayed et al. (2003) found that the virus was sap transmissible, its TIP is 46°C, DEP is 4x10-3 and LIV is from 5 to15 hrs at room temperature. Bidari and Reddy (1985) studied preservation and storage mannersof chilli viruses found on Capsicum plants. Tissue dehydration and preservation in gelatin capsules over calcium chloride was the best method of preserving most of tested viruses, followed by lyophilization of standard extract, storage of freshly infected leaves at – 20 °C and the standard extract in phosphate buffer sealed in glass tubes kept at – 20 °C. They also mentioned that, most of the viruses retained an infectivity of 32.5% for up to 450 days under the tissue dehydration method, with the exception of tomato spotted wilt and tobacco ring spot viruses which lost their infectivity more rapidly. Tobacco mosaic virus was the most stable, followed by cucumber mosaic, pepper vein banding, pepper veinal mottle, tobacco etch and potato virus Y.

Transmission A) Mechanical Transmission Mechanical transmission of TSWV could be carried out by gentle rubbing the plants with infectious sap prepared by grinding infected leaves in inoculation buffers (potassium phosphate pH 7.07.2 and 0.05 M β-mercaptoethanol or sodium sulfite as reducing agent as described by Bald and Samuel (1934). Gonsalves and Trujitto (1986), observed a new disease of pawpaw caused by TSWV in Hawaii They mentioned that the disease was reproduced

by mechanically inoculating pawpaw plant seedlings by leaf extracts from TSWV-infected lettuce. B) Seed transmission The spread of the virus through seeds or pollens from infected plants does not seem to be high (Norris, 1946; Best and Gallus, 1955; Ie, 1971; Reifschneider et al. 1989; Peters et al. 1991 and Brown et al., 1998). C) Insect transmission Sakimura, (1963) suggested that acquisition and transmission of TSWV by insect larvae is not a general phenomenon in the dispersion of Bunyaviridae. A peculiar characteristic of this transmission is that the virus can only be acquired by larvae upon feeding on infected plants, while the adults cannot acquire the virus (Sakimura, 1963; Ullman et al. 1992).

Best (1968) and Ananthakrishnan (1984) considered that Thrips tabaci was the main vector of TSWV, followed in importance by Frankliniella schultzei. Allen and Matteoni (1991) showed that indicator plants such as petunia, gloxinia, and Nicotiana glutinosa, have been used successfully as monitors for the presence of viruliferous insects. They reported that indicator plants would be useful for monitoring the persistence of viruliferous thrips in greenhouses before introducing successive crops. Symptoms of TSWV were varied with the host, the age of the plant, the time of infestation, nutrition level and environmental conditions. Transmission of tospovirus was closely linked to the life history and development of the thrips on plants (Cho et al. 1987; Adam and Keglar 1994; Kaminska and Korbin 1994; Wijkamp et al. 1995; and Jenser et al 1996). Wijkamp et al. (1993) reported that approximately 80 % of the thrips that transmitted the virus when they were still larvae. Most of these insects resumed transmission after emergence as adults. The median latent period for the infecting larvae ranged from 80 to 170 hr

when they were kept at either 27 or 20oC, respectively. Wijkamp et al. (1995) listed the most frequently reported vector species to TSWV. They belong to the order Thripidae which includes Frankliniella occidentalis (the Western flower thrips), F. schultzei (the cotton bud thrips), and Thrips tabaci (the onion thrips). Other virus vectors in this order were F. fusca, T. setosus, T. palmi, and F. intonsa. Kritzman et al. (2002) determined the transmission efficiency of tomato spotted wilt virus in different Thrips spp. during their development. The virus was initially detected in the proximal midgut region in larvae of both species, and then in the second and third midgut regions, foregut, and salivary glands. Mortiz et al. (2004) studied the acquisition of tospoviruses by thrips vectors. They were restricted to a well-defined time period during the first and early second larval stages, when there was a temporary association between mid-gut, visceral muscles and salivary glands. This association was the result of a displacement of the brain into the prothoracic region by enlarged cibarial muscles. The subsequent loss of this association leads to a strong input of virus particles into the malpighian tubules via the haemocoel. Mechanical transmission through excrement and oviposition by adults was a possible alternative mode of virus transmission that requires investigation.

Host range and symptoms Holmes (1946), Allen and Broadbent (1986), Cho et al. (1986), Halliwell and Philley (1974) and Matteoni and Allen (1989) reported that spotted wilt is a viral disease of world wide distribution. TSWV has reached every continent and many oceanic islands and it causes serious diseases in a number of economically important crops worldwide. Reddy and Wightman (1988), Berling et al. (1990), De

Avila et al. (1992), Nuez et al. (1992), Schuster and Halliwell (1994), and Diez et al. (1999) studied the host range of TSWV. They reported up to 550 species belonging to more than 70 botanical families as TSWV host plants. Edwarson and Christie (1986) listed hosts of TSWV. It includes 271 species in 34 dicotyledonous and seven monocotyledon families. Species of Asteraceae, Fabaceae and Solanaceae account for over 100 of the recorded hosts of TSWV. Weeds were hosts as well as cultivated plants (Jorda et al. 1995), but Van Os et al. (1993) doubt whether they played an important role in epidemiology or not. Green et al. (1988) showed that plants of Stephanotis floribunda in a commercial green house planting established 20 years ago in Oregon began showing disease symptoms (wilting and die back of terminal growth, leaf spots, leaf mottling, chlorotic and necrotic lesions on flowers) in 1987. Bioassays on Chenopodium quinoa, Gomphrena globosa, cucumber and tomato, electron microscopy of extracts and sections and a DAS-ELISA confirmed the presence of TSWV in all symptomatic Stephanotis floribunda samples. Marchoux et al. (1991) isolated TSWV from Capsicum, tomato, aubrgine, faba bean, lettuce, Ocimum basilicum, Chrysanthemum, aster, anemone and gloxinia plants. They identified the virus based on host range vector transmission, serology and E.M. Bitterlich and MacDonald (1993) detected TSWV in four perennial weeds (Trifolium spp, Cirsium arvense , Rumex acetosella and Oxalis spp) one biennial weed (Cirsium vulgar), three winter annuals (Stellaria media, Senecio vulgaris ,Capsella bursa –pastoris) and five annual weeds (Caramineoli gosperma, Medicago lupulina , Galium spp., Geranium molle and Sonchus oleraceus ) . Adam and Keglar (1994) mentioned that, TSWV had an extensive plant host range of more than 360 different species. They also mentioned that, the nature of symptoms is stated to depend on the tospovirus species, the virulence of the virus strain

and the environmental conditions and examples are provided for 13 plant species including tomatoes, peas and tobacco. Hobbs et al. (1994) revealed that, tomato spotted wilt tospovirus (TSWV) isolate from tomato in New Roads in Southern Louisiana, USA, produced chlorotic lesions on inoculated leaves of Capsicum chinense followed by systemic movement and mosaic symptoms. Rosello et al. (1994) studied the symptoms of TSWV on tomato. The appearance and severity of the infection depend on the genotype, the plant development stage at the time of infection, the virus isolate and the environmental conditions. Jenser et al. (1996) reported that tomato spotted wilt tospovirus caused significant yield losses in tobacco, pepper and tomato plantations in Hungary. Singh et al. (1996) reported that, an unusual disease of watermelon (Citrullus lanatus) in India, with streaks on veins, shortened internodes, upright branches and necrosis and dieback of the buds was caused by the watermelon strain of tomato spotted wilt tospovirus (TSWV–W ) . Latham and Jones (1998) determined the incidence of TSWV in native plants, weeds, vegetables and flowering ornamentals in Australia. Leaf or petal samples were tested for TSWV by ELISA. Results indicated that TSWV was found in 59 samples belonging to 16 different species. Among these, the highest virus infection levels at individual locations were in Arctotheca calendula (cape weed; 15 %) and Sonchus asper (Sowthistle; 32 %). TSWV was found in 309 samples of vegetables including capsicum, celery, aubergine, globe artichoke, lettuce, paprika, potato and tomato. At individual locations, capsicum and tomato crops were sometimes 100% infected. TSWV was found in flowering ornamentals and from those found to be infected with TSWV were alstroemeria, calendula, Chinese aster, chrysanthemum, cosmos dahlia, delphinium, gladiolus,

snapdragon, startice and zinnia. The highest incidences were in alstroemeria (32 %) and astere (81 %). Typical symptoms on tomato plants were collected from Viiramin region in Tehran. Symptoms appeared as brownish local lesions on stem and leaves, concentric ring spots; tissue necrosis, vein clearing, and yellow discoloration on fruits; wilting, stunting and plant collapse (Mohammadi et al. 2000). The virus has a wide host range, the symptoms varied from necrotic local lesions, chlorotic local lesions, systemic infection, stunting, wilting and mosaic mottling (AlKhazindar, 1999 and ElSayed et al. 2003). Branch and Fletcher (2001) reported the presence of TSWV in peanut (Arachis hypogaea L.). USA growers currently need disease- and insect-resistant cultivars to lower input production cost to increase net returns and enhance their competitiveness in the global market. Salomone et al. (2003) reported Euphorbia eritrea and Asclepias curassavica as new hosts for TSWV in Liguria, Italy. Euphorbia eritrea showed chlorotic and necrotic spots in the stems, starting from the attachment point of true leaves.

Serology Gonsalves and Trujitto (1986) purified the lettuce isolate of TSWV and produced antiserum and they concluded that, the antiserum was effective in detecting TSWV in leaf tissue by SDS agar gel immunodiffusion tests and by direct and indirect ELISA. Wang and Gonsalves (1990) and Cho et al. (1998) used enzyme– linked immunosorbent assay (ELISA) technique to detect TSWV in individual thrips. Resende et al. (1991) tested different polyclonal antisera and enzyme-linked immunosorbent assay ELISA procedures to detect TSWV. This virus could efficiently be detected in high dilutions of sap from infected plants, and at low concentrations of purified virus

and nucleocapsid protein preparations in the cocktail ELISA and DAS-ELISA. Differences in the detection level were observed using nucleocapsid protein antiserum (anti-N-serum) and the antiserum against intact virus particles (anti-TSWV-serum), but both antisera showed to be powerful sera for the detection of TSWV. Hsu and Lawson et al. (1991) compared direct tissue blotting with ELISA and a dot-blot immunoassay (DBIA) for the detection of TSWV. TSWV was readily detected in tissue blots of infected Nicotiana benthamiana leaves using biotinylated mouse monoclonal antibodies. The results found that the DBIA was nearly eight times more sensitive than ELISA for the detection of TSWV in extracts from infected N. benthemiana leaves. Boiteux and Nagata (1993) studied the susceptibility of Capsicum Chinese PI 159236 to TSWV isolates in Brazil. Virulent and avirulent isolates were shown to be related but serologically distinct by immunodiffusion. Adam and Keglar (1994) stated that based on the reaction of TSWV isolates with N–specific polyclonal antisera, 3 serogroups have been established. The most frequently used technique for serologically based diagnosis of tospoviruses is double antibody sandwich ELISA with N–specific or pre-adsorbed antisera against the complete virus. Vicchi and Bellardi (1996) concluded that detection and diagnosis of TSWV had basically been solved, since ELISA was more sensitive than any other serological technique and can be more readily applied than the other methods. Jenser et al. (1996) identified tomato spotted wilt virus isolated from tobacco, pepper and tomato by symptoms on test plants, double antibody sandwich ELISA and electron microscope (EM). Alexandre et al. (1997) studied 118 leaf samples from 23 commercial crops showing variable symptoms by DAS–ELISA. Data showed that TSWV was found inducing indistinguishable symptoms in affected cultivars, including the most widely used commercially. Mohammadi et al. (2000) prepared an antiserum against

partially purified TSWV). The produced antiserum, however, was of low titer. Heinze et al. (2001) synthesized a stretch of 24 amino acids at the C terminus of the non-structural protein NSs with a highly conserved sequence YFLSKTLEVLPKNLQTMSYLDSIQC was used to raise antisera in two rabbits. The specificity of the antisera against NSs from infected plants was confirmed with Western blots and by immunogold labelling and electron microscopy. These antisera detected tospovirus isolates of serogroup I to III in antigencoated plate ELISA and Western blots but failed to detect isolates of serogroup IV. Epitope scanning using overlapping octopeptides composing the peptide suggested that the antisera contained antibodies against two different epitopes. Inoue et al, (2004) examined the presence of TSWV in second instar larvae and adults of two thrips genera, Frankliniella and Thrips. A triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) showed that large amounts of the TSWVnucleocapsid (N) protein were present in the ELISA-positive larvae of each species, with the exception of T. palmi. Snippea et al. (2005) expressed the nucleocapsid (N) protein of tomato spotted wilt virus as a fusion protein with either cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP).

Electron Microscopy Martin (1964), Kitajima (1965) and Ie (1971), studied the E.M. of TSWV. They showed that TSWV virions were spherically shaped with diameter ranges from 80-110 nm. Each viral particle consists of a granular core bounded by an envelope with surface projections. Mohamed (1981) mentioned that, particles of TSWV were membrane–bound, isometric, and about 80 nm diameter. Adam and Keglar (1994) mentioned that tospoviruses have quasispherical particles of 85-nm diameter.

Milne (1970), Law and Moyer (1990), Urban et al. (1991), Kitajima et al. (1992), Singh et al. (1996), AlKhazindar (1999) and El-Sayed (2003) demonstrated that TSWV particles

accumulated in the cisternae of the endoplasmic reticulum in the cytoplasm of infected cells. Formations of amorphous materials with aggregates of more dense material as well as fibres and fibrous structures were observed. The electron dense masses have been identified as groups of non-enveloped nucleocapsids. The structural fibres were made up of non-structural proteins (NSs). De Avila et al. (1991) observed a large number of spheroidal particles 80 -100 nm in diameter in leaf–dip preparations from infected plants with TSWV isolated from pepper. They added that, the virus particles occurred in clusters between membranes and the viral genome is three segments of RNA (L, M and S).

Molecular characterization and detection De Haan et al. (1991); German et al. (1992); Kormelink et al. (1992) and Adkins et al. (1995) stated that the genus Tospovirus, family Bunyaviridae, includes virus particles limited by a lipid bilayer envelope containing glycoproteins. Inside the envelope, besides the viral polymerase, three genomic RNAs were designated based on their size as: L-RNA, M-RNA, and S-RNA. The L-RNA, of negative polarity, contained one open reading frame (ORF) that encoded the RNA-dependent RNA polymerase (RdRp, 331.5 kDa). The M-RNA, of ambisense polarity, contained two ORFs responsible for the production of the non-structural protein (NSm) (33.6 kDa) in an ORF near the 5’ terminus of the viral sense RNA. The second ORF, the envelope glycoproteins G1/G2 (127.4 kDa) was an ORF in the viral complementary sense located near the 3’ terminus of the viral RNA. The S-RNA, also had an ambisense coding strategy with two ORFs and was responsible for production of the nonstructural protein (NSs) (52.4 kDa) of unknown function. It was encoded in a viral sense ORF from the 5’ region, and the nucleocapsid protein (NP) (28.8 kDa) encoded in an ORF in the viral complementary sense near the 3’ terminus of the viral RNA.

Francki et al. (1991); Adam et al. (1993); De Avila et al. (1993 a); Pang et al. (1993) and Yeh and Chang (1995) demonstrated that there were several other distinct tospoviruses, including impatiens necrotic spot virus (INSV), tomato chlorotic spot virus (TCSV). Ronco et al. (1989); Huguenot et al. (1990) and Rice et al. (1990) proposed the use of cDNA probes and riboprobes in the identification and diagnosis of TSWV. Nolasco et al. (1993); Mumford et al. (1994) and Weekes et al. (1996) developed a PCR-based assay based on reverse transcription polymerase chain reaction (RT-PCR) and immunocapture RT- PCR. German et al. (1992) showed that the detection and diagnosis of tospoviruses could be achieved by a variety of different means. The use of PCR as a highly sensitive technique for the detection of plant pathogens offers a number of advantages over other existing techniques (Henson and French, 1993). De Avila et al. (1993a) and Pappu et al. (1998a) stated that the isolates of TSWV had a high degree (94-100 %) of nucleotide sequence identity in the N gene and appeared to be grouped phylogenetically according to the geographic location. Dewey et al. (1996) and Mumford et al. (1996) identified TSWV using reverse transcription-polymerase chain reaction RT-PCR followed by gelbased for the discrimination of tospoviruses using primers designed from sequences in the L and S-RNAs. Mostly, regions of the N gene and 3´ untranslated region had been targeted, giving rise to amplicons of 450-871 base pairs (bp).Both species-specific primers and serogroup specific primers (primers with specificity to members of more than one serogroup) had been used, and restriction enzyme digestions of PCR products had enabled differentiation between the virus species. Jain et al. (1997) and Pappu et al. (1998a) enabled the amplification of the entire N gene from peanut tissue by using specific primers designed in the conserved regions. Roberts et al. (1999) developed a real-time reverse transcription-polymerase chain

reaction assay based on TaqMan chemistry for the detection and quantification of tomato spotted wilt virus (TSWV). Okuda and Hanada (2001) detected multiple species of tospovirus from plant tissues by using RT-PCR procedures. An upstream primer was designated from the 3' untranslated sequences of the S-RNA from the degenerated sequences of the nucleocapsid protein of TSWV. Approximately 450 bp DNA fragments were detected when TSWV or impatiens necrotic spot virus (INSV) - infected tissues were examined. Approximately 350 bp DNA fragments were detected when watermelon silver mottle virus (WSMoV) or melon yellow spot virus (MYSV)-infected tissues were examined. Mason et al. (2003) studied the capability of RT-PCR to detect the transmission of TSWV by thrips. Fukuta et al. (2004) developed a new detection method for TSWV from chrysanthemum plants. This method enabled sensitive, reproducible and specific detection of TSWV from chrysanthemum plants. In this method, TSWV genomic RNA was amplified under isothermal (65o C) conditions within 1 h. The resulting amplicons were detected by the measurement or observation of the turbidity of the reaction mixture without gel electrophoresis. The described method was 100 times more sensitive than IC/RT-PCR.

Molecular cloning and sequencing Goldbach and Kuo (1996) stated that in order to become a distinct species of the Tospovirus genus, the N protein sequence of the tested virus should show not less than 90 % amino acid sequence identity with that of any other tospovirus species that has been described. Dewey et al. (1997) employed a cladistic analysis using parsimony of TSWV genus with RNA sequences of 450 nucleotides of the N gene from 14 new Argentinean isolates and 4 previously described isolates. The genus Tospovirus was thought to be composed only of the tomato spotted wilt virus (TSWV), but now at

least four Tospovirus species have been proposed based on molecular data. A classification of Tospovirus has been proposed taking into account global similarities of the N gene and N protein sequences of 7 isolates of Tospovirus. Heinze et al. (2001) determined the genetic heterogeneity of the N protein gene and the intergenic region (IGR) of the S-RNA from tomato spotted wilt virus (TSWV) isolates, collected in Bulgaria, and compared with isolates from other parts of the world. The results revealed the highly conserved nature of the N protein.

III. MATERIALS AND METHODS The present study was carried out at the Molecular biology laboratory, Virus and Phytoplasma Research department, Plant Pathology Research Institute, ARC and the greenhouse at the Faculty of Agriculture, Ain Shams University from 2003 to 2006. The experiments aimed to identify tomato spotted wilt virus (TSWV) isolate from naturally infected plants (Part I) and to study its molecular characterization (Part II) Part I Isolation and Identification A. Virus Isolation Tomato fruits showing symptoms suspected to be due to TSWV infection were collected from Shalakan farm, Faculty of Agriculture, Ain shams University, Kalubia Governerate and used for mechanical inoculation of Nicotiana rustica L. and Petunia hybrida

Vilm and kept under greenhouse conditions (25-30°C). The virus was purified biologically through two consecutive passages onto the local lesion host, Petunia hybrida Vilm, followed by one passage on Nicotiana rustica L. The resulting local concentric ringspots were singly back inoculated onto tomato plants. Inoculated tomato plants were kept in the green house and used as a source of infection in the following experiments. B. Identification Identification of the isolated virus was focused on studying its mode of transmission, host range, symptomatology, and stability. Identity was further insured by electron microscopy. As shown later, molecular and serological studies also ensured the identity of the virus under study.

B. 1. Virus transmission B. 1.a. Mechanical transmission Infected tomato leaves were homogenized in a sterilized mortar in the presence of few drops of Solvent 4 buffer solution pH 7.2 (0.1 M Na2HPO4-NaH2PO4, pH 8.3, 0.02 M of Na2SO3 and 0.02 M EDTA) as described by Roggero and Pennazio (1997). The extracted sap was expressed through cheesecloth and used to inoculate the test plants (Apium graveolens, Eminium spiculatum, Eruca sativa, Chenopodium murale L., Chenopodium amaranticolor L., Lactuca sativa L., Sonchus oleraceus, Convolvulus arvensis L., Cucurbita pepo L., Arachis hypogaea L., Lupinus termis L.,Phaseolus vulgaris L. cv. Giza 6., Pisum sativum L. cv. Little Marvel., Vicia faba L. cv. Giza 2., Pelargonium spp., Malva parviflora, Cidrella toona, Anagalis arvensis, Antirrhinum majus, Capsicun annuum L. cv.California wander, Datura metel L., D. stramonium, Lycopersicon esculentum Mill. cv. Money Maker, Nicotian glutinosa L.,N. rustica L.,.N. tabacum cvs. Kentuckey, N. tabacum cv. White Burley, Physalis peruviana L., Petunia hybrida Vilm., Solanum albicaule, S. nigrum, Hordium vulgaris, and Gomphrena glubosa L). Leaves to be inoculated were slightly dusted with carborandum (600-mesh) before inoculation. Inoculated test plants were rinsed slightly with tap water, kept under greenhouse conditions and observed for two to three weeks after inoculation for symptom expression. Some naturally infected plants (Vitis, Bougainvillea glabra, Cidrella toona, Eminium spiculatum, Phaseolus vulgaris, Solanum nigrum, Sonchus oleraceus, Convolvulus arvensis, Urtica urens, and Solanum albicaule) found to be exhibiting symptoms similar to that produced by TSWV infection were collected from the experimental farm, Faculty of Agriculture, Cairo University and tested against TSWV using Indian TSWV/AS in DBIA. B.1.b .Insect transmission Virus-free colony of specific vector, Thrips tabaci, kindly provided by Dr. Manal El-Shazly (Virus and Phytoplasma

Research Dept, Plant Pathology Research Institute, ARC), was used in this experiment. Infected tomato plants, showing typical symptoms of TSWV, were used as a source of infection in this experiment. Immature thrips insects were left to feed for one day on infected tomato plants in insect-proof cages, and then transferred to healthy tomato plants (20 plants) A control of healthy tomato plants received the same number of non-viruliferous insects. Two days later, all plants were sprayed with insecticide (2 % Malathion) and symptoms continuously observed till the end of the experiment. TSWV Indian Antiserum was used to verify the developed symptoms as TSWV by DBIA. B.2. Host range and Symptomatology Thirty three plant species belonging to sixteen different families were mechanically inoculated with TSWV isolated from tomato plants using Solvent buffer 4, pH 7.2. Inoculated plants were kept under greenhouse conditions for 7 to 21 days and symptoms were recorded afterwards. The inoculated plants were serologically tested by indirect ELISA method using TSWV-AS (Kindly provided by Prof. Dr. H.S. Savithri, Dept of Biochemistry, Indian Institute of science, India). B.3. Virus stability Dilution end point (DEP), thermal inactivation point (TIP), and longevity (LIV) of the isolated virus isolate were determined according to Noordam (1973). B.3.1. Determination of thermal inactivation point (TIP) Thermal inactivation point ( TIP ) of the isolated viruses from naturally infected tomato leaves was determined by exposure normal test tubes containing the crude sap of the isolated virus (each tube contains 1 ml ) to ten minutes in thermally controlled water path to 40 , 45 , 55 , 60 ,65 , 70 , 75 , 80 , 85 , 90 , 95 and 98ºC respectively in preliminary experiments . After treatment, the tubes were cooled immediately under tap water. The isolated virus was assayed by

inoculation in healthy seedlings of Datura metel (six plants were used per each treatment). Untreated crude sap of the isolated virus was injected also into healthy seedlings of Datura metel as control. B.3.2. Determination of the dilution end point (DEP) To determine the dilution end point (DEP) of the isolated viruses ten fold dilution of the crude sap was prepared up to 10 -1 to 10-7 were used as inoculums. The infectivity of isolated viruses was assayed by back mechanical inoculation on healthy D. metel leaves. Dilution end point of the isolated viruses was determined as the highest dilution which the virus lost its infectivity after it. B.3.3. Longevity in vitro (LIV) Longevity in vitro (LIV) of the isolated viruses was also determined. Infectious crude sap was kept in small eppendorf tubes (1.5 ml per tube). Tubes (30 tubes) were stored at room temperature (25-30ºC). The stored sap was assayed in a narrow time scales, starting from 0 time, 2, 4, 6, 8, 10, 12, and 14 hours by back inoculation mechanically on the healthy D. metel leaves. The above experiment was repeated but in a narrower time scales, starting from 1 to 6 hours, at one hour interval. The number of local lesion appeared on D. metel leaves was recorded. The aging of viruses was determined as the longest period or time that the virus lost its infectivity after it. B.4. Serological studies The viral nucleoprotein of TSWV produced in E.coli via molecular cloning and expression of the viral NP gene during this study as shown later on Part II, was used in immunization experiment to produce polyclonal antibodies against TSWV- Egyptian isolate. The reactivity of the purified NP was examined against Indian TSWV antiserum before immunizing the rabbits. B.4.1. Reactivity of TSWV nucleoprotein (NP) against Indian antiserum The purified NP was examined against Indian TSWVantiserum by dot-blot immunoassay. The purified nucleoprotein was

diluted 100 fold in TBS buffer (20 mM Tris-HCl pH7.4, 150 mM NaCl) and only five microliter was spotted onto nitrocellulose membrane and the test was performed as described by De Àvila et al. (1990). Indian TSWV-AS at dilution 1-4000 was used in this assay to detect the presence of TSWV purified nucleoprotein (native and denatured form). The second antibody was an alkaline phosphataseconjugated goat anti -rabbit IgG (Sigma) and was diluted 1:7500 in blocking buffer before reacting with the membrane blot. The color reaction started by incubating the membrane in 5-bromo-4-chloro-3indolyl phosphate/nitro bluetetrazolium (BCIP/NBT) substrate for alkaline phosphatase until the signals of interest has reached the desired intensity. The reaction was stopped by washing the membrane in deionized water for several minutes. The membrane was air dried on a filter paper and photographed. B.4.2. Antiserum production A polyclonal antiserum for TSWV/NP was raised through applying six weekly consecutive intramuscular injections of expressed TSWV nucleoprotein (1.0 mg/ml) each into a New Zealand rabbit. In the first injection, the fusion nucleoprotein was emulsified with complete Freund’s adjuvant and injected subcutaneously (1.0 mg NP emulsified with an equal volume of complete Freund’s adjuvant). The immunization schedule was done in the animal house, Faculty of Agriculture, Cairo University according to the protocol applied in Veterinary Clinical Services, Yale Animal Resources Center, Yale University. In boosting, five intramuscular injections with incomplete Freund’s adjuvant were done. The rabbit was bled at weekly intervals for 6 weeks. The final bled was started two weeks after the last injection. B.4.2. a. Determination of Antiserum titer The Egyptian antiserum produced during this study was titrated against the purified nucleoprotein of TSWV (NP/6xHis) expressed in E.coli using indirect ELISA. The obtained antiserum (TSWV-AS-EG) was diluted 1:250, 1:500, 1:1000, 1: 2000, 1:4000,

and 1:6000 in PBS pH 7.4. The NP/6xHis was diluted 1:1000 in coating buffer, pH 9.6) and 100 µl/well was incubated in the microtitre plate O/N at 4oC . The optimum dilution of TSWV-ASEG which reacted specifically with the denatured nucleoprotein (NP/6xHis) and gave the highest O.D. readings values was determined. The absorbance values are measured at 405 nm and the data was tabulated.

The negative control used was the bacterial proteins only.

B.4.2.b. Separation of TSWV/ IgG The collected blood was left at 37˚C in an incubator for 1 hour, then kept at 4˚C overnight. Antiserum was separated by centrifugation at 8,000 xg for 10 min. IgG fractions of TSWV antiserum was then separated according to the technique described by (Mckinney and Parkinson, 1987) through precipitation of other globulins than IgG with caprylic acid (Sigma, C2875), then precipitation of IgG with ammonium sulfate as described by Perosa et al. (1990) with some modifications. One volume of antiserum was treated with four volumes of 60 mM acetate buffer , pH 4.0, and the pH was readjusted to 4.5 with 0.1 N NaOH. For each ml antiserum, 25 μl caprylic acid were added drop wise under stirring for 30 min/250C. The insoluble materials were separated by centrifugation (4000 g/20 min/ 40 C). The supernatant was collected through filtration and mixed with 10 x concentrated phosphate buffer saline, PBS pH 7.4, (0.137 M NaCl, 1.5 mM KH2PO4, 8 mM Na2HPO4, 2.7 mM KCl, 0.02% NaN3) using 10 parts supernatant to one part PBS. The pH was adjusted to 7.4 with 5.0 N NaOH. The supernatant was cooled to 4oC, and then ammonium sulfate (0.277 g/ml) was added under stirring at 4oC for 2 hr (or overnight at 4oC). IgG was then precipitated at a low speed. The pellet was suspended in 1 x PBS (using the same volume of the original used antiserum). The concentration of IgG was adjusted with 1/2 strength of PBS to read approximately 1.44 O.D. at 280 nm which is equal to 1mg protein. The measured IgG was frozen at -20 oC. The IgG fraction extracted

from the antiserum for the nucleoprotein was termed TSWV-IgG-EG. B.4.2.C. Titration of TSWV / IgG-EG The titer of the purified IgG was determined by indirect ELISA method. The infected and healthy leaves were extracted in 0.05 M coating buffer pH 9.6 diluted to 1:1000 (W/V) and then centrifuged for 5 min at 10,000 g. The IgG was diluted from 1/250 to 1/4000 in PBST-PVP buffer pH 7.4. The antigen was detected with anti-rabbit alkaline phosphatase conjugate (Sigma), followed by the addition of pNpp substrate. Absorbance reading was recorded at 405 nm after incubation with substrate for 2 hrs. B.4.3. Determination of dilution end-point of TSWV infected sap by indirect ELISA method The dilution end point of crude sap extracts from different plants that found to be frequently infected by TSWV during this study such as Pelargonium spp., Tomato, Petunia, and Nicotiana rustica were determined by indirect ELISA. The indirect ELISA method was carried out as described by Converse and Martin (1990) with some modifications. The virus crude sap of all infected plants were diluted from 10-1 to 10-5 in ELISA coating buffer pH 9.6 and 150 µl/well of each extract was incubated in the microtitre plate overnight at 4 °C. Wells were washed 3 times with PBST by shaking for 3 min each time. The microtitre plates were dried after the last wash by inverting the plates on a stack of paper towels by hand then blocking agent (3% BSA) dissolved in PBST-PVP was applied for 30 min at room temperature. The plates were dried without washing and 150 µl/well of TSWV/ IgG – EG was then added at dilution of 1/1000. Plates were then incubated for 3 hrs at 37 °C or overnight at 4°C and then washed and dried. The antigen was detected with antirabbit alkaline phosphatase conjugate (Sigma) diluted at 1-7500 in conjugate buffer, pH 7.4, added to the plates using 150 µl/well and incubated for 3 hrs at 37 °C or O/N at 4 °C. The plates were washed and dried and Sigma Fast tablet of pNpp (1.0 mg/ml) dissolved in

5ml of substrate buffer pH 9.8 was added. Absorbance readings at 405 nm were taken after incubation with substrate for 2 hr. B.4.4. Evaluation of Egyptian TSWV-IgG by dot- blot Immuno binding assay (DBIA) Total proteins extracted from selected 30 different TSWV host plant (Table 1) and five microliters of each extract was spotted onto a nitrocellulose membrane as described by Whitfield et al. (2003). The membrane was blocked in TBS (20 mM Tris-HCl pH7.4, 150 mM NaCl containing 1% BSA and 0.05% tween 20) for 30 minutes. The blot was incubated with the Egyptian TSWV-IgG as primary antibody diluted (1:500) in blocking buffer for three hours and washed three times for 5 minutes each in TBS containing 0.1% BSA. The second antibody was an alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma Chemical Co.) and was diluted 1:7500 in blocking buffer before reacting with the membrane blot. Three 5minute washes were performed with TBS containing 0.5% tween 20, and the blots were rinsed briefly with water. The color reaction was started by incubating the membrane in 5-bromo-4-chloro-3-indolyl phosphate/nitro bluetetrazolium (BCIP/NBT) substrate for alkaline phosphatase until the bands of interest have reached the desired intensity. The reaction was stopped by washing the membrane in deionized water for several minutes. The membrane was air dried on a filter paper and photographed. B. 5. Electron Microscopy Samples of mechanically inoculated tomato leaves (100 gm) were macerated in a mortar with an equal volume of buffer 4 pH 7.2. The extracted sap was filtered through cheesecloth and centrifuged for 20 min at 5000 xg in SigmA 2K15 centrifuge. The supernatant was concentrated by centrifugation at 30000 rpm for 120 min. The pellets were resuspended in an amount of 0.01 M Na2SO3 equal to 1/10 of the original weight of tissue (Black et al., 1963). Partially purified suspension of the TSWV was examined by electron microscope, (Philips E.M. 401) at the Electron Microscope

Unit, Specialized Hospital Ain Shams University using negative staining (2 % Uranyle acetate) technique as described by (Noordam, 1973). Part II Molecular Studies II.1.Total RNA isolation Total RNA was extracted from TSWV-infected Lycopersicon esculentum Mill. cv. Money Maker, Nicotiana rustica L., Datura metel L., and Pelargonium spp., plants using the two following methods: 1) High Pure RNA Tissue Kit (Roche) as described by the manufacturer and; 2) Cetyl trimethyl ammonium bromide method (CTAB) as described by Gibbs and Mackenzie (1997) as follows: A. High Pure RNA tissue kit Total RNAs were prepared from young leaves of the above mentioned infected plants as described by manual of High Pure RNA tissue kit from Roche diagnostics GmbH, Germany. 50 mg leaf tissues were homogenized in 400 µl lysis/Binding buffer (4.5M guanidine-HCl, 100 mM sodium phosphate, pH 6.6). The lysate was centrifuged for 2 min at 14,000 xg in a micro centrifuge and 200 µl of absolute ethanol was added to the lysate supernatant. The high pure filter tube and the collection tube were combined and the sample was pipetted in the upper reservoir and centrifuged for 30 sec at maximal speed (14,000 xg). The flowthrough was discarded and 500 µl of wash buffer 1 (5 M guanidine-HCl, 20 mM Tris-HCl, pH 6.6, in ethanol) was added to the upper reservoir, and centrifuged for 15 sec at 8000 xg. The flowthrough was discarded again and 500 µl wash buffer II (20 mM NaCl, 2 mM Tris-HCl, pH 7.5 in ethanol) was added to the upper reservoir of the filter tube and centrifuged 15 sec at 8000 xg. After the flowthrough was discarded, 100 µl of Elution buffer (nuclease–free, sterile, double dist. water) was added to the upper reservoir of the filter tube and centrifuged at 8000 xg. The eluted RNA was stored at -80 oC for later analysis.

B. Cetyl trimethyl ammonium bromide method (CTAB) TSWV infected Lycopersicon esculentum Mill. cv. Money Maker, Nicotiana rustica L., Datura stramonium L., and Pelargonium spp. leaves were prepared by grinding 50 mg fresh leaf tissue homogenenized in liquid nitrogen to a fine powder and 500 µl of wash buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 2 M NaCl) was added to the powdered leaves before adding CTAB buffer (2% w/v CTAB, 1.4 M NaCl, 0.1 M Tris-HCl, pH 8.0, containing 0.5 % β-mercaptoethanol) to get rid of the polysaccharides. The mixture was centrifuged at 14,000 xg for 5-10min. Supernatant was removed and 600 µl of CTAB buffer was added. The mixture was mixed and incubated at 55°C for 15-30 min and 400 µl Chloroform: isoamyl alcohol (24:1, v/v) was added to the mixture and vortexed for emulsification. The mixture was centrifuged at 14,000 g for 10 min and the aqueous top phase was transferred to a clean Eppendorf tube and 1 /10 volumes of 7.5 M NH4OAC and 1 volume of isopropanol were added. The tubes were mixed well and placed in the freezer 510 min. Nucleic acid was pelleted by centrifugation at a maximum speed (14,000 xg) for 5-10min. Supernatant was poured off and 1 ml of 70 % ethanol was added and the tubes were centrifuged for 1 min and the pellet was air dried. The pellet was resuspended in 50-100 µl of sterile nuclease-free H2O on ice or overnight in the fridge at 4°C. The nucleic acid pellet was stored at-20°C. The isolated RNA was used as a template in reverse transcription PCR reaction (RT-PCR) in order to synthesize cDNA to be amplified by PCR. II. 2. RT-PCR amplification A modification method of Pappu et al. (1998a) was used for synthesizing cDNA strand by using Retrotools Reverse Transcriptase (from Biotools, Biotechnological & Medical Laboratories, S.A Madrid, Spain). 7 µl of total nucleic acid primed with 50 pm/µl of plus sense primer tSW1 (5' ATGTCTAAGGTTAAGCTC 3') in a total volume 20 µl were placed in a water bath at 70oC for 5 min. The reaction contained 4 μl of 5x RT buffer (Biotools,

Biotechnological & Medical Laboratories, S. A. Madrid, Spain), 1 µl of 10 mM dNTPs, 1 µl of enhancing buffer, and 1.5 μl of Retrotools Reverse Transcriptase enzyme. The reaction was performed at 70oC for 45-60 min. For PCR, 50 pm/µl of each amplification primer (forward primer) TSW1 corresponding to nucleotide position 762781 (5'-ATGTCTAAGGTTAAGCTC-3') and (reverse primer) tSW2 was complementary to nucleotide position 1064-1083 (5' – TTAAGCAAGTTCTGTGAG- 3') (Jain et al., 1997) were selected based on TSWV nucleoprotein gene (NP) sequence, located at the 3' end of RNA-S (Jan et al. 2000). 5 µl of each cDNA reaction, and 5 U/µl of High Expand Fidelity DNA polymerase (Roche) were used in a 5x Standard DNA buffer containing 20 mM Tris HCl, pH 8.2, 10 mM KCl, 6 mM (NH4)2 SO4, 2 mM MnCl2, 0.1% Triton X-l00 and l0 µg/ml of nuclease-free BSA. The amplification reaction was carried out in a total volume of 50 µl using PCR thermal cycler, UNOII from Biometra and using 0.2 ml micro Amp PCR tubes. Hard denaturation of the DNA was performed at 95oC for 2 min followed by 35 cycles of amplification with denaturation at 94oC for 30 sec, annealing at 45oC for 45 sec, and extension at 72oC for 1 min. A single tailing cycle of long extension at 72oC for 7 min was carried out in order to ensure flush ends on the DNA molecules. Finally, the amplification reactions were hold at 4°C. The amplified DNA was electrophoresed on 1 % agarose gel and photographed using gel documentation system (Biometra). II.3. Molecular Cloning The generated DNA fragment of TSWV nucleoprotein genes (NPs) obtained after PCR amplification were purified using QiAquick Gel Extraction Kit (Qiagen) according to Instruction manual (Fig. 13 A) and ligated into pBAD-Topo TA expression vector 4.1 kb. Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3´ ends of PCR products. The linearized vector supplied in this kit has

single, overhanging 3´ deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector. Ligation reaction was incubated for 5 minutes at room temperature that contained in equimolar amounts of PCR product (NP insert) and (pBAD-Topo) plasmid vector. Ligation reaction was conducted in 6 µl total volume as following: 1 µl of salt solution (1.2 M NaCl, 0.06 M MgCl2), 4 µl of fresh PCR product and 1 µl of TOPO vector. Protein expression in pBAD-Topo 4.1 kb plasmid vector was driven by the araBAD promoter and is induced by addition of very minute amount (0.02 mM) of 20 % L-arabinose. II.3.A. Preparation of E. coli competent cells The E.coli BL21-DE3 competent cells were prepared according to Sambrook et al. (1989). Single colony of BL21-DE3 cells strain from Promega was inoculated into 5 ml of Lauria Bertani (LB) media (1% Bactotryptone, 0.5% Bacto yeast extract and 0.5% NaCl, pH 7.0) and incubated at 37°C over night with vigorous shaking (200 rpm) for cell aeration. The over night culture was diluted (1:100) with LB broth and incubated with shaking at 37°C until the culture O.D. reached 0.3-0.4 at wave length 600 nm. The cells were transferred into 250 ml centrifuge bottles and chilled on ice for 10 min. The cells were precipitated by centrifugation at 10,000 xg for 7 min at 4°C (centrifuge J2- MC Beckman). The supernatant was discarded and the bacterial cells pellet were resuspended in 1020 ml of ice cold freshly prepared and sterilized triturating buffer (100 mM CaCl2, 70 mM MgCl2 and 40 mM sodium acetate pH 5.5) and centrifuged at 10,000 g for 7 min at 4°C. The pellet was resuspended in 2 ml of the same buffer for each 50 ml of the original culture. The cells were incubated on ice for 1 hr and the cells were dispensed into aliquots in sterile Eppendorf tubes and mixed with glycerol to a final concentration of 15 % (v/v) and stored at -80°C. II.3.B. Bacterial Transformation The protocol described by Hanahan and Meselson, (1983) was used for bacterial transformation of pBAD-TOPO-NP ligation

reaction. To an aliquot of 100 µl competent BL21-DE3 cells, 2 µl of pBAD-TOPO-NP ligation reactions were added and mixed gently by tapping and incubated on ice for 30 min. Cells were heat shocked for 45 sec at 42°C water bath incubator to increase the transformation efficiency. The tubes were then placed on ice for 1 min to cool down. 900 µl of LB medium was added to each tube and shacked gently at 37°C for 1 hr for cell recovery. A 100µl aliquot of the culture was plated on solidified agar medium (LB with Ampicillin 50 mg/ml) and incubated at 37°C overnight for selecting the transformed cells II.3.C. Rapid screening of the transformed colonies by PCR Validation of cloning took place by PCR to select the transformed colonies with recombinant pBAD-TOPO vector as recommended by Sambrook et al. (1989). A loopfull of the white colonies was picked into PCR tubes containing 50 µl of PCR reaction mixture (1x PCR buffer, 2 mM MgCl2, 200 µM dNTPs, 1 µM each primer TSW1 and TSW2) and 1.25 units of Taq DNA polymerase. The reaction proceeded in the thermocycler (UnoII, Biometra) for 35 cycles under the following conditions: Hard denaturation for 3 min (One step), denaturation at 94°C for 45 sec, annealing at 45°C for 1 min, and extension at 72°C for 90 min (35 cycles), additional final .extension step was performed at 72 °C for 7 min II.3.D. Preparation of recombinant plasmids High pure plasmid isolation kit (Roche) was used to isolate pure super-coiled plasmid DNA with high yields (~15 µg) according to the instruction manual and without using expensive equipments such as ultracentrifuges or expensive and hazardous materials such as CsCl, phenol, chloroform, or ethidium bromide. An overnight culture (3 ml LB medium containing 50 µl /ml ampicillin) inoculated with single transformed bacterial colony containing the recombinant plasmid were centrifuged at 10,000 xg under cooling centrifuge for 2 minutes. Bacterial pellets were completely resuspended in 250 µl of cell suspension solution containing RNase (50 mM Tris-HCl, 10 mM EDTA, pH 8.0, 2.5 mg RNase A) without leaving any clumps. To

lyse the cells, 250 µl of cell lysis solution (0.2 M NaOH, 1% SDS) were added and the mixture was incubated at room temperature for 5 minutes. The mixtures were inverted gently about 4-6 times without vortex in order to avoid shearing of genomic DNA. To obtain a cleared plasmid preparation, 350 µl of binding buffer (4 M guanidine HCl, 0.5 M potassium acetate, pH 4.2) were added and the mixture was incubated for 5 min on ice and centrifuged at 14,000 xg in a microcentrifuge for 10 minutes. The supernatant (cleared lysate) was transferred onto the upper reservoir of High pure filter tube assembly containing two layers of glass fiber fleece (Roche) and centrifuged for 60 sec at 14,000xg. The High pure filter tube was detached from the minicolumn and the flowthrough solution was discarded. The column was washed with 500 µl of column wash buffer I (5 M guanidine HCl, 20mM Tris-HCl, pH 6.6, in Ethanol) and of High pure filter tube was inserted again into the column and centrifuged for 60 sec at 14,000 xg. The wash buffer was decanted the minicolumn was transferred to 1.5-ml micro centrifuge tube, and Wash buffer II was added to the upper reservoir, centrifuged at 14,000g for 1 min. The wash buffer II was decanted and the column was reassembled again and centrifuged again to remove any residual wash buffer. Elution buffer (50μl) was applied to the minicolumn and the plasmid DNA was eluted by centrifugation at 14,000 xg for 30 sec. Plasmid yield and purity were checked by spectroscopy at 260nm and 280nm. II.3.E. Agarose gel electrophoresis Agarose gel electrophoresis was performed in DNA electrophoresis submarine mini-cell. Agarose concentration was selected according to DNA size of PCR products and the electrophoresis was performed in 1X TAE buffer (0.04 M Tris acetate, 0.001 M EDTA, pH 8.0). DNA samples were mixed with 6 x gel loading dye (10mM Tris-HCl (pH 7.6), 0.03% bromophenol blue, 0.03% xylene cyanol FF, 60% glycerol, 60mM EDTA). DNA was stained with ethidium bromide added both to the gel and to the buffer at a concentration of 0.5 g/ml. DNA was visualized on a UV-Transilluminator (254 nm) and

photographed with photo documentation system from UVP-CCD Camera, Laboratory products, Epichemi, 11 Darkroom, 3 UV Transilluminator, Pharmacia). II. 4. Southern hybridization After agarose gel electrophoresis, the gel was soaked into a DNA denaturing buffer (1.5 M NaCl, 0.5 N NaOH) with constant agitation to denature the DNA. The gel was neutralized by soaking for 30 min in neutralization solution (1 M Tris pH 7.4, 1.5 M NaCl) at room temperature with gentle agitation. The nitrocellulose membrane was wetted with deionized water and then immersed in the transfer buffer 20xSSC (3M NaCl 0.3 M odium Citrate-2H2O). The wetted nitrocellulose membrane was placed on top of the gel, 2 pieces of 3 MM paper (cut exactly the same size as the gel) was placed on top of the wet nitrocellulose membrane. A stack of paper towels was placed on the 3 MM papers and 500 gm weight was also placed above this setup. The DNA transfer was allowed to proceed for 8-24 hours as described by (Southern, 1975). The membrane was then washed with 6X SSC and placed between two sheets of 3 MM paper till dry. The DNA was cross-linked under UV light for 2 min between 2,500 and 10,000 µJoules/cm2. The membrane was subjected to hybridization according to Boehringer Mannheim guide manual. II.4.A. TSWV/NP DNA probe preparation Digoxigenin-11-dUTP–labeled DNA probe, corresponding to TSWV/NP were prepared by using 10x DNA labeling nucleotide mix (Roche, Boehringer Mannheim, Indianapolis). Digogxigenin-11dUTP nucleotide mix was incorporated into the PCR cocktail instead of the normal nucleotide mix using the protocol described under the technical bulletin (Roche, Boehringer Mannheim, Indianapolis). The PCR reaction was performed in 50 µl total volume reaction containing 50 pmole of each amplification primer flanking the TSWV/NP coding sequence, 5 µl TSWV/DNA clone, 2 µl 10x diglabeled dNTPs and 5 U/ µl of the High Expand Fidelity DNA polymerase (Roche) were used in a buffer containing 20 mM Tris-

HCl, pH 8.2, 10 mM KCl, 6 mM (NH4)2SO4, 0.2 mM MgCl2, 0.1 % triton x-100 and 10 µg/ml of nuclease- free BSA. The amplification reaction was carried out using the UNOII thermal cycler from Biometra using 0.2 ml micro Amp PCR tubes. Hard denaturation of the DNA was performed at 95°C for 3 min followed by 35 cycles of amplification with denaturation at 94°C for 45 sec, annealing at 45°C for 1 min, and extension at 72°C for 90 min. A single tailing cycle of long extension at 72°C for 7 min was carried out in order to ensure .flush ends on the DNA molecules II.4.B. Dot Blot Hybridization Assay Tissue extracts of tomato plants collected from El-Fayoum and El-Menia regions were prepared for dot blot hybridization according to Loebenstein and Akad (1997) with some modifications. The leaf tissue (0.5 g) was ground in 2.5 ml of denatured solution 8x SSC containing (150 mM NaCl, 15 mM Na acetate, pH 7.0) with 10 % formaldehyde . The mixture was heated to 60°C for 15 min and was kept on ice. 10 µl of the supernatant extract was spotted onto presaturated nitrocellulose membrane with 2x SSC. The membrane was then crosslinked using the U.V irradiation for 2 min, between 2,500 and 10,000 µJoules/cm2 followed by nucleic acid hybridization. II.4.C. Hybridization technique The membrane was subjected to hybridization according to Boehringer Mannheim crop protocol. The prehybridization, hybridization, and colourimetric detection procedures were carried out using "Genius II DNA labeling and detection kit" (Boehringer Mannheim IN). Nitrocellulose membrane then soaked in 25 ml of prehybridization solution (6.25 ml of 20x SSC, 0.25ml of 10% Nlaurysacrosine, 0.025 ml of 20% SDS, 1.25 gm blocking reagent) overnight at 65ºC in hybridization oven (Techne Hybridizer HB-ID). The TSWV Dig-labeled DNA/probe was denatured by boiling in water bath for 5 min at 100ºC then placed immediately on ice. The membrane then bathed in hybridization solution containing 5 µl of 10ng freshly denatured TSWV/NP DNA probe overnight at 65ºC.

The membrane was washed three times at room temperature for 5 min in 2x SSC and two times for 15 min each in 0.1x SSC containing 0.1% SDS at room temperature. Nitrocellulose membrane was washed briefly for 1 min in buffer I (0.1M maleic acid, 0.15M NaCl, pH 7.5) and incubated for 30 min at 25 ˚C with 100 ml buffer 2 (100 ml buffer (I) and 1 gm blocking reagent). The membrane was incubated for 30 min at 25˚C with anti-dig-alkaline phosphatase conjugate diluted 1-5000 in buffer II. The unbound antibodies were removed and the membrane was equilibrated by washing 2 times for 15 min with 100 ml of buffer (I), then washed for 2 min with 20 ml of buffer 3 containing 100 mM Tris-HCl, 100 mM NaCl, and 50 mM MgCl2 pH 9.5) at 25 ˚C. II.4.D. Colorimetric detection The membrane was introduced for color detection system in 10 ml color solution using 35µl of 5-bromo-4-chloro-3-indolylphosphate (BCIP) and 45µl of Nitro blue tetrazolium (NBT) to visualize the fragments. The membrane was incubated for 15 min in a suitable clean box in the dark. The reaction was stopped when desired signals were obtained using Genius buffer IV (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) for 5 min. The membrane was air dried and stored at room temperature. II. 5 .Immunocapture – Reverse Transcription – Polymerase Chain Reaction (IC RT-PCR) IC–RT–PCR method was performed as described by (Nolasco et al. 1993). Infected Lycopersicum esculentum were ground (1:10 w/v) in 500 µl Tris-HCl (pH 8.2) containing 2 % PVP-40, 1 % PEG 6000, 140 mM NaCl, 0.05 % Tween 20 and 3 mM NaN 3. The mixture was centrifuged at 5000 xg for 3 min and serially diluted 101, 10-2, 10-3, 10-4 and 10-5 in the same buffer. Thermo-resistant polypropylene PCR tubes were coated with 100 µl of Indian TSWVAS diluted 1 to 4000 in TBS buffer (4 mM NaHPO 4, 1.8 mM KH2PO4, 2.7 mM KCl, 137 mM NaCl) and incubated overnight at 4°C. Equal volume of the TSWV leaf sap extracts in TBS buffer was

added to each tube. The tubes were then washed twice with PBS-T (4 mM NaHPO4, 1.8 mM KH2PO4, 2.7 mM KCl, 137 mM NaCl, 0.05 % tween 20, pH 7.4) and once with MILIQ water. Extra care was taken to avoid cross contamination between the tubes. The RT reaction was carried out by adding 20 µl of the reverse transcription mixture (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 1 mM each dNTPs, 40 unit ribonuclease inhibitor, 1 µM TSW2 plus sense primer, 200 unit of Retrotool reverse transcriptase (RTase) enzyme. The mixture was incubated for 30 min at 70ºC. The PCR was then carried out by adding 30 µl amplification mixture to reach the final concentration (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 4 mM MgCl2, 200 µM dNTPs, 0.2 µM of each primer (tws2) and (tsw1), 2.5 units of Taq DNA polymerase. The amplification proceeded in the thermocycler (UnoII, Biometra) for 35 cycles under the following conditions: One step hard denaturation at 94ºC for 5 min, denaturation at 94ºC for 45 sec, annealing at 45ºC for 1 min, and ºC for 7 72extension at 72ºC for 90 min, and final extension step at min, followed by holding at 4°C. II.6. Nucleotide sequencing of TSWV-nucleoprotein gene The partial nucleotide sequence of TSWV-NP gene from infected tomato isolate was performed at the Plant Pathology Research Institute. Agriculture Research Center., by using CLIP tower automated DNA sequencer from Visible Genetics Inc. The nucleotide sequence of TSWV nucleoprotein was determined by the using Cy5.5 Dye terminator kit (Pharmacia). The resulting PCR product of about 700 bp was subsequently purified by Qiagen spin column and then 50 ng was used in cycle sequencing reaction as follows: 8 µl of DNA template, 3 pm/λ of either forward or reverse specific primer, 1µl of sequenase enzyme, 2.5 µl reaction buffer, the volume was completed with nuclease free water up to 32 µl. Seven microliters of the reaction was added to 4 tubes labeled (G) (A) (T) (C) each of them containing 1 µl of CY5.5 Dye terminators ddATP, ddGTP, ddCTP, ddTTP. The four tubes were subjected to thermal

cycling with 94°C for 1 min., 45°C for 1 min, and 72°C for 120 sec. for 30 cycles followed by holding at 4°C. After cycle sequencing, each tube was precipitated using 7.5 M ammonium acetate and 2.5 volume ethanol and centrifuged for maximum speed 14,000 xg for 30 minutes. The pellets were washed with 70% ethanol and air dried. 4 µl of stopping dye (provided in the kit) was added to each tube and boiled at 99 °C for 3 min before loading into the sequencing gel. Sequence comparisons were performed using the published nucleotide sequences of TSWV nucleoprotein from gene bank under the accession numbers; AB088385, AB038341, AB038342, AJ242772, AY611529, Z36882, AB175809, and X94550. II.7. Production of recombinant TSWV/NP-6xHis fusion protein via gene expression BL21-DE3 bacterial cells transformed with pBAD-Topo construct were induced by adding 0.02 mM of 20 % L-arabinose to the culture when its optical density reached 0.6 to 0.8 at 600 nm. The cultures were incubated for an additional 4 hours before the cells were harvested by centrifugation (10,000 xg for 10 min) Sambrook et al. (1989). The effect of nucleoprotein expression on E. coli cells was examined by comparing the growth rates of E. coli cells transformed with pBAD-NP construct or pBAD non-recombinant vector control in the presence and absence of the gratuitous inducer (L-arabinose). II.7.A. Rapid screening of small cultures (Mini-prep) Screening of small expression cultures was performed under denaturing conditions in order to isolate the 6xHis-tagged recombinant NPs regardless of its solubility in the cell. The bacterial cells were harvested by centrifugation in a microcentrifuge tube, for 1 minute at 15,000 xg. The cell pellet from 3 ml bacterial culture was resuspended in 200 µl of lysis buffer (8 M urea, 0.1 M NaH 2PO4, 0.01 M Tris-Cl, pH 8.0) and the cells were lysed by gentle vortexing to avoid foaming. The cell lysate was centrifuged for 10 min at 15,000 xg and 50 µl of 50 % slurry of nickel-nitrilotriacetic acid (Ni-

NTA) resin was added to the supernatant. The contents were mixed gently for 30 minutes at room temperature. The tubes were centrifuged at 15,000 xg for 10 sec to pellet the resin (containing the 6xHis-tagged nucleoprotein). To check for binding efficiency, 10 µl of the supernatant was transferred to a fresh tube and subjected to SDS-PAGE analysis, and the remaining supernatant was discarded. The resin was washed twice with 250 µl of washing buffer (8 M urea, 0.1 M NaH2PO4, 0.01 M Tris-Cl, pH 6.3) with centrifugation for 10 sec at 15,000 xg between each wash. The 6xHis-tagged nucleoprotein of TSWV was eluted three times from the resin with 25 µl of elution buffer (8 M urea, 0.1 M NaH2PO4, 0.01 M Tris-Cl, pH 4.5) and submitted to SDS-PAGE analysis. II.7.B. Large scale production of TSWV/NPs A single bacterial colony transformed with pBAD-NPs construct was inoculated into 25 ml LB media supplemented with 50 µg/ml ampicillin and grown overnight at 37ºC with shaking (225-250 rpm). The 25 ml culture was diluted into 250 ml LB media and the expression of nucleoprotein was started after the OD has reached 0.3 to 0.6 at 600 nm wave length. The nucleoprotein was induced by adding 0.02 mM of 20 % L-arabinose with shaking (225 rpm) at 37 °C for 4 hours. The cells were harvested by centrifugation (7,000 xg for 10 min) and each cell pellet was resuspended in 5 ml lysis buffer (8 M urea, 0.1 M NaH2PO4, 0.01 M Tris-Cl, pH 8.0). The cell suspension was incubated for 2 h at 37°C with gentle shaking and then subjected to sonication by using either ultrasonic disintegrator for 2-4 min or syringe needle. The cell lysates were centrifuged at 11,000 xg for 15 min and the soluble fraction (supernatant) was kept to be analyzed by SDS/PAGE. II.7.C. SDS- PAGE Electrophoresis of nucleoprotein Proteins were analyzed by denaturing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli (1970). The separating gel was 12 % acrylamide-bisacrylamide, 0.37 M Tris-Cl (pH 8.8), 0.1 % SDS, 0.05 % (w/v)

ammonium persulphate, and 0.05 % (v/v) TEMED. The stacking gel was 4.0 % acrylamide-bis-acrylamide, 0.125 M Tris-Cl (pH 8.8), 0.1 % SDS, 0.05 % (w/v) ammonium persulphate, and 0.1 % (v/v) TEMED. The protein samples were diluted at least 1:4 with sample buffer and heated at 95°C for 5 minutes prior loading the gel. The purified nucleoprotein polypeptide was resolved by electrophoresis onto a 12% SDS -PAGE on a midi protein II XI system (Biometra). The protein gel was stained for 60 min in a solution of 50 % (v/v) methanol, 10% (v/v) acetic acid and 0.25% (w/v) Coomassie brilliant blue R-250 (BDH, Merck Biochemicals) at room temperature with shaking. Thereafter, the gel was destained as recommended (Harlow and Lane 1988) using a solution of 5% (v/v) Ethanol and 7.5% (v/v) acetic acid for 6 hours. II.7.D. Purification of 6xHis-tagged TSWV-nucleoprotein under denaturing conditions: Purification of 6xHis-tagged NPs was performed under denaturing conditions by nickel-nitrilotriacetic acid (Ni-NTA) batch chromatography. The cell pellet from 200 ml of induced bacterial culture was resuspended in 4 ml of lysis buffer (8 M urea, 0.1 M NaH2PO4, 0.01 M Tris-Cl, pH 8.0). The cells were lysed and the cell lysate was centrifuged for 10 min at 15,000 xg to remove the cellular debris. To the supernatant, 1 ml of 50% Ni-NTA slurry was added and the lysate-resin mixture was loaded into an empty column. Endogenous proteins with histidine residues were washed out of the matrix twice by 4 ml of washing buffer (8 M urea, 0.1 M NaH 2PO4, 0.01 M Tris-Cl, pH 6.3). The 6xHis-tagged nucleoprotein was eluted 4 times with 0.5 ml of elution buffer (8 M urea, 0.1 M NaH 2PO4, 0.01 M Tris-Cl, pH 4.5). The 6xHis-tagged nucleoprotein was dissociated from the Ni-NTA resin at the acidic pH. II.7.E. Purification of 6xHis-tagged TSWV-nucleoprotein under native conditions: Purification of 6xHis-tagged NPs was performed under native conditions as described by (Jacquet et al. 1998) by washing the cell

pellet from 200 ml of induced bacterial culture twice in 10 ml cold STE buffer (20 % sucrose, 100 mM Tris pH 8.0, 10 mM EDTA) and re suspended in 5 ml of the same buffer containing 1 mg/ml lysozyme. The cells were then incubated for 20 min at 4 °C and centrifuged again (5000 xg, 10 min). The spheroplasts were suspended in 3 ml lysis buffer (100mM Tris pH 8.0, 5 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 0.2 % Sarkosyl), gently shake for 5 min and fractionated by syringe's needle on ice. RNase and DNase were added (1µg/ml) for 20 min at room temperature before centrifugation (15 min, at 11,000 xg). The supernatant was collected and centrifuged again, the pellet was discarded and the supernatant was concentrated in a Speed-Vac.

IV. RESULTS

Part I Isolation and Identification of tomato spotted wilt virus (TSWV) A. Isolation TSWV was isolated from tomato fruits exhibiting symptoms suspected to be due to TSWV infection from the experimental farm, Faculty of Agriculture, Ain Shams University, Kalubia Governorate. The purity of the virus isolate was verified by several mechanical inoculations onto Petunia hybrida Vilm followed by Nicotiana

rustica plants. The isolated virus was maintained on tomato plants for symptoms development under the greenhouse conditions. B. Identification and characterization Identification of the isolated virus was based on studying the mode of transmission, host range, symptomatology, virus stability, electron microscopy (Part I) and characterized by some molecular and serological means as follows: B1. Transmission B.1.a. Mechanical transmission Infectious sap was prepared as described before. Obtained data revealed that TSWV was transmitted by mechanical inoculation to tomato seedlings in the greenhouse. Symptoms appeared from 14 to 21 days after inoculation. Inoculated plants showed thickening of young leaves, especially the veins, sometimes accompanied by concentric rings. Leaflets tended to curl downwards and inwards and started bronzing (Fig.1A-a2). Infected plants were stunted and the early infection killed the young seedlings. The later stages of the diseased leaves showed yellowish mosaic mottling and distortion. Fruit symptoms often appeared as pale or yellow areas in the normal red skin of the ripe tomato fruits. The pale areas appeared as irregular mottling or distinct concentric circles (Fig.1A-a2). B.1.b.Insect transmission (Thrips transmission) Thrips tabaci were used to transmit TSWV from infected to healthy tomato seedlings. The insects were maintained on infected tomato plants to feed on the virus for one day and then transferred to the healthy tomato plants. Fourteen out of twenty tomato plants (70%) showed symptoms of TSWV infection after 14 - 21 days from inoculation. These symptoms were verified as TSWV by using TSWV Indian Antiserum in DBIA (Fig. 1B) B2. Host range and symptomatology Thirty three plants belonging to 31 Genus and sixteen families Amaranthacea, Apiaceae, Araceae, Asteracea, Brassicaceae, Chenopodiaceae,

Convolvulaceae,

Cucurbitaceae,

Fabaceae,

Geraniaceae, Malvaceae, Meliaceae, Poaceae, Primulaceae, Scrophulariaceae, and Solanaceae were mechanically inoculated with the isolated virus by the infected crude sap, to study the interaction between them and the isolated virus. These plants are presented in Table 1 and Fig. 1. A. Uniform seedlings of each host were inoculated. Controls of corresponding seedlings were inoculated with the extraction buffer alone. Symptoms were recorded daily up to 30 days. Some other plant reservoirs (Fig. 2 A) were found to be naturally infected by TSWV in the open field collected from the experimental farm, Faculty of Agriculture, Cairo University were photographed and confirmed positive to TSWV by DBIA. However, some plants such as Vitis may harbour other viruses in mixed infection with TSWV and these plants couldn’t be tested for all the viruses due to the lake of specific antiserum but confirmed positive to TSWV by DBIA Fig. (2 B). The plants displaying symptoms typical to TSWV were checked by in-direct ELISA as shown in Table 1.

Table (1): Host range, symptoms, and ELISA readings of tomato spotted wilt virus (Tospovirus) after mechanical inoculation. Family & Tested Plants

Observed Symptoms

Absorbance value

1

Apiaceae: L.

2

NLL

+

0.374

BR

+

0.352

Brassicaceae: Eruca sativa

NLL

+

0.337

Chenopodiaceae: Chenopodium murale L.

NLL

+

0.324

Chenopodium amaranticolor L.

NLL

+

0.430

Apium graveolens

Araceae: Eminium spiculatum 3 4

5

Asteracea:

6

Lactuca sativa L. Sonchus oleraceus Convolvulaceae: Convolvulus arvensis L. Cucurbitaceae:

7

LD NLL, NR

+ +

0.442

NLL, NR

+

0.605

O

-

0.050

Arachis hypogaea L. Lupinus termis L. Phaseolus vulgaris L. cv. Giza 6

CR NLL, NR NLL, NR

+ NT +

0.141 NT 0.156

Pisum sativum L. cv. Little Marvel Vicia faba L. cv. Giza 2 Geraniaceae: Pelargonium sp.

NLL, NR NLL

+ +

0.321 0.310

CLL

+

0.631

CLL

+

0.476

CL

+

0.587

NLL

+

0.476

NLL, NR

+

0.412

CR

+

0.375

Cucurbita pepo L. 8

0.361

Fabaceae:

9 10 11 12 13

14

Malvaceae: Malva parviflora Meliaceae: Cidrella toona Primulaceae: Anagalis arvensis Scrophulariaceae Antirrhinum majus Solanaceae: Capsicun annuum L. cv.California wander

Datura metel L. D. stramonium

NLL,CNR NR

+ +

0.504 0.562

Lycopersicon esculentum Mill. cv. Money Maker Nicotian glutinosa L. N. rustica L. N. tabacum cvs. Kentuckey

BR, R, ST, B

+

0.585

NLL, CNR NLL, NR,

+ + +

0.634 0.487 0.593

N. tabacum cv. White Burley Physalis peruviana L. Petunia hybrida Vilm

SNR NLL, NR, SNR

+ + +

0.445 0.521 0.483

Solanum albicaule S. nigrum

NLL, CNR M, M

+ +

0.430 0.651

O

-

0.054

NLL

+

0.505

NLL, CNR NLL, CNR CNR 15

16

Poaceae: Hordium vulgaris Amaranthaceae: Gomphrena glubosa L.

B= blistering, BR= bronzing, CL= chlorosis, CLL=chlorotic local lesions, M, M= mosaic and mottling, CNR= concentric necrotic rings, CR= chlorotic ring spot, NLL= necrotic local lesion, NR= necrotic rings, R= rolling, O= No symptoms, SN= systemic necrosis, ST= stunting, LD= leaf discoloration. (+)= Positive reaction ≥ 0.300, (-) = Negative reaction (healthy leaves from each host) ≤ 0.054. NT=not tested. Indian As= TSWV-AS.

Fig(1.A1) : Symptoms expression on mechanically inoculated plants. (a1): Lycopersicon esculentum Mill. cv. Money Maker fruit (virus source) showing yellow areas scattered along the red skin of the fruit (fingerprint like shape). (a2) Tomato leaves showing thickening of young leaves especially the veins and the newly formed leaves start bronzing accompanied by wrinkling of leaves. (b): Arachis hypogaea leaves showing chlorotic ring spot. (c): Nicotiana glutinosa leaf showing local lesions which gradually increase in size forming spots of concentric necrotic zones. (d) Datura metel leaf showing necrotic local lesions developed into concentric necrotic rings. (e): Petunia hybrida plant showing necrotic local lesions. (f): Nicotiana tabacum cv. Kentuckey leaves showing necrotic local lesions,

Fig (1. A2) : Symptoms expression on mechanically inoculated plants. (g): Datura stramonium leaves showing necrotic rings. (h): Physalis peruviana showing mosaic and mottling. (i): Nicotiana tabacum cv. White Burley showing necrotic local lesions. (j): Lactuca sativa plant showing light marginal wilting and leaf discoloration, the plants grow from one side. (k): Pelargonium sp. Leaf showing chlorotic local lesions.

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

Fig (1 B) DBIA of twenty tomato plants exposed to viriluferous Thrips tabaci insects and showed symptoms after 14 - 21 days from inoculation. Fourteen out of twenty tomato plants exhibited symptoms of TSWV gave positive signals when TSWV Indian Antiserum was used.

Fig (2 A1): Symptoms expression on some naturally infected plants. (a, b and c): vitis plants showing necrotic areas (d): Bougainvillea glabra showing necrotic lesions (e): Cidrella toona:

showing chlorosis. f: Eminium spiculatum showing necrotic lesions.

Fig (2. A2) : Symptoms expression on some naturally infected plants. g: Phaseolus vulgaris: showing necrotic local lesions. h: Solanum nigrum showing brown necrotic areas. i: Sonchus oleraceus showing brown leaf spotting. j: Convolvulus arvensis showing marginal necrotic areas k: Urtica urens showing vein necrosis. l: Solanum albicaule showing necrotic zones A

b

c

d

E

f

g

h

i

j

k

l

Fig (2 B): DBIA showing the reactivities of naturally infected

reservoir plants collected from the experimental farm, Fac of Agric, Cairo Univ. nominated (a, b, c, d, e, f, g, h, I, j, k, and l) with Indian TSWV/AS. B3. Virus stability: Results in Tables (2, 3 and 4) indicated that the virus was sap transmissible. Its thermal inactivation point was 45-50ºC; the dilution endpoint was 10-3 and the virus was completely inactivated after incubation for 5-6 hours at room. Table (2): Determination of thermal inactivation point (TIP) of TSWV in crude sap. Temp

Number of Local Lesions

in °C

Number of plants 3 4

1

2

30 35

95 90

105 82

91 103

40 45 50 55

45 18 0 0

34 12 0 0

60

0

0

Mean 5

6

102 89

65 105

35 24

82.6 82.1

50 6 0 0

48 4 0 0

40 5 0 0

10 3 0 0

37.8 8 0 0

0

0

0

0

0

Table (3): Determination of the dilution end point (DEP) of TSWV in crude sap. Dilution of Virus 1

Number of Local Lesions Number of plants 2 3 4 5

Mean 6

Undiluted

130

95

120

69

49

38

83.5

10-1 10-2 10-3 10-4

150 90 30 0

104 87 18 0

100 45 12 0

75 89 15 0

40 85 5 0

17 20 11 0

81 69.3 15.16 0

10-5 10-6

0 0

0 0

0 0

0 0

0 0

0 0

0 0

10-7

0

0

0

0

0

0

0

Table (4): Determination of the longevity in vitro of TSWV in crude sap. First experiment from 0 to 12 hrs. Incubation Period

Number of Local Lesions Number of plants

Mean

(hrs.)

1

2

3

4

5

6

0 2 4

135 100 50

133 85 42

145 115 30

180 30 35

130 40 35

140 55 15

143.8 70.8 34.5

6 8 10 12

0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 0

Second experiment from 0 to 6 hrs Incubation Period

Number of Local Lesions Number of plants

Mean

(hrs.)

1

2

3

4

5

6

1 2 3

137 89 76

95 69 85

105 49 62

75 30 81

60 55 50

45 62 75

86.1 59 71.5

4 5 6

43 10 0

23 13 0

27 5 0

15 8 0

12 5 0

40 9 0

26.6 8.3 0

Mean: Number of local lesions / Total number of inoculated seedlings (Datura metel plant).

B.4.Serological studies B.4.1. Reactivity of TSWV nucleoprotein (NP) against Indian antiserum TSWV polyclonal antibodies were produced against the Egyptian isolate tomato spotted wilt viral nucleoprotein via molecular biological techniques as shown latter under the Molecular studies

(Part II). The reactivity of the purified NP was examined against Indian TSWV antiserum before immunizing the rabbits. The antigenicity of the purified recombinant nucleoprotein was tested by using DBIA. The results showed high specific reactions between TSWV/NP purified under native conditions and TSWV polyclonal antibodies (India) as shown in Fig. (3). NP/6xHis was spotted onto nitrocellulose membrane and probed with Indian TSWV/AS at dilution (1-4000) as primary antibody and the anti-rabbit alkaline phosphatase conjugate was used at dilution (1-7500) as secondary antibody. The results indicated that the obtained nucleoprotein of TSWV successfully reacted with TSWV/AS. Pooled purified native and denatured fusion nucleoprotein (NP/6xHis) expressed and purified from E.coli was injected intramuscularly into white NewZealand rabbit for six injections as described under Materials and Methods. Serologic studies indicated also that the Egyptian antiserum reacted specifically with the denatured fusion nucleoprotein (NP/6xHis) in in-direct ELISA (Table 5). CL Flow-through

Wash 1

Wash 2 E1 Non-induced culture E2 Fig (3) : DBIA showing the reactivity of TSWV/NP expressed in E.coli using Indian antiserum (dil 1/4000) as primary antiserum. Nitrocellulose membrane shows the crud preparation from bacteria expressing TSWV/NP (CB). CL: cleared lysates of bacteria expressing the NPs. Flow-through (Fth), Wash 1 (W1), Wash 2 (W2), E1 and E2: purified 6x-His/NPs fusion protein purified under native conditions. Anti rabbit IgG alkaline

phosphatase-conjugated (Sigma) was used as second antibody at dilution 1-7500.

B 4.2. Antiserum Production B 4.2.a. Determination of TSWV antiserum titer Absorbance values of the Egyptian antiserum produced against TSWV fusion nucleoprotein (NP/6xHis) using indirect ELISA indicated that the best optimum dilution of TSWV-AS-EG which reacted specifically with the denatured nucleoprotein (NP/6xHis) and gave the highest O.D. readings values was 1-500. The absorbance values are measured at 405 nm and the data was tabulated as shown in Table 5 and presented in Fig. (4). Table 5. Antiserum titration produced against TSWV ELISA O.D. readings at 405 nm TSWV/AS dilution B (-ve) NP /6xHis 1/250 1/500

0.251 0.360

1.501 1.840

1/1000 1/2000 1/4000 1/6000

0.370 0.351 0.240 0.171

1.741 1.650 1.351 1.201

B: Bacterial proteins (negative control). Absorbance values: mean of two wells. NP/6xHis: Purified nucleoprotein of TSWV expressed in E.coli. TSWV-AS-EG: Egyptian TSWV antiserum

TSWV Antiserum Dilution

2

ELISA O.D. readings at 405 nm B (-ve)

1.8 1.6 1.4 1.2 1

ELISA O.D. readings at 405 nm NP /6xHis

0.8 0.6 0.4 0.2 0

1/250

1/500 1/250

1/1000 00-‫يناير‬ 1/500 1/1000 00-‫يناير‬ 1/2000 00-‫يناير‬ 1/4000 TSWV/AS dilutiion

1/6000

Fig (4): Titration of TSWV antiserum (TSWV-AS-EG) against purified NP expressed in E.coli. B: Bacterial proteins (negative control). TSWV/NP: Purified nucleoprotein of TSWV expressed in E.coli. B.4.2. b. Separation of TSWV/IgG: IgG fraction of TSWV/NP antiserum was purified and adjusted to the concentration of 1.0 mg/ml (A280 nm=1.4 O.D.). Result in Fig. (5) showed that the purified IgG had Amax at 280 nm and Amin at 250 nm

Fig (5): Ultraviolet spectrum of purified IgG from TSWV/NP-AS.

B.4.2.c. Titration of TSWV/IgG/EG and its reactivity with different infected tissues by in-direct ELISA Egyptian TSWV/IgG was capable to detect TSWV in different sap extracts from Pelargonium spp., Tomato, Petunia, and Nicotiana rustica by using indirect ELISA till the dilution 1/4000 as shown in Fig. (6). However, the highest absorbance values obtained were 0.375 and 0.257 with tomato and Pelargonium spp respectively, when 1/1000 dilution of antibodies were used. While the highest absorbance values obtained were 0.221 and 0.405 with Petunia and TSWV/NP respectively, when 1/500 dilution of antibodies were used whereas the best antibody dilution which gave the highest O.D. value (0.216) was 1/250 when N. rustica was used. Table 6. Titration of TSWV/IgG/EG with four TSWV infected plants: Pelargonium spp., Tomato, Petunia, and Nicotiana rustica using indirect plate trapping ELISA. Each crude extract (antigen) was plated at dilution 10 -3. The titers against each antigen were calculated as absorbance values represented on Y axis. Dilutio n of TSWV /IgG 1/250 1/500 1/1000 1/2000 1/4000

Pelarg. spp

Tomato

Petunia

O.D. values of infected tissues N. rustica TSWV/NP

I

H

I

H

I

H

I

H

NP+

B

0.207 0.195 0.257 0.236 0.199

0.039 0.037 0.035 0.030 0.029

0.230 0.308 0.375 0.236 0.188

0.057 0.059 0.054 0.038 0.030

0.194 0.221 0.182 0.161 0.112

0.034 0.039 0.037 0.040 0.049

0.216 0.186 0.182 0.166 0.186

0.038 0.039 0.040 0.045 0.049

0.321 0.405 0.327 0.224 0.231

0.049 0.056 0.055 0.053 0.049

Healthy: (calculated mean of 2 wells) B: bacterial protein (negative control) NP: TSWV nucleoprotein expressed in E.coli

Pelarg I

Titration of TSWV/IgG-EG

Pelarg H

0.45

Tomato I Tomato H

0.4

Petunia I Petunia H

0.35

N.rust I

0.3 A 405 nm

N.rust H NP+

0.25

B

0.2 0.15 0.1 0.05 0 1/250

1/500

1/1000

1/10000

1/100000 1/2000

1/4000

TSWV/IgG dilution

Fig (6): Schematic diagram showing the absorbance values of indirect ELISA measured at 405 nm for determination of approximate working dilution of Egyptian TSWV/NP IgG against different sap extracts from four plants: Pelargonium spp, Tomato, Petunia, and Nicotiana rustica infected with TSWV NP. Positive and negative controls are included. B.4.3. Determination of dilution end-point of TSWV infected sap Different plants that were found to be frequently infected by TSWV during this study such as Pelargonium spp., Tomato, Petunia, and Nicotiana rustica were used to detect the appropriate end point dilution of infected sap extracts that can be used in indirect ELISA. The virus was detected reliably in crude sap of all infected plants at dilutions between 10-2 and 10-3. TSWV/IgG/EG was used at dil (1: 1000) and the highest I/H ratio were at 10-3 dilution of the plant sap (Table7 and Fig 7).

Table (7): In direct plate trapping ELISA showing the dilution end point of TSWV infected crude sap extracts. Sap dilution Pelargonium I/H O.D. O.D. (mean) value H

I 10-1

0.625 0.735 1.689

(0.049+0.043)/2=0.046 (0.045+0.064)/2=0.054 (0.071+0.061)/2=0.066

0.625/0.046=13.6 0.735/0.054=13.5 1.689/0.066=25.6

10-4 0.327 10-5 0.070

(0.065+0.046)/2=0.055 (0.047+0.043)/2=0.045

0.327/0.055=5.95 0.070/0.045=1.56

10-2 10-3

Sap dilution

Tomato O.D.

O.D. (mean)

I/H value

H I 10-1 10-2 10-3 10-4

1.372 0.795 1.445 0.537

(0.059+0.063)/2=0.061 (0.048+0.054)/2=0.051 (0.045+0.078)/2=0.061 (0.043+0.067)/2=0.055

1.372/0.061=22.4 0.795/0.051=15.5 1.445/0.061=23.6 0.537/0.055=9.76

10-5 0.199

(0.074+0.034)/2=0.054

0.199/0.054=3.68

Sap dilution

Petunia O.D.

O.D. (mean)

I/H value

H I 10-1 0.349 10-2 0.318

(0.049+0.043)/2=0.046 (0.045+0.064)/2=0.054

0.349/0.046=7.6 0.318/0.054=5.9

10-3 0.323 10-4 0.154

(0.071+0.061)/2=0.066 (0.065+0.046)/2=0.055

0.323/0.066=4.9 0.154/0.055=2.8

10-5 0.089

(0.047+0.043)/2=0.045

0.089/0.045=1.97

Sap dilution

N .rustica O.D.

I/H value

O.D. (mean) H

I 10-1

0.792

(0.058+0.056)/2=0.057

0.792/0.057=13.9

10-2 10-3 10-4 10-5

0.755 0.626 0.256 0.172

(0.059+0.060)/2=0.059 (0.056+0.053)/2=0.054 (0.055+0.056)/2=0.055 (0.063+0.053)/2=0.058

0.755/0.059=12.8 0.626/0.054=11.6 0.256/0.055=4.67 0.172/0.058=2.96

Sap dilution

NP (Pos) O.D.

I/H value

O.D. (mean) H

I 10-1 1.311 10-2 1.952

(0.063+0.052)/2=0.057 (0.064+0.059)/2=0.061

10-3 2.436 10-4 1.022 10-5 0.329

(0.059+0.057)/2=0.058 2.436/0.058=42 (0.055+0.050)/2=0.052 1.022/0.052=19.65 (0.057+0.055)/2=0.056 0.329/0.056=5.87

1.311/0.057=22.9 1.952/0.061=32

Dilution of crude sape

I/H Pelarg. sp

I/H Tomato

I/H Petunia

I/H N.rustica

I/H NP (Pos)

1/10 1/100

13.6 13.5

22.5 15.6

7.6 5.9

13.9 12.8

23 32

1/1000 1/10,000

25.6 5.9

23.7 9.78

4.9 2.8

11.6 4.67

42 19.67

1/100,000

1.56 I: infected

3.69 H: healthy

1.98

2.98

5.89

I/H: calculated as shown above.

In-Direct ELISA Pelarg. spp

45

Tomato 40

Petunia N.R.

35

NP ( Pos)

30 25

I/H 20 15 10 5 0

1/10

1/100

1/1000

1/10,000

1/100,000

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B.4.4. Evaluation of the Egyptian TSWV/NP antiserum by dot – blot Immunoassay (DBIA) TSWV was identified by DBIA test and indirect ELISA using the Egyptian TSWV/NP IgG produced during this study. In dot-blot immunoassay (Fig. 8), TSWV-infected leaf saps from different host plant chosen from Table 1 were detected when the nitrocellulose membrane was probed with Egyptian polyclonal antibody (TSWV/NP/IgG) diluted 1:1000 in TBS-T buffer as primary antibody and the anti-rabbit alkaline phosphatase conjugate diluted (1-7500) was used as secondary antibody (Fig 8 B). The colored signals indicated the high reactivity of Egyptian TSWV/ IgG with infected saps as well as the Indian TSWV/AS (Fig 8 A). The results of both antibodies (Egyptian and Indian) indicated that each antibody has its own specificity and reactivity against the virus isolate that belongs to. So, the reactivity of each antibody with the tested samples is not the same and this is clearly obvious in Fig 8 (A&B). Healthy control showed No colored signals. (H) 1

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Fig (8): DBIA showing the serologic reactivities of Indian (A) TSWV/AS (1-4000) and Egyptian (B) TSWV/NP/IgG (1:1000) with different tested protein sap preparations from 1 to 30 different TSWV infected host plant as indicated in Table 1. Healthy sap (control)

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showing No colored signal. The nitrocellulose membrane was color developed using NBT/BCIP. Fig. (A) Showing that sample numbers 8, 21, 23, 25, 27 and 29 (Arachis hypogaea L., Lupinus termis L., Vicia faba L. cv. Giza 2, Phaseolus vulgaris L. cv. Giza 6, Hordium vulgaris and Cucurbita pepo L.) could not be detected by Indian TSWV/AS. While in Fig (B) sample numbers 1, 16, 17, 29 and 30 (Apium graveolens L, Sonchus oleraceus, Antirrhinum majus, Cucurbita pepo L., and Pisum sativum L. cv. Little Marvel) could not B.5. Electron microscopy Electron microscopic examination of negatively stained partially purified preparations of the virus under test with 2% Uranyle acetate (Fig. 9) revealed the presence of quasi-spherical particles ranging from 80-100 nm in diameter.

Fig (9): Electron micrograph of partially purified virus preparation stained with Uranyl acetate. Magnification power is printed on the photo.

Part II Molecular Studies of TSWV 1. RNA extraction Total RNA was purified from mechanically infected Lycopersicum esculentum, Datura metel, Pelargonium sp., by using two methods, i.e. High Pure RNA Tissue extraction Kit (Roche) and CTAB method as described under Materials and Methods. The integrity and quantity of the purified RNA were confirmed by gel electrophoresis (Fig. 10 A & B) and UV spectrophotometery. The two methods used were successful and showed the characteristic 2:1 ethidium bromide staining ratio of 28S to 18S ribosomal RNA indicating no significant RNA degradation.

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Fig (10): 1% agarose gel electrophoresis showing the total RNA extraction from different tissues infected with TSWV. Lane 1: T-RNA isolated from Lycopersicum esculentum infected with TSWV. Lane 2: T-RNA isolated from Datura metel infected with TSWV. Lane 3: T-RNA isolated from Pelargonium sp. infected with TSWV. The arrows indicate the presence of 18S and 28S ribosomal RNA. (A) T-RNA extraction using CTAB method. (B) T-RNA extraction using High pure RNA tissue Kit (Roche).

2. Detection of TSWV by RT-PCR 2.A. RT-PCR of TSWV/NPs The total RNAs prepared by using High pure RNA tissue Kit (Roche) and purified from mechanically infected Lycopersicum esculentum Pelargonium sp., Datura metel, and Nicotiana rustica, were reverse transcribed using Retrotools Reverse Transcriptase (Spain, Madrid) and tsw1 (plus sense primer) (Fig. 11A) and the cDNAs were amplified by PCR using the oligonucleotides TSW1 and TSW2 as PCR primers. RT-PCR was used to amplify a fragment of about 780 bp from the nucleoprotein gene (NP) S-RNA. The size of the PCR product amplified from Lycopersicum esculentum, and Pelargonium sp was estimated by comparing its electrophoretic mobility with those of standard DNA marker as shown in Fig. 11 A and B. The size of the amplified DNAs was in agreement with the expected size calculated (~780 bp) from the positions of the primers and also with the published nucleotide sequence of NP gene S-RNA (Accession number; AB038341; Kato and Hanada, 2000). The authenticity of the resulting PCR product (~780 bp) was verified by direct DNA sequencing. However, the size of the PCR products amplified from mechanically infected Datura metel and Nicotiana rustica as differential hosts were less than the expected size (~600bp). No signal was detected in the negative control (Fig. 11 B).

agagcaatcg tgtcaatttt tattcaaacc ttaacactca gtcttacaaa tcatcacatt 60 aaaaccctaa gaaacgactg cggaatacag agttgtactt ttgcaccttg aattacatac 120 ggtcaaagca tataataact tctgtgatca tcatgtctaa ggttaagctc actaaggaaa 180 acattgttgc tttgttgaca caaggcaaag accttgaatt tgaggaagat cagaatctgg 240 tagcattcaa cttcaagact ttttgtctgg aaaaccttga ccagatcaag aagatgagca 300 ttatttcgtg tctgacgttc ctaaagaatc gtcagggtat aatgaaggtt attaagcaaa 360 gtgattttac ttttggtaaa attaccataa agaaaacttc aaacaggatt ggagccactg 420 acatgacctt cagaaggctt gatagcttga tcagggtcag gcttgtcgag gaaactggga 480 attctgagaa tctcaatact atcaaatcta agattgcttc tcaccctttg attcaagcct 540 atggattacc tcttgatgat gcaaagtctg tgaggcttgc cataatgctg ggaggtagct 600 tacctcttat tgcttcagtt gatagctttg agatgatcag tgttgtcttg gctatatatc 660 aggatgcaaa atacaaagat ctcgggatcg acccaaagaa gtatgacact agggaagcct 720 taggaaaagt ttgcactgtg ctgaaaagca aagcatttga aatgaatgaa gatcaggtga 780 agaaaggaaa agagtatgct gctatactta gctccagcaa tcctaatgct aaaggaagta 840 ttgctatgga acattacagt gaaactctta acaagttcta tgaaatgttc ggggttaaaa 900 acaggcaaaa cttgcagaac ttgcttaaaa gcag 934

tsw1 Fig (11A): Nucleotide sequence of the nucleoprotein gene of TSWV infecting Chrysanthemum isolate accession number (AB038341) Kato and Hanada (2000). The specific primers tsw1 (+) and tsw2 (-) designed for RT-PCR were boxed. The initiation (ATG) and stop (TAA) codons are highlighted.

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Fig (11 B): 1 % agarose gel electrophoresis showing the results of RT-PCR products, performed using tsw1 and tsw2 primers to amplify about the full length NPs gene (~780 bp and 600 bp), using RNA extracted from Lycopersicon esculentum (lane 1), Datura metel (lane 2), Pelargonium spp. (lane 3) , and Nicotiana rustica (lane 4) infected with TSWV. Lane 5: Negative control (No RNA template). Lane M: DNA Molecular weight Marker XVI (Roche, Applied Science).

2. B. Southern Blot Analysis of the PCR products Southern blot hybridization was used to confirm the authenticity of the PCR products of TSWV/NP gene. PCR products were resolved on 1% agarose gel and transferred onto nitrocellulose membrane as described by Southern (1975). Hybridization was performed using 10 ng of TSWV-NP probe labeled with digoxigenin11- dUTP as described under Materials and Methods. Fig. 12 shows that the DNA probe successfully hybridized with all PCR products of TSWV/NPs (780 bp and 600 bp).

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Fig (12): Southern hybridization of TSWV PCR products amplified from different infected tissues using TSWV-NP DNA-probe labeled with Dig-11 dUTP. Nitrocellulose membrane showing the hybridization signal of PCR products (Lane 1) from Lycopersicon esculentum, Lane 2: Datura metel, Lane 3: Pelargonium spp. Lane 4: Nicotiana rustica. The hybridization signals appeared after addition of Anti-Dig-Ap conjugate diluted (14000) and the chromogenic substrate NBT/BCIP. The DNA- probe of TSWV/NP cross- reacted with all PCR products.

3. Molecular Cloning of partial sequence of TSWV/NP gene in E.coli The strategy used for cloning of TSWV/NPs gene into E.coli was based on direct cloning of the generated DNA fragments after gene clean using QiAquick Gel Extraction Kit (Qiagen) eluted in 50 μl of autoclaved water and quantified by a spectrophotometer and gel electrophoresis (Fig. 13 A and B). The DNA concentration was checked by measuring the OD at 260/280 nm (at 260 nm, 1 OD corresponds to 50 µg/ml of double-stranded DNA) and the ratio 260/280 nm provides an estimate of the purity of the cleaned PCR product. The purity of the cleaned PCR product was 1.8. The PCR product was ligated into prokaryotic expression vector pBAD-Topo (Invitrogen, life technologies) (Fig. 14). The ligation reactions were

transformed into competent E. coli BL 21- DE3 cells. The NP DNA sequence was inserted downstream from the araC promotor. The obtained white colonies resistant to ampicillin containing recombinant plasmids were further selected for isolation of DNA plasmids containing the TSWV/NPs gene by plasmid minipreparation using High pure plasmid DNA preparation kit (Roche) as shown in Fig. 15. Gel slice (100 mg)

Add 300 ul of QG buffer

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Fig (13 A): Schematic diagram showing the steps used in cleaning of the TSWV/NP- PCR product from Agarose gel band using QiAquick Gel Extraction Kit (Qiagen).

13 B: Cleaned PCR product (TSWV/ NP gene) after QiAquick purification of the correct size (780 bp).

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Fig (14): Genomic organization of TSWV (L, M, and S RNAs); L: Large minus sense RNA dependent RNA Polymerase which has replicas activity, M: coding for nonstructural protein (NSm) which is plus sense RNA and Glycoprotein genes G1 & G2 which are minus sense RNA; S: small RNA

coding for Non-structural protein (NSs) which is plus sense RNA and Nucleocapsid protein (Nc) which is minus sense RNA. The partial restriction map of pBAD-Topo expression vector (Invitrogen life technologies, Catalog nos. K4300-01, K4300-40). T overhangs used for cloning of TSWV/NP gene after PCR amplification.

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Fig (15): Plasmids preparations of pBAD-Topo containing the NP insert. Plasmid DNAs were isolated from 5 ml overnight LB bacterial culture using the High Pure Plasmid isolation kit (Roche). 10 µl of each was run onto 1% Agarose gel electrophoresis stained with Ethidium bromide. The recombinant plasmid DNAs were validated by PCR to check for the presence of NP insert in the right orientation. The arrows indicate the promising recombinant clones (Lanes 2, 3 and 4).

4. Validation of Cloning of TSWV/NPs gene by PCR Plasmid mini-preparations were used to screen random colonies from about 55 tansformants using High pure plasmid preparation kit (Roche) as described under the Materials and Methods. Randomly selected clones were validated by PCR to confirm the presence of TSWV-NPs gene insert in the right orientation. The PCR amplification was performed on selected clones by using tsw1 and tsw2 primers identified several colonies containing the NP insert with the expected size (780 bp) Fig. (16 A). After verification by DNA hybridization a strong and specific reaction was obtained (Fig. 16 B) and the cloned TSWV/NP fragment was used for the expression experiment.

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5. Dot Blot Hybridization Assay Lycopersicon esculentum showing necrosis and bronzing symptoms collected from Fayoum and El-Menia regions were tested by dot blot hybridization technique in order to test for the presence of TSWV in these regions and also to test the efficiency of TSWV/NP DNA probe in the detection TSWV in field survey. The TSWV/NP DNA probe successfully hybridized with eight samples out of eighteen collected from Fayoum region (44 %) and with nine samples out of twelve collected from El-Menia region (75 %). Negative controls show no signals. On the other hand, the DNA probe gave no signals with extracts from healthy tissues as shown in Fig. 17.

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Fig (17): Dot blot hybridization assay performed on sap extracts from Lycopersicon esculentum samples collected from Fayoum region (18 samples) and El-Menia region (12 samples). The extracted saps were spotted onto nitrocellulose membrane and eight samples out of eighteen collected from Fayoum region gave positive signals (44 %) and nine samples out of twelve collected from El-Menia region gave positive signals (75 %). Negative controls show no signals. Anti-dig alkaline phosphatase conjugate was diluted 1/5000 and the color was developed after 15 min using BCIP and NBT substrate.

6. Immune Capture Reverse Transcription Polymerase

Chain Reaction (IC- RT-PCR) IC-RT-PCR successfully detected TSWV Tomato isolate in infected tissues. Results in Fig. 18 showed the sensitivity of IC-RT-PCR to detect TSWV in diluted cDNAs. On the other hand IC-RT-PCR showed no amplification product in the Healthy control.

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Fig (18): Gel electrophoresis showing the sensitivity of IC-RT-PCR for detection of TSWV in different Tomato tissues infected with TSWV. TSWV/ cDNAs were diluted (10-1, 10-2, 10-3, 10-4, 10-5) and subjected to PCR. The optimum dilution of cDNA that produce a strong amplified product was (10-3) but the method was capable to detect TSWV in infected tissues till 10-4 dilution as shown above. M: Molecular weight DNA Marker XVI (Roche, Applied Science). Negative control (No cDNA) showing no PCR product.

7. Nucleotide sequencing analysis A DNA fragment of approximately 780 nucleotide in length, corresponding to the sequence of nucleocapsid (NPs) gene was amplified from TSWV tomato isolate, and partially sequenced from forward direction (Accession number DQ479968) by using CLIP Automated DNA sequencer as described under Materials and Methods. The Egyptian TSWV/NPs partial nucleotide sequence was

translated into the corresponding amino acid sequence and aligned with the amino acid sequences of eight published nucleoproteins from the genus Tospovirus. Tomato isolate NP amino acid sequence showed conservation of 81 % identity among the published isolates Fig. 19. Phylogenetic tree of the deduced amino acid sequence of (TSWV/NP-EG) (Fig 20) showed 81% sequence homology with the nucleoprotein amino acid sequences recorded under the accession numbers: AB088385, AB038341, AB038342, AJ242772, AY611529, Z36882, AB175809, and X94550.

Fig (19): Multiple sequence alignment of deduced amino acid sequences encoding the Nucleoprotein (NP) gene of TSWV-EG (DQ479968) isolate with that recorded in the gene bank with. Z36882: TSWV/NP gene (Italy) (Vaira et

al. 1995), AB038341: TSWV/NP gene (Kato and Hanada, 2000), AB038342: TSWV/NP gene Tospo-G isolate (Kato and Hanada, 2000); AB088385: TSWV/NP gene (Takeda et al., 2002); AB175809: TSWV/NP gene Korean isolate (Kim et al. 2004); AJ242774: TSWV/NP gene (isolate 873) (Roberts et al. 1999); AY611529: TSWV/NP gene (Thomas et al. 2004); and X94550: TSWV/NP gene (Guerra et al. 1995). The alignment was generated using DNAMAN V 5.2.9 package (Madison, Wisconsin, USA). Identical amino acid sequences are shown.

Fig (20): Phylogentic tree of the deduced amino acid sequences of TSWV/NP Tomato isolate (DQ479968) and published sequences of TSWV isolates recorded in gene bank under the accession numbers shown above. The Phylogenetic analysis was based on genetic distances (DNAMAN V 5.2.9) package, Madison, Wisconsin, USA). The NP of TSWV isolate under study has 81 % amino acid sequence homology with that published under the accession numbers: AB088385, AB038341, AB038342, AJ242772, AY611529, Z36882, AB175809, and X94550.

8. Expression of TSWV nucleocapsid gene in E.coli The nucleoprotein construct, pBAD-Topo-NP; i.e. NP cloned into the expression vector pBAD-Topo under pBAD promoter and the tight regulator AraC.

8. A. Rapid screening of small cultures Screening of small (3 ml) expression cultures was performed under denaturing conditions in order to identify the ~ 28 kDa polypeptide band representing 6xHis-tagged rNP. NiNTA affinity chromatography and SDS-PAGE analysis revealed the presence of a prominent band with an estimated molecular weight approximately 28 kDa in the total proteins isolated from induced cultures. Other major cell proteins were also present (Fig. 21).

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expressed in E.coli. Lane IC: crude preparation from induced bacteria culture expressing NPs (IC). E1 and E2: First and second Elution purified NP in fusion with 6x-His protein purified with NiNTA resin.

8. B. Purification of TSWV 6xHis-tagged rNP under denaturing conditions NiNTA batch chromatography was used to purify the 6xHistagged rNP from E.coli. The final yield of the recombinant NP was estimated as 5-7 mg/l culture, which corresponds to approximately 4.75% of the total proteins of E.coli (Fig. 22). SDS-PAGE and western blot analysis of the purified recombinant nucleoprotein (rNP) revealed the presence of a major protein with an estimated molecular mass ~ 28 kDa. The different aliquots (total cell lysates, flow – through (unbound), wash, and eluted rNP) collected during NiNTA batch chromatography were subjected to SDS-PAGE and western blot analysis as presented in Fig. 21 (A and B). The eluted 6xHistagged rNP (~28 kDa) protein was highly reactive with Indian TSWV/AS (diluted 1-4000). M

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V. DISCUSSION The current study aimed to identify and determine different biological and molecular characterization of tomato spotted wilt virus in Egypt (TSWV-EG). TSWV is the type species of the genus Tospovirus in the family Bunyaviridae, a large family of RNA viruses most of which infect vertebrate and/or invertebrate hosts. TSWV is one of the most destructive plant viruses infecting several hundred different species of plants. Many of these are important food and oil crops. As the name indicates, this virus does infect tomato, and causes serious damage to tomato crops. It is not, however, just a tomato virus. This conclusion was built on results of comparatively extensive experiments dealing with biological, serological, and molecular investigations on the virus under study as well as on different isolates of TSWV infecting some ornamental plants. TSWV-EG tomato isolate was mechanically transmitted from diseased to healthy tomato plants. Inoculated plants showed chlorotic and necrotic ringspots followed by systemic mosaic, bronzing or stunting on successive leaves. Symptoms appeared after 21 days post inoculation (Fig. A 1&2) were similar to those described by Reddy et al. (1992), De Angelis et al. (1994). In nature, TSWV is transmitted by at least eight species of thrips (Mound, 1996; Ullman, 1996; Groves et al., 2003), in a circulative and propagative manner (Wijkamp et al. 1993; Ullman et al. 1992). Recently, factors involved in the determination of vector competence have been identified in Frankliniella occidentalis and Thrips tabaci (Best, 1968; Ananthakrishnan, 1984; Nagata et al. 2002). The present results showed that immature Thrips tabaci insects were able to acquire and transmit TSWV from infected to healthy tomato seedlings under greenhouse conditions. This result was similar to the results obtained by Wijkamp et al. (1993) and

Wijkamp et al. (1995) who indicated that Thrips tabaci (the onion thrips) is one of the most frequently reported vector species that transmit TSWV in order to be spread in host plant populations. Tomato spotted wilt virus (TSWV) was found to infect a wide range of host plants that resemble those reported by many investigators (Reddy and Wightman, 1988; Berling et al. 1990; De Avila et al. 1992; Nuez et al. 1992; Schuster and Halliwell, 1994; Diez et al. 1999; Parrella et al. 2003). Plant species capable of being locally and/or systematically infected by TSWV shown in (Fig. 1) and (Table 1) belongs to 31 Genus and 16 Family including including Amaranthacea, Apiaceae, , Araceae, Asteracea, Brassicaceae, Chenopodiaceae, Convolvulaceae, Cucurbitaceae, Fabaceae, Geraniaceae, Malvaceae, Meliaceae, Poaceae, Primulaceae, Scrophulariaceae, and Solanaceae. The induced symptoms on the tested hosts ranged between mosaic, mottling, chlorotic spots, necrotic lesions, necrotic ring spots, concentric necrotic rings, yellowing or bronzing of leaves, and leaf drop or wilting. Leaf distortion and plant stunting were also observed. Systemic symptoms were generally more severe, including mosaic, mottling, chlorotic or necrotic ring spots, leaf deformation and stunting as reported by Hean (1940) and Best (1968). However, some differences in host reactions were also observed. For instances, no symptoms were observed with inoculated Cucurbita pepo L. nor with Hordium vulgaris. However, the expression of symptoms appeared on tested host plants may varied when compared with the previously described symptoms indicating some differences in host reactions, age, nutrition and particularly with environmental conditions, especially temperature. Petunia hybrida is one of the most useful diagnostic host plant (Allen and Matteoni, 1991) because of the rapidity with which the brown local lesions develop, within 2-4 days under favorable conditions, without systemic virus spread. Nicotiana tabacum, and N.

glutinosa show large local necrotic lesions followed by systemic mosaic and necrosis. Sap transmission of TSWV is usually efficient if young test plants are used. However, difficulties are sometimes encountered in transmitting the virus from old infected plants, and transmission efficiency can be greatly increased using neutral phosphate buffers containing a reducing agent (AlKhazindar, 1999). Thirty three host plants showing the characteristic symptoms of TSWV were selected for ELISA test Table 1. ELISA results were capable to identify the infected hosts by using Indian TSWV-AS From the above results, it was deduced that the virus under study is an Egyptian isolate of tomato spotted wilt virus. This result was ascertained since the virus under study reacted positively in indirect ELISA test. Stability of the isolated virus in sap indicated that, the thermal inactivation point (10 min) is between 45-50ºC and this result was in accordance with Best (1946) who stated that the thermal inactivation point of TWSV is 46±1. In contrary with our results, Bald and Samuel (1931) and Best (1961) showed that the thermal inactivation point of the virus is 41–42ºC. The dilution end point is 10-3 and longevity in vitro at room temperature was between 5 and 6 hours. Similar data were obtained by Adam and Kegler (1994) who mentioned that, the thermal inactivation point in crude plant sap is 40-50ºC (10 min), longevity in vitro were 2-5 at room temperature and dilution end point ranges between 2x10-2 and 10-4. An Egyptian polyclonal antibodies raised against viral nucleoprotein of TSWV expressed and purified from E.coli was locally produced during this study via recombinant protein expression technology as shown latter under the molecular studies (Part II). Pooled preparations of purified fusion nucleoprotein (denatured and native forms) (500 µg per injection) mixed with incomplete Freund's adjuvant (Difco Laboratories, Detroit, USA) and injected into a NewZealand rabbit intramuscularly for six times at weekly intervals

as reported by Varia et al. (1996). When the native nucleoprotein extracted from infected tomato plants examined against TSWV-AS-EG using DBIA (Fig. 3), the results indicated that the obtained antiserum can recognize the native form of nucleoprotein of TSWV at serial dilution of antigen till 10-4. Additionally, the absorbance values of the induced antisera (TSWV-ASEG) against the denatured form of the recombinant nucleoprotein (rNP) purified from E.coli (Table 5 & Fig. 4) indicated high specific reactivity of the Egyptian antiserum against denatured 6x-His recombinant NP. The results indicated that the obtained antiserum successfully recognized both forms of nucleoprotein of TSWV in a specific way. Purified γ-globulin (IgG) fraction of TSWV/NP antiserum had properties of protein spectrum with Amax at 280 nm and Amin at 250 nm, respectively. This IgG fraction was used in Dot-blot immunoassay (Fig. 5 A&B) and indirect-ELISA (Fig. 6). During the evaluation of locally produced TSWV antiserum by comparing its cross reactivity with that one from India using DBIA on different host plants infected with TSWV, it was found that the Egyptian antiserum was as sensitive as Indian TSWV/AS in the detection of TSWV in infected tissues (Fig. 5) However, each antiserum had its own specificity according to the isolate of the virus that raised against.

Egyptian TSWV/IgG was capable to detect TSWV in different sap extracts from Pelargonium spp., Tomato, Petunia, and Nicotiana rustica by using indirect ELISA till the dilution 1/6000 as shown in Fig. (6). However, the highest absorbance values were obtained with tomato and Pelargonium spp. respectively, when 1/1000 dilution of antibodies were used. While the highest absorbance values obtained with Petunia and TSWV/NP respectively, when 1/500 dilution of antibodies were used whereas the best antibody dilution which gave the highest O.D. value was 1/250 when N. rustica was used. The results obtained were in agreement with that reported by Varia et al. (1996). The appropriate end point dilution of infected sap extracts that can be used in indirect ELISA was detected reliably in crude sap of

the same previous plants (Fig. 7). The highest I/H peaking value was at 10-3 dilution. These data are in agreement with that reported by Varia et al. (1996). Theses results indicated that the Egyptian antiNP-serum could be used in the detection of TSWV in highly diluted extracts of different infected hosts, and also in leaf extracts or intact tissues stored for about one month in deep freezer which mean that the recombinant viral nucleoprotein (rNP) purified from E.coli remains antigenic for long periods. The virus under study was partially purified from Nicotiana rustica L of 21 days old, mechanically infected with TSWV (Tomato isolate) as described by Black et al. (1963). Electron microscopic examination of negatively stained partially purified preparations of the virus under test with uranyle acetate (Fig. 9) revealed the presence of quasi-spherical particles ranging from 80-100 nm in diameter. This result is in agreement with Black et al. (1963); Mohamed and Randles (1972); Honda et al. (1989) Adam et al. (1990) and De Angelis et al. (1994). The isolation of RNA for use in RT-PCR by the CTAB method (Gibbs and Mckenzi, 1997) was used. The protocol recommended to use a small samples of tissue (e.g., 5-100 mg), providing no detectable decrease in the quantity and/or quality of the RNA was observed as measured by spectrophotometric readings (A 260/280 was 1.3 µg/µl), and agarose gel electrophoresis (Fig. 10 A &B). When TSWV- infected tissues (Lycopersicum esculentum, Datura metel, and Pelargonium sp) were examined with this protocol (as described under Materials and Methods), some tissues (Pelargonium sp) didn't give optimal results due to the presence of some phenolic and/or polysaccharides compounds in the infected tissues. In cases where the results were less than optimal, another standard method (High pure RNA Tissue Extraction System (Roche) for RNA isolation) was used. In reverse transcription reaction, the results indicated the

successful isolation of undegraded RNA from TSWV infected tissues using both methods as well as the specific priming of efficient RT reactions. The nucleocapsid (NPs) protein gene was isolated by reverse transcription-polymerase chain reaction (RT-PCR) from total nucleic acid extracts from TSWV-infected plants and the primer pair specific for the NPs gene of the TSWV was used in order to amplify a PCR product of about 780 bp from the samples tested which was in agreement with the results reported by Whitfield et al. (2003) and Nervo et al. (2003). The specific primers TSW1 and TSW2 (Jain et al. 1998) were capable of detecting and nucleoprotein gene (NP) of (Lycopersicum esculentum Mill. the isolated NP gene sequence

amplifying about 780 bp of the TSWV isolated from Tomato Cv. Money Maker) indicating that was amplified from an authentic

isolate of TSWV-EG. The amplification of nucleoprotein DNA fragment from the nucleoprotein cistron allowed the identification of TSWV as definite species of the Tospoviruses group. The fragment covering the conserved part of the nucleoprotein was unique in size (~780 bp) and was amplified from all TSWV isolates. Priming of cDNA synthesis with the plus sense primer (TSW1) generated fragments of sufficient length to allow amplification of ~780 bp fragments indicating that the average length of cDNA molecules is about 1 Kb. Priming was therefore done with the plus sense primer TSW1 because the genomic RNA is negative sense RNA. On this study, not all tested host especially Datura metel and Nicotiana rustica gave amplification of the same fragment size, which suggested that either cDNA synthesis was incomplete or that primer annealing during PCR was suboptimal or due to the differences between the viral sequences in the tested samples (Fig. 11 B ). Southern hybridization techniques have been used to confirm the RT-PCR amplification of TSWV (Fig. 12) and it was shown to be

more sensitive and more specific than serological tests. Such positive results were in agreement with those obtained by Hahm et al. (1993). Hybridization using cDNA probes (Ronco et al., 1989) and riboprobes (Huguenot et al. 1990) were also applied to TSWV detection. DNA hybridization with digoxigenin-labeled probes was used for plant viruses other than Tospoviruses (James et al., 1999). In seeking a broad spectrum detection method, digoxigenin-labeled probes directed at the S, M and L RNAs should be developed. Obtained results with the NPs gene probe (780 bp fragment) were virus-specific and were in agreement with the results reported by De Ávila et al. (1990 and 1993b). Successful cloning of 780 bp of TSWV (Tomato isolate) was carried out by using the pBAD-Topo vector that is provided with T-overhang for direct cloning and expression of the PCR products. The PCR products obtained after purification from Agarose gel band using QiAquick Gel Extraction Kit (Qiagen) (Fig 13 A&B) were subsequently cloned into pBAD-Topo vector and used for the expression of the nucleoprotein gene of TSWV. In the presence of L-arabinose, expression from pBAD is turned on while the absence of L-arabinose produces very low levels of transcription from pBAD (Lee, 1980; Lee et al.1987). By varying the concentration of L-arabinose, protein expression levels can be optimized to ensure maximum expression of soluble protein. In addition, the tight regulation of pBAD by AraC is useful for expression of potentially toxic or essential genes (Carson et al. 1991; Dalbey and Wickner, 1985; Guzman et al., 1992; Russell et al., 1989; San Millan et al. 1989). The presence of 6xHis-tag at the N-terminus of the produced protein made the screening and verification of rNP expression more easier if the anti-6x His antibody was used in western blotting assays.

The nucleoprotein gene was cloned in frame with both the gene III secretion signal and the C-terminal peptide in order to guarantee the correct expression of recombinant nucleoproteins (Fig. 14). The initiation ATG of the secretion signal is correctly spaced from the optimized RBS to ensure optimal translation. The plasmids can be stored for long periods and the production of bacterial expressed viral proteins can be used as a good strategy to provide uniform immunogenes for antibody production as it avoids laborious

virus purification methods and the need for expensive equipments Chen et al. 2002. The NP gene constructs were verified by PCR and Southern hybridization after plasmid DNA preparation using tSW1 and tSW2 specific primer pair (Fig. 16 A & B). Such positive results of amplification by using primers specific for TSWV to verify the successful cloning of TSWV/NP gene from purified plasmids from E.coli indicated the successful cloning of the TSWV nucleoprotein gene. The percentage of TSWV infection on tomato, in this study, was not actually estimated since no real survey has been done to cover all fields cultivating tomato plants in Egypt. However, few samples collected from two fields of tomato belonging to Upper Egypt (El-Menia and El-Fayoum Governorates) were surveyed and tested using Dot-blot hybridization method. Results indicated that eight samples out of eighteen collected from Fayoum region gave positive signals and nine samples out of twelve collected from ElMenia region gave positive signals (Fig. 17). The total percentage of infection was 44.4% and 75% in Fayoum and El-Menia Governorate, respectively. Dot blot hybridization technique was used to detect TSWV in Lycopersicon esculentum showing necrosis and bronzing symptoms collected from Fayoum region and El-Menia region. And also to test the efficiency of TSWV/NP DNA probe in the detection TSWV in field survey. Successful hybridization with the infected tomato samples indicated the presence of TSWV in some infected tomato plants. On the other hand, the DNA probe gave no signals with extracts from healthy tissues as shown in Fig. (17). This level of sensitivity and easiness has been previously described for the detection of TSWV (Ronco et al. 1989; Rice et al. 1990; Huguenot et al. 1990). This non-radioactive hybridization technique considered as a good alternative technique for routine diagnosis of TSWV even in different hosts. It has been applied in a large scale analysis of apricot samples. When it was used in the nonisotopic molecular hybridization assay, it resulted in 10% more samples being scored as positive as when DAS-ELISA was used (Dominguez et al., 1997).

Immunocapture RT-PCR amplification is a method described for the detection of plant viral and subviral pathogens that combines immunocapture of the pathogen on a microtiter plat or eppendorf tube, reverse transcriptional – polymerase chain reaction, and agarose gel electrophoresis of the amplified products. Immunocapture RTPCR successfully detected TSWV (Tomato isolate) in infected tissues Fig. (18). The results obtained showed the sensitivity of IC-RT-PCR to detect TSWV in diluted cDNA until 10-4 dilution. Our results were in agreement with the Immunocapture PCR which has enabled amplification of the entire N gene from peanut tissue (Jain et al. 1997; Pappu et al. 1998b). The method is designed so that the whole procedure can be carried out in a microtiter plate or in the eppendorf tube, by the combination of its four main steps. It does not require any previous handling of pant tissue A partial nucleotide sequence of the NP gene of TSWV-EG (Accession number DQ479968) was compared to eight published Tospovirus sequences. Comparison of the partial nucleotide sequence of TSWV-EG showed an extensive conservation (~72.11 % identity) among the published isolates. However, the deduced amino acid sequence of the NP-EG showed 81% homology (Fig. 19 & 20) when amino acid sequence alignment was done with other eight amino acid sequences of Tospovirus published in the gene bank under the accession numbers Z36882: TSWV/NP gene (Italy) (Vaira et al. 1995), AB038341: TSWV/NP gene (Kato and Hanada, 2000), AB038342: TSWV/NP gene Tospo-G isolate (Kato and Hanada, 2000); AB088385: TSWV/NP gene (Takeda et al. 2002); AB17809: TSWV/NP gene Korean isolate (Kim et al. 2004);AJ242774: TSWV/NP gene (isolate 873) (Roberts et al. 1999); AY611529: TSWV/NP gene (Thomas et al. 2004); and X94550: TSWV/NP gene (Guerra et al. 1995). These results were in agreement with that reported by Vaira et al. (1996) who stated that the GRSV N protein has an amino acid sequence homology of about 77% with the N protein of TSWV (De Avila et al. 1993b). The amplified NP gene was cloned and expressed into E. coli expression vector (pBAD-Topo Expression vector, Invitrogen). The final construct that encodes the NP (in pBAD-Topo) is referred to as pBAD-NP. In this expression construct, NP was expressed in Escherichia coli BL21 (DE3) cells under a powerful pBAD promoter and the tight regulator AraC, recognized by E. coli RNA polymerase, and a double lac operator system inducible by L-arabinose.

The good level of expression of the recombinant 6x-His-Nprotein and stability during purification allowed producing enough immunogen for six injection cycles from a single 250 ml bacterial culture. The rNP expressed in E. coli appears to be near full length, residues (279 amino acid residues). SDS-PAGE analysis of rapid lysates from L-arabinose -induced cultures revealed a major band with the estimated molecular weight of 28 kD (Fig 21 A). This result was in agreement with that reported by Sherwood et al. (1989); Wang and Gonsalves (1990); Huguenot et al., (1990); Adam et al. (1990). The recombinant NP is approximately 1 kD shorter than the native NP isolated from plants (29 kD) because the rNP neither resemble the full length of the native NP (29 kDa) which contains the full amino acids residues (279 amino acids) at the carboxy terminus nor the conformational structure of the native protein (protein folding). SDS-PAGE analysis suggested that the recombinant NP accumulated in the bacterial cytoplasm. The insoluble fractions of E. coli lysates were separated by centrifugation and resolved by SDSPAGE (Fig 22 A). NP was detected in the insoluble fraction when TSWV polyclonal antibody (Indian Institute of science, India) was used (Fig 22 B). This result was consistent with that reported by Snippea et al. (2005). In this study, the purification of rNP was accomplished by using Ni-NTA affinity chromatography under denaturing conditions in order to maximize the recovery of rNP from the bacterial pellets. The E.coli expression system used in this study allows generating about 5-7 mg/l of recombinant NP (i.e. ~ 3% of the total protein). This value was similar to those reported by Snippea et al. (2005). SDS-PAGE and western blot analysis of the purified recombinant nucleoprotein (rNP) revealed the presence of a major protein subunit with an estimated molecular mass ~ 28 kDa. This was within the

values reported by Barbier et al. (1992); Varia et al. (1996); and Cortez et al. (2001). The different aliquots of nucleoproteins collected during NiNTA batch chromatography were subjected to SDS-PAGE and western blot analysis as presented in Fig. 22 (A and B). The eluted 6xHis-tagged rNP (~28 kDa) protein was highly reactive with Indian TSWV polyclonal antibody. In conclusion, the antibodies obtained in this study proved to have good specificity for TSWV-NP and were useful for biological characterization and field diagnosis. They recognized the N protein in western blots, in- DBIA and in different versions of ELISA, in laboratory and field samples, without needing preabsorption with healthy plant components. Since the immunogen was not prepared form plant material, there was no risk of injecting plant immunogens and raising antibodies to these. In contrast with antibodies obtained by immunizing with purified virus, these antibodies also present no risk of reaction with other viral structural proteins (the glycoproteins), present in whole virus preparations and sometimes also contaminating nucleocapsid preparations. The cost of producing a polyclonal antiserum is much lower than for monoclonals and any molecular biology laboratory could grow bacteria containing the appropriate expression plasmid and purify enough protein for immunization. With antisera obtained from a few bleeding it is possible to prepare kits for about one million test. The method is therefore suitable for laboratories without virus facilities or without the license to propagate a foreign virus. Finally, the bacterial strain is more easily stored than a viable virus isolate. All these considerations are relevant in developing countries, which might need to prepare low – cost antibodies to economically important viruses.

VI. SUMMARY The isolated virus was identified as tomato spotted wilt virus on the basis of biological, serological and molecular means. The results can be summarized as follows:

Part (1) 1 . isolation and identification 1.1.Virus isolation TSWV was isolated from tomato fruits exhibiting symptoms suspected to be due to TSWV infection from the experimental farm, Faculty of Agriculture, Ain Shams University, Kalubia Governorate. The causal virus induced thickening of young tomato leaves, especially the veins, sometimes accompanied by concentric rings.

Leaves tended to curl downwards and inwards and started bronzing. The later stages of the diseased leaves showed yellowish mosaic mottling and distortion. Fruit symptoms often appeared as pale or yellow areas in the normal red skin of the ripe tomato fruits. The virus was biologically purified through several passages on local lesion hosts of Petunia hybrida (which produce local lesions after 2-4 days post inoculation), Datura metel, and Nicotiana tabacum cv. Kentuckey and was mechanically transmitted onto tomato plants for virus propagation. 1 . 2 virus identification 1.2. 1 Virus transmission: A . Mechanical transmission: TSWV-EG was easily mechanically transmitted thirty three plants belonging to sixteen families in the greenhouse by using solvent buffer 4. B . Insect transmission (Thrips transmission): Trial made for transmitting TSWV-EG by Thrips tabaci and the results found that the insects were able to transmit the virus from infected to healthy tomato seedlings after 14-21 days post inoculation. Fourteen out of twenty tomato plants (70%) showed symptoms of TSWV infection after insect inoculation. 1.2. 2 Host range studies Host range studies revealed that TSWV-EG had a wide host range. It infects at least thirty three plants belonging to 31 Genus and sixteen families. Infectivity was verified by indirect ELISA test. 1.2. 3 .Virus stability Results indicated that the virus is sap transmissible. Its thermal inactivation point is 45-50 ºC; the dilution endpoint is 10-3 and the virus was completely inactivated after incubation for 5-6 hours at room.

1. 2. 4 Serological studies A. indirect ELSA The mechanically infected host range that displaying typical symptoms of TSWV were checked by in-direct ELISA during this study. B. Dot – blot Immunoassay (DBIA) Naturally infected reservoir plants collected from the experimental farm, Fac. of Agric, Cairo Univ. that displaying symptoms looks like that occurred by TSWV were confirmed positive to TSWV by using DBIA. 1.2. 5. RT-PCR The PCR products amplified from mechanically infected Lycopersicon esculentum confirmed that the amplified amplicon of the expected size (~780 bp) was authentic sequence of TSWV nucleoprotein gene.

1.2 .6 . Electron microscopy Electron microscopic examination of negatively stained partially purified preparations of the virus under test revealed the presence of quasi-spherical particles ranging from 80-100 nm in diameter.

Part (2( Molecular Studies: A . Production of TSWV antiserum 1. RNA extraction Two methods were used successfully in the isolation of total RNA from infected tissues; the High Pure RNA Tissue Kit and CTAB method. The integrity and quantity of the purified RNA were confirmed by gel electrophoresis and UV spectrophotometer

2. RT-PCR After reverse transcription reaction using Retrotools Reverse Transcriptase enzyme, the two oligonucleotides TSW1 and TSW2 designed by Kato and Hanada (2003) were able to amplify a fragment of about 780 bp from nucleoprotein gene (NP) S-RNA from all infected tissues however, the PCR products amplified from mechanically infected Datura metel and Nicotiana rustica as differential hosts, the size of the amplified PCR products were less than the expected one (~600 bp). ). The authenticity of the resulting PCR product (~780 bp) was verified by Southern hybridization and direct DNA sequencing. 3 . Cloning The PCR products of TSWV nucleoprotein gene (NP) was directly cloned into prokaryotic expression vector pBAD-Topo. The ligation reaction was transformed into competent E. coli BL 21- DE3 cells. The recombinant plasmids containing the NP gene of TSWVEG was isolated by plasmid mini-prep in the screening experiment. 4 . Validation of Cloning by PCR The recombinant plasmids carrying the NP gene (780 bp) were validated using the two primers TSW1 and TSW2 in the PCR. Theses results were verified with Southern blot hybridization and the selected recombinant clones were used for the protein expression. 5 . Expression of TSWV nucleocapsid gene in E.coli The NP gene cloned into the expression vector pBAD-Topo under pBAD promoter and the tight regulator AraC was expressed in BL 21 DE3 E.coli strain using L-arabinose to ensure maximum expression of soluble protein and to produce homogeneous and unlimited amount of expressed fusion nucleoprotein to be used as a strong immunogen in the immunization experiment for polyclonal antibody production. These steps were as follows:

6. Purification of fusion nucleoprotein under denaturing conditions NiNTA batch chromatography was used to purify the 6xHistagged rNP from E.coli. SDS-PAGE and western blot analysis of the purified recombinant nucleoprotein (rNP) revealed the presence of a major protein with an estimated molecular mass ~ 28 kDa. The fusion nucleoprotein was highly reactive with Indian TSWV antiserum. 7. Reactivity of TSWV nucleoprotein (NP) against Indian antiserum The results indicated that the obtained recombinant nucleoprotein of TSWV successfully reacted with Indian TSWV antiserum under native conditions. 8. Production of TSWV-EG antiserum Polyclonal antibodies produced against TSWV-EG nucleoprotein (NP) by injecting pooled purified native and denatured fusion nucleoprotein into a Newzealand rabbit was successfully used in serologic studies indicated high specific reactivity of the Egyptian antiserum when examined with native nucleoprotein extracted from infected tomato plants using DBIA and also reacted specifically with denatured 6x-His recombinant NP (expressed and purified from E.coli) in-direct ELISA at dilution 1-1000. 9. Determination of TSWV antiserum titer The best titer of TSWV/AS used in DBIA was at dilution 11000 to detect the native protein of the virus in the infected samples. On the other hand, TSWV-AS was able to detect the denatured nucleoprotein till dilution 1- 4000 in indirect ELISA. 10. Purification of TSWV/IgG Purification of IgG fraction of TSWV/NP antiserum was done by using caprylic acid method and the results showed that the purified

IgG had Amax at 280 nm and Amin at 250 nm. 11. Titration of TSWV/IgG/EG and its reactivity with different infected tissues Titer of TSWV/IgG-EG was measured by indirect plate trapping ELISA. Egyptian TSWV/NP IgG was capable to detect TSWV in different sap extracts till 1/4000 dilution. However, the best dilution of TSWV/IgG/EG that can be used in detection was 1/1000 dilution. 12. Determination of dilution end-point of TSWV infected sap TSWV/IgG-EG was used to detect the appropriate dilution of infected sap extract that can be used in indirect ELISA. The virus was detected reliably in crude sap of all infected plants at dilutions between 10-2 and 10-3 when 1-1000 dilution of Egyptian IgG was used.

13. Evaluation of the Egyptian TSWV/NP antiserum by dot – blot Immunoassay (DBIA) TSWV was detected in thirty tested hosts by DBIA test using the Egyptian TSWV/NP IgG. The results indicated the high reactivity and specificity of both Egyptian TSWV/ IgG and Indian TSWV/AS in detecting TSWV in infected crude saps. B. Molecular characterization of TSWV 1. Automated DNA sequencing: Direct DNA sequencing of the PCR product was performed using CLIP Automated DNA sequencer (Plant Pathology Research Institute, sequence published translated

Agriculture Research Center). The Partial nucleotide of the nucleoprotein gene submitted to gene bank and at NCBI under the Accession number DQ479968 was into its deduced amino acids and aligned with eight

sequences of TSWV/NP published in the gene bank which showed conservation of 81 % identity among the published isolates. 2. Phylogentic tree Phylogenetic tree of the obtained amino acid sequences (Accession number DQ479968) showed also conservation of 81 % identity among the published isolates. The tree analysis was done using the software (DNAMAN V 5.2.9 package, Madison, Wisconsin, USA).

VII. REFERENCES Abd-El Nazeir, N. 1999. Isolation, characterization and preservation of some plant viruses. M.Sc. Thesis, Faculty of Agriculture, Ain Shams University. 85pp. Abdel-Monem, E. M. 2004. Arab Republic of Egypt, Ministry of Agriculture and Land Reclamation, Economic Affairs Sectors. Agriculture Statistics, Summer and Nili Crops Book part 2: 218-262 pp. Adam, G. and Keglar, H. 1994. Tomato spotted wilt virus and related tospoviruses. Archives of Phytopathology and Plant Protection 28: 483-504. Adam, G.; Lesemann, D.E. and Vetten, H.J. 1990 . Monoclonal antibodies against tomato spotted wilt virus: characterization and application. Annals of Applied Biology 118: 87-104. Adam, G.; Yeh, S.D.; Reddy, D.V.R. and Green, S.K. 1993. Serological comparison of Tospovirus isolates from Taiwan and India with impatiens necrotic spot virus and different tomato spotted wilt isolates. Arch. Virol. 130: 237-250. Adkins, S.; Quadt, R.; Choi, T. J.; Ahlquist, P.; and German, T. L. 1995. An RNA-dependent RNA polymerase activity associated with virions of tomato spotted wilt virus, a plantand insect-infecting bunyavirus. Virology 207:308-311. Alexandre, M.A.V.; Guzzo, S.D.; Harakava, R.; Noronha, A. B. 1997. Partial purification of a virus inhibitor from Silene schafta leaves. Fitopatol. Brazil. 22 (2): 171-177. AlKhazindar, M. 1999 . Studies on a Virus Naturally Infecting Physalis Peruviana. M.Sc. Thesis, Faculty of Science, Cairo University. 225pp.

Allen, W. R. and Broadbent, A. B. 1986.. Transmission of tomato spotted wilt virus in Ontario Canada greenhouses by Frankliniella occidentalis. Can. J. Plant Pathol. 9:33-38. Allen, W.R. and Matteoni, J.A. 1991. Petunia as an indicator plant for use by growers to monitor for thrips carrying the tomato spotted wilt virus in greenhouses. Plant Disease 75: 78-82. Ananthakrishnan, T.N. 1984. Bioecology of Thrips. Indira, Oak Park, Michigan, 233 pp. Aramburu, J. and Marti, M. 2003. The occurrence in north-east Spain of a variant of Tomato spotted wilt virus (TSWV) that breaks

resistance in tomato (Lycopersicon esculentum) containing the Sw-5 gene. Plant Pathology 52: 407. Baker, J. R. and Jones. R. K. 1988. Update on western flower thrips and tomato spotted wilt virus. N. C., Flower Growers Bulletin 33 (2):6-9. Bald, J. G. and Samuel, G. 1931. Investigation on “spotted wilt” of tomatoes. II. Austr. Counc. Sci. & Indus. Res. Bull. 54. 24pp . Barbier, P.; Takahashi, M.; Nakamura, I.; Toriyama, S. and Ishihama, A. 1992. Solubilization and promoter analysis of RNA polymerase from rice stripe virus. Journal of Virology 66: 6171-6174. Bellardi, M.G. and Vicchi V. 1990. TSWV: una nuova insidia per la produzione agricola italiana. Informatore Fitopatologico 40 (3): 17. Berling A.; Llanas-Bousquet, W.; Malezieux, S. and Gebre Selassie, K. 1990. Tomato spotted wilt virus. Connaître le problème pour enrayer l'épidémie. Phytoma no. 422: 46-50. Best, R. J. 1946. Thermal inactivation of tomato spotted wilt virus. Part 1. Aust. J. Exp. Biol. 24:21-25. Best, R. J. 1961. On maintaining the infectivity of a labile plant virus by storage at -69°C. Virol. 14:440-443.

Best, R. J. 1968. Tomato spotted wilt virus. Advances in Virus Research 13: 65-146. Best, R. J. and Palk, B.A. 1964. Electron microscopy of strain of tomato spotted wilt virus and comments on its probable synthesis. Virol. 23: 445-460 Best, R.J. and Gallus, H.P. 1955. Further evidence for the transfer of character-determinants (recombination) between strains of tomato spotted wilt virus. Enzymologia. 15;17(4):207-21. Bidari, V. B. and Reddy, H. R. 1985. Preservation and storage of chilli viruses . Indian phytopathology 38 (3): 488 – 491. Bitterlich I. and MacDonald L.S. 1993. The prevalance of tomato spotted wilt virus in weeds and crops in southwestern British Columbia. Canadian Plant Disease Survey 73: 137-142. Black, L.M.; Brakke, M.K. and Vatter, A.E. 1963. Purification and electron microscopy of tomato spotted-wilt virus. Virology. 20:120-30. Boiteux L.S., and Nagata T. 1993. Susceptibility of Capsicum Chinese PI159236 to tomato spotted wilt virus isolates in Brazil. Plant Disease 77: (2) 210. Branch, W.D. and S.M. Fletcher. 2001. No-pesticide preliminary yield trials in peanut. Peanut Sci. 28:21–24. Brittlebank, C.C. 1919. Tomato diseases. Journal of Agriculture, Victoria 27:231-235. Brodsgaard, H.F. 1989. Frankliniella occidentalis (Thysanoptera; Thripidae) a new pest in Danish glasshouses. A review. Tidsskrift Planteavl. 93:83-91. Brown, S.; J.W. Todd; A.K. Culbreath; J. Baldwin; J. Beasley and H.R. Pappu. 1998. Tomato spotted wilt virus risk index. University of Georgia, Extension Bulletin No. 1165, 12 pp. Carson, M. J.; Barondess, J.J. and Beckwith, J. 1991. The FtsQ protein of E.coli: membrane topology, abundance, and cell

division phenotypes due to overproduction and insertion mutations. J. Bacteriol. 173: 2187-2195. Converse, R.H. and Martin, R.R. 1990. ELISA methods for plant viruses. pp 179-196, in: Serological Methods for Detection and Identification of Viral and Bacterial Plant Pathogens. R. Hampton, E. Ball, and S. De- Boer, (eds.), American Phytopathological Society, St. Paul, MN. Cortez I, Saaijer J, Wongjkaew KS, Pereira AM, Goldbach R, Peters D, Kormelink R. 2001. Identification and characterization of a novel tospovirus species using a new RTPCR approach. Arch Virol. 146(2):265-278 Cortez, I., Pereira, A., Goldbach, R., Peters, D. and Kormelink R. 1999. RT-PCR procedure to amplify S RNA sequences of iris yellow spot virus, a distinct tospovirus. Petria 9:187-190. Cuadrado, I.M.; Juan, E.; Moreno, R.; and Sez, E. 1991. Detecciel virus del bronceado del tomate (TSWV) en cultivos de pimiento y tomate bajo invernadero en el poniente Almeriense. Estudios de fitopatologia 216-221. Culbreath, A.K.; Todd, J.W.; and Brown, S.L. 2003. Epidemiology and management of tomato spotted wilt in peanut. Annu Rev Phytopathol. 41:53-75. Chen, Y.K.; Prins, M.W.; Langeveld, S.A. and Goldbach, R. 2002. Alstroemeria-infecting Cucumber mosaic virus isolates contain additional sequences in their RNA3 segments. Acta Horticulture 568:93-102 Cho J. J.; Mau R.F.L.; Gonsalves D. and Mitchell W.C. 1986. Reservoir weed hosts of tomato spotted wilt virus. Plant Disease 70:1014-1017. Cho J. J., Mau R.F.L., Hamasaki R.T. and Gonsalves D. 1998. Detection of tomato spotted wilt virus in individual thrips by enzyme-linked immunosorbent assay. Phytopathology 78: 1348-1352.

Cho, J. J.; Mau, R.F.L.; Mitchell, W.C.; Gonsalves, D. and Yudin, L.S. 1987. Host list of plants susceptible to tomato spotted wilt virus (TSWV) Research Extension Series, Hawaii, 10 pp. Da Graca-J. V; Trench, T. N and Martin, M. M. 1985. Tomato spotted wilt virus in commercial Cape gooseberry (Physalis peruviana) in Transkei. Plant Pathology. 34 (3): 451-453 Dalbey, R. E., and Wickner, W. 1985. Leader Peptidase catalyzes the release of exported proteins from the outer surface of the Escherichia coli plasma membrane. J. Biol. Chem. 260:1592515931. De Angelis, J. D.; Sether, D. M. and Rossignol, P. A. 1994. Transmission of impatiens necrotic spot virus in peppermint by western thrips (Thysanoptera: Thripidae). J. Econ. Entomol. 87 (1): 197-201. De Avila A. C.; Huguenot, C.; Resende, R.D.; Kitajima, R., Resende, R. de O., Goldbach, R., and Peters, D. 1992. Characterization of a distinct isolate of tomato spotted wilt virus (TSWV) from Impatiens sp. in the Netherlands. J. Phytopathol. 134: 133–151. De Avila, A. C.; De Haan, P.; Kitajima, E.W.; Kitajima, E.W.; Goldbach, R. W. and Peters, D. 1990. Serological differentiation of 20 isolates of tomato spotted wilt virus. J. gen. Virol. 71: 2801-2807. De Avila, A. C.; Pena, L.; Kitajima, E. W.; de Resende, R.; Diaz Mugica, M. V.; Diaz Ruiz, J. R. and Peters, D. 1991. Characterization of tomato spotted wilt virus (TSWV), isolated from Capsicum annuum L. in the Canary Islands. Phytopathol. Mediterr. 30 (1): 23-28. De Ávila, A.C.; De Haan, P.; Kormelink, R.; Resende, R.O.; Kitajima, E.W.; Goldbach, R.W.; and Peters, D. 1993a.

Classification of tospoviruses based on phylogeny of nucleoprotein gene sequences. J. gen. Virol. 74:153-159. De Ávila, A.C.; De Haan, P.; Smeete, M.L.L.; Resende, R.O.; Kormelink, R.; Kitajima, E.W.; Goldbach, R.W.; and Peters, D. 1993b. Distinct levels of relationship between tospovirus isolates. Arch. of Virol.128:211-227. De Haan, P.; De Avila, A. C.; Kormelink, R.; Westerbroek, A.; Gielen, J. J. L.;Peters, D., and Goldbach, R. 1992. The nucleotide sequence of the S-RNA of Impatiens necrotic spot virus, a novel Tospovirus. FEBS Lett. 306: 27–32. De Haan, P.; Kormelink, R.; de Oliveira Resende, R.; van Poelwijk, F.; Peters, D. and Goldbach, R. 1991. Tomato spotted wilt virus L RNA encodes a putative RNA polymerase. J. Gen. Virol. 72:2207–2216. Dewey, R.A.; Semorile, L.C.; and Grau, O. 1996. Detection of Tospovirus species by RT-PCR of the N-gene and restriction enzyme digestions of the products. J. Virol. Methods 56, 19– 26. Dewey, R.A.; Semorile, L.C.; Grau, O.; and Crisci, J.V. 1997. Cladistic analysis of Tospovirus using molecular characters. Mol Phylogenet Evol. 8(1):11-32. Diez, M.J. Rosello, S., Nuez, F.; Costa, J.; Lacasa, A.; and Catala, M.S. 1999. Afalfa mosaic virus RNAs serve as cap donors for tomato spotted wilt virus transcription during coinfection of Nicotiana benthamiana. J. Virol. 73, 5172-5175. Dominguez, S; Aparicio, F.; Sanchez-Navarro, J. A.; Cano, A.; Garcia-Bruntom, J. and Pallas, V. 1997. Studies on the incidence of Ilarviruses and Apple chlorotic leaf spot virus (ACLSV) in apricot trees in the Murcia Region (Spain) using serological and molecular hybridization methods. 17th International Symposium on Virus and Virus like Disease of Temperate Fruit Crops, Bethesda, MD, USA.

Edwarson, J.R. and Christie, R.G. 1986. Tomato spotted wilt virus. Viruses infecting forage legumes, Vol. III. Florida Agricultural Experiment Stations Monograph Series no. 14, 563-579 El-Sayed, E.T.A.; Habib, H.; Ismail, M. and AlKhazindar, M.M. 2003. Identification of a virus naturally infecting Physalis peruviana. Egypt J. Biotechnol. 13: 328-343. EPPO/CABI, 1997. Tomato spotted wilt tospovirus In: Quarantine Pests for Europe (2nd ed), pp. 1379-1387. CAB International, Wallingford (GB). Francki, R.I.B.; Fauquet, C.M.; Knudson, D.L. and Brown, F. 1991. Classification and nomenclature of viruses: Fifth report of the International Committee on Taxonomy of Viruses. Arch. Virol. Suppl.2 Fukuta, S.; Ohishi, K.; Yoshida, K.; Mizukami, Y.; Ishida, A.; and Kanbe, M. 2004. Development of immunocapture reverse transcription loop-mediated isothermal amplification for the detection of tomato spotted wilt virus from chrysanthemum. J Virol Methods. 121(1):49-55. German, T.L.; Ullman, D.E. and Moyer, J.W. 1992. Tospoviruses: diagnosis, molecular biology, phylogeny and vector relationships. Annu. Rev. Phytopathol. 30: 315-348. Gibbs, A. and Mackenzie, A. 1997. A primer pair for amplifying part of the genome of all potyvirids by RT-PCR. J. Virol. Methods. 63:9-16. Goldbach, R. and Kuo, G. 1996. Introduction (Tospoviruses and Thrips). Acta Horticulturae, 431, 21-26. Goldbach, R. and Peters, D. 1996. Molecular and biological aspects of tospoviruses. In Elliot, ZR.M. (Ed). The Bunyaviridae, ed. Elliott, R. M. (Plenum, New York), pp. 129–157.

Gonsalves, D. and Trujitto, E. E. 1986. Tomato spotted wilt virus in papaya and detection of the virus by ELISA. Plant Dis. 70:501-506. Green, J.L.; Allen, T.C. and Fischer, S. 1988. Symptoms of stephanotis infected with tomato spotted wilt virus. Ornamentals Northwest Newsletter 12:10-11. Groves, R.L.; Walgenbach, J.F.; Moyer, J.W. and Kennedy, G.G. 2003. Seasonal dispersal patterns of Frankliniella fusca (Thysanoptera: Thripidae) and tomato spotted wilt virus occurrence in central and eastern North Carolina. J Econ Entomol. 96(1):1-11. Guerra-Sanz, J. M.; De Both, M. and Cuadrado, I. M. 1995 . Tomato spotted wilt virus nucleoprotein gene. National Center for Biotechnology Information (www.ncbi.nih.gov/index.html), Sequence Viewer v2.0, GenBank Accession number: X94550 (VERSION X94550.1). Guzman, L.M.; Barondess, J. J. and Beckwith, J. 1992. FtsL, an Essential Cytoplasmic Membrane Protein Involved in Cell Division in Escherichia coli. J. Bacteriol. 174:7716-7728. Hahm, Y.I.; Choi, K.S.; Kim, Y.S.; Choi, J.K.; Bae, S.W. and Hwang, Y.S. 1993. Hybridization detection of potato viruses using digoxigenin-labeled RNA probe. RDA-Journal of Agricultural Science 35:239-244. Halliwell, R. S. and Philley, G. 1974. Spotted wilt of peanut in Texas. Plant Dis. Rep. 58:23-25. Hanahan, D. and Meselson, A. 1983. Studies on transformation of Echerichia coli with plasmids. J. Mol. Biol. 166:557. Harlow, E. and Lane, D. 1988. In Antibodies, A laboratory Manual, Cold Spring Harber Laboratory press. 649pp. Hassan, S. 1995 . Investigation on virus diseases of tomato in Malak and Pakistan. Sarchad – Journal of Agriculture 11 (No .1): 89 – 96.

Hean, A. F. 1940 . Kromnek in South Africa. Its host range and distribution. Emg S. Afr.X V, 388-390. Heinze C., Letschert B., Hristova D., Yankulova M., Kauadjouor O., Willingmann P., Atanassov A., and Adam, G. 2001. Variability of the N-protein and the intergenic region of the S RNA of tomato spotted wilt Tospovirus. Microbiologica 24: 175-187. Henson, J.M. and French, R. 1993. The polymerase chain reaction and plant disease diagnosis. Annu. Rev. Phytopathol. 31: 81-109. Hobbs H.A., Black L.L., Johnson R.R, and Valverde R.A. 1994. Differences in reactions among tomato spotted wilt virus isolates to three resistant Capsicum chinense lines. Plant Disease 78 (12):1220. Holmes, F. O. 1946. Acomparison of the experimental host ranges of Tobacco-etch and tobacco-mosaic virus, Phytopathology 36: 643-659. Honda,Y.; Kameya-Iwaki, M.; Hanada, K.; Tochihara, H. and Tokashiki, I. 1989. Occurrence of Tomato spotted wilt virus in Watermelon in Japan. Techn. Bull. ASPAC Food & Fertilizer Techn. Center 114: 14-19. Hsu, T.H. and Lawson, H.R. 1991. Plant Interaction of Tomato Spotted Wilt Virus. Proceedings USDA Workshop, United States Department Agriculture Research and Service ARS-87; Beltsville, Maryland 1-14. Huguenot, C.; Van Den Dobbelsteen, G.; De Haan, P.; Wagemakers, C.A.; Drost, G.A.; Osterhaus, A.D. and Peters, D. 1990 . Detection of tomato spotted wilt virus using monoclonal antibodies and riboprobes. Archives of Virology 110: 47-62. Hull, R. 2002. Matthews' Plant Virology. Fourth edition, Academic Press , UK. ISBN: 0-12-361160-1. pp. 1001

Ie, T. S. 1971. Electron microscopy of developmental stages of tomato spotted wilt virus in plant cells. Virology. 43(2):468-79. Inoue, T.; Sakurai, T.; Murai, T.; and Maeda, T. 2004. Specificity of accumulation and transmission of tomato spotted wilt virus (TSWV) in two genera, Frankliniella and Thrips (Thysanoptera: Thripidae). Bull Entomol Res. 94 (6):501-7. Jacquet, C; Delecollle, B.; Raccah, B.; Lecoq, H.; Dunez, J.; and Ravelonandro, M. 1998. Use of modified plumpox virus coat protein genes developed to limit heteroencapsidation associated risks in transgenic plants. J. gen. Virol. 9:1509-1517. Jain, R.K.; Pappu, H.R.; Pappu, S.S.; Reddy, M.K. and Vani A. 1998. Watermelon bud necrosis tospovirus is a distinct virus species belonging to serogroup IV. Arch Virol. 143(8):1637-44. Jain, R.K.; Pappu, S.S.; Pappu, H.R.; Culbreath, A.K. and Todd, J.W. 1997. Detection of tomato spotted wilt tospovirus infection of peanut by immunocapture RT-PCR. Int. Arachis Newslett. 17, 38–39. James, D.; Jelkmann, W. and Upton, C. 1999. Specific detection of Cherry Mottle Leaf Virus using Digoxigenin-labeled cDNA probes and RT-PCR. Plant Disease 83:235-239. Jan, F. J.; Fagoaga, C.; Pang, S.Z.; and Gonsalves, D. 2000. A minimum length of N gene sequence in transgenic plants is required for RNA-mediated tospovirus resistance J. gen. Virol. 81:235–242. Jenser, G.; Gaborjanyi, R.; Vasdinnyei, R.; Almasi, A. and Kuo, G.G. 1996. Tospovirus infections in Hungary. Acta Horticulture 431: 51-57. Jorda, C.; Ortega, A. and Juarez, M. 1995 . New hosts of tomato spotted wilt virus. Plant Disease 79: 538. Kaminska, M. and Korbin, M. 1994. New natural hosts of tomato spotted wilt virus. Acta Horticulturae 377: 123-128.

Kato, K. and Hanada, K. 2000. A necrotic disease of chrysanthemum caused by Tomata spotted wilt virus in Japan. Ho Kyushu Byogaichu Kenkyukai 46:61-65. Kim, J. H.; Choi, G.S.; Kim, J.S. and Choi, J.K. 2004. Tospovirus Infecting Paprika in Korea. National Center for Biotechnology Information (www.ncbi.nih.gov/index.html), Sequence Viewer v2.0, GenBank Accession number: AB175809 (VERSION AB175809.1). Kitajima, E.W. 1965. Electron microscopy of Vira-Cabeca virus (Brazilian tomato spotted wilt virus) within the host cell. Virology. 26:89-99. Kitajima, E.W.; De Ávila, A.C.; Resende, R.O.; Goldbach, R.W. and Peters, D. 1992. Immuno-electron microscopical detection of tomato spotted wilt virus and its nucleocapsids in crude plant extracts. Journal of Virological Methods 38:313-322. Kormelink, R.; De Haan, P.; Meurs, C.; Peters, D. and Goldbach, R. 1992a. The nucleotide sequence of the M RNA segment of tomato spotted wilt virus, a bunyavirus with two ambisense RNA segments. J. Gen. Virol. 73:2795-2804. Kovalenko, O.H. and Shepelevych, V.V. 2004. Pathogenesis and induced virus resistance in tobacco plants affected by tomato spotted wilt virus. Mikrobiol Z. 66(2):81-85. Kritzman, A.; Gera, A.; Raccah, B.; van Lent, J. W. M.; and Peters, D. 2002. The route of tomato spotted wilt virus inside the thrips body in relation to transmission efficiency. SpringerVerlag Wien . 147 (11): 2143 – 2156. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 Latham, L.J. and Jones, R.A.C. 1998. Selection of resistance breaking strains of Tomato spotted wilt tospovirus. Annals of Applied Biology 133: 385-402.

Law, M. D. and Moyer, J.W. 1990. A tomato spotted wilt-like virus with a serologically distinct N protein. J. gen. Virol. 71, 933-938. Law, M. D.; Speck, J.; and Moyer, J. W. 1991. Nucleotide sequence of the 3' non-coding region and N gene of the S RNA of a serologically distinct tospovirus. J. gen. Virol. 72: 25972601. Lee, N. 1980. Molecular Aspects of ara Regulation. In The Operon, J. H. Miller and W. S. Reznikoff, eds. (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory), pp. 389-410. Lee, N.; Francklyn, C. and Hamilton, E. P. 1987. ArabinoseInduced Binding of AraC Protein to araI2 Activates the araBAD Operon Promoter. Proc. Natl. Acad. Sci. USA 84:8814-8818. Loebenstein, G. and Akad, F. 1997. Improved detection of potato leaf roll luteoviruses in leaves and tubers with a digoxigeninlabeled cRNA probe. Plant Disease 81: 489-491. Maiss, E.; Ivanova, L.; Breyel, E.; and Adam, G. 1991. Cloning and sequencing of the S RNA from a Bulgarian isolate of tomato spotted wilt virus. J. gen. Virol. 72, 461-464. Marchoux, G.; Gebre-Selassie, K. and Villevielle, M. 1991. Detection of tomato spotted wilt virus and transmission by Frankliniella occidentalis in France. Plant Pathology 40:347-351. Martin, M. M. 1964. Purification and electron microscope studies of tomato spotted wilt virus (TSWV) from tomato. Virology. 22:645-648. Mason G.; Roggero P. and Tavella L. 2003. Detection of Tomato spotted wilt virus in its vector Frankliniella occidentalis by reverse transcription-polymerase chain reaction. J. Virol. Methods. 109(1):69-73.

Matteoni, J.A. and Allen, W.R. 1989. Symptomatology of tomato spotted wilt virus infection in florist's Chrysanthemum. Can. J. Plant Pathol. 11:373-383. Mckinney, M. M. and Parkinson, A. 1987. A simple, nonchromatographic procedure to purify immunoglobulins from serum and ascites fluid. J. Immunol. Methods. 96: 271-278 Milne, R. G. 1970. An electron microscope study of tomato spotted wilt virus in sections of infected cells and in negative stain preparations. J. Gen. Virology 6: 267-276. Mohamed, N. A. and Randles, J.W. 1972. Effect of tomato spotted wilt virus on ribosomes, RNA and Fraction I protein in Nicotiana tabacum leaves. Physiol. Plant Parthol. 2:235-245. Mohamed, N.A. 1981. Isolation and characterization of subviral structures from tomato spotted wilt virus. J. Gen. Virol. 53: 197−206. Mohammadi, M.L; Esmaeeli-far, A.; Zad, J.; Mossahebi, G. and Okhovat, M. 2000. Serological detection and symptomology of tomato spotted wilt virus in Tehran Province. Irananian Journal of Agricultural Science Technology. 2: 107-117 Mortiz, G.; Kumm, S. and Mound L. 2004. Tospovirus transmission depends on thrips ontogeny. Virus Res. 100:143–149 Mound, L.A. 1996. The Thysanoptera vector species of Tospovirus. Acta Hort. 431: 298-307. Moyer, J. W. 1999 . Tospoviruses. Encyclopedia of Microbiology 4:592-597. Mumford, R.A.; Barker, I. and Wood K.R. 1994. The detection tomato spotted wilt virus using the polymerase chain reaction. Journal of Virological Methods 46: 303-311. Mumford, R.A.; Barker, I.; and Wood, K.R. 1996 . An improved method for the detection of Tospoviruses using the polymerase chain reaction. Journal of Virological Methods, 57, 109-115.

Nagata, T.; Inoue-Nagata, A.K.; van Lent, J.; Goldbach, R. and Peters, D. 2002. Factors determining vector competence and specificity for transmission of Tomato spotted wilt virus. J. gen Virol. 83 (3):663-71 Nervo, G.; Cirillo, C.; Accotto, G.P. and Vaira, A.M. 2003 . Characterisation of two tomato lines highly resistant to tomato spotted wilt virus following transformation with the viral nucleoprotein gene. JPP:5 (3). Nolasco, G.; de Blas, C.; Torres, V. and Ponz, F. 1993. A method combining immunocapture and PCR amplification in a microtiter plate for the detection of plant viruses and subviral pathogens. J.Virol. Meth. 45: 201-218. Noordam, D. 1973. Identification of Plant Viruses. Methods and Experiments. Cent. Agric. Publ. Doc., Wageningen, the Netherlands. 207 pp. Norris, D. 1946. The strain complex and symptom development of tomato spotted wilt virus. Bull. Aust. Council Sci. Ind. Res. 202. Nuez, F.; Fernández de Córdova, P.; and Díez, M.J. 1992. Collecting vegetable germplasm in the Iberian Peninsula. Plant Genetic Resources Newsletter 90, 31-33. OEPP/EPPO 1999a. Data sheets on quarantine organisms no. 291. Impatiens necrotic spot tospovirus. Bulletin OEPP/EPPO Bulletin 29: 473-476. OEPP/EPPO 1999b Data sheets on quarantine organism’s no. 294. Watermelon silver mottle tospovirus. Bulletin OEPP/EPPO Bulletin 29: 489-492. Okuda, M. and Hanada, K. 2001. RT-PCR for detecting five distinct Tospovirus species using degenerate primers and dsRNA template. J. Virol. Methods 96: 149–156. Pang, S. Z.; Slightom, J. L. and Gonsalves, D. 1993 . Different mechanisms protect transgenic tobacco against tomato spotted

wilt and impatiens necrotic spot Tospoviruses. Biotechnology 11: 819-824. Pappu, H.; Pappu, S.; Jain, R.; Bertrand, P.; Culbreath, A.; McPherson, R. and Csinos, A. 1998a. Sequence characteristics of natural populations of tomato spotted wilt tospovirus infecting flue-cured tobacco in Georgia. Virus Genes. 17 (2):169-77. Pappu, S.S.; Pappu, H.R.; Chang, C.A.; Culbreath, A.K. and Todd, J. W. 1998b. Differentiation of biologically distinct peanut stripe potyvirus strains by a nucleotide polymorphismbased assay. Plant Disease 82: 1121-1125. Parrella, G.; Gognalons, P.; Gebre Selassie, K.; Vovlas, C. and Marchoux, G. 2003. An update of the host range of tomato spotted wilt virus. Journal of Plant Pathology. 85(4): 227-264. Perosa, F.; Carbone, R. Ferrone, S. and Dammacco, F. C. 1990. Purification of human immunoglobulin by sequential precipitation with caprylic acid and ammonium sulphate. J. Immunol. Methods. 128:9-16. Peters, D.; de Avila, A.C.; Kitajima, E.W. Resende, R. de O.; de Haan, P. and Goldbach, R. 1991. An overview of tomato spotted wilt virus. In: Hsu, H.T. and Lawson, R.H. (Eds.) Proc. USDA workshop Virus-Thrips-Plant Interactions of Tomato Spotted Wilt Virus. (Beltsville, MD, USA) pp. 1-14. Pitblado, R. E., Allen, W. R., Matteoni, J. A., Garton, R., Shipp, J. L., and Hunt, D. W. A. 1990. Introduction of the tomato spotted wilt virus and western flower thrips complex into field vegetables in Ontario, Canada. Plant Dis. 74:81. Reddy, D.V.R. and Wightman, J.A. 1988. Thrips transmission and control In Advances in Disease vector Research, ed.KF Harris, 8:203-20 Reddy, D.V.R., Ratna, A.S., Sudarshana, M.R., Poul, F. and Kumar, I.K. 1992. Serological relationships and purification of

bud necrosis virus, a tospovirus occurring in peanut (Arachis hypogaea L) in India. Ann. Appl. Biol. 120, 279–286. Reifschneider, F.J.B.; Café, A. C.; Dusi, A. N.; and Kitajema, E. W. 1989. Brown pod, a disease caused by tomato spotted wilt virus on peas in Brazil. Tropical-Pest Management, 35 (3): 304-306. Resende, R. de O., Avila, A.C. de, Goldbach, R.W. and Peters, D. 1991. Detection of tomato spotted wilt virus using polyclonal antisera in double antibody sandwich (DAS) ELISA and cocktail ELISA. J. Phytopathol. 132: 46−56. Rice, D. J., German, T. L., Mau, R. F. L. and Fujimoto, F. M. 1990. Dot blot detection of tomato spotted wilt virus RNA in plant and thrips tissues by cDNA clones. Plant. Dis. 74:274276. Roberts, C.A., Heelan, L., Persley, D., Maclean, D.L., and Dietzgen, R.G. 1999 . TaqMan™ RT-PCR realtime fluorescent detection of tomato spotted wilt tospovirus. Abtracts of the XIth International Congress of Virology, Sydney, p. 369, VP65.46. Rodriguez, D.R. 1990. [Frankliniella occidentalis in protected cultivation in Almería] In: 1st Symposium on Frankliniella occidentalis. Valencia (ES) (in Spanish). Roggero, P. and Pennazio, S. 1997. Thermal inactivation of tomato spotted wilt tospovirus in vivo. Physiological and Molecular plant pathology. 51, 35-40. Roggero, P.; Ciuffo, M.; and Dellavalle 1999. Additional ornamental species as hosts of Impatiens Necrotic Spot Tospovirus in Italy. Plant Disease 83, 967. Ronco, A.E.; Dal Bo, E.; Ghiringhelli, P.D.; Medrano, C.; Romanovski, V.; Sarachu, A.N.; and Grau, O. 1989. Cloned cDNA probes for the detection of tomato spotted wilt virus. Phytopathology 79:1309-1313.

Rosello, S.; Jordá C.; and Nuez, F. 1994. El virus del bronceado de tomate (TSWV). I. Enfermeda des y epidemiologia. Phytoma España 62: 21-33. Russell, C. B.; Stewart, R. C.; and Dahlquist, F. W. 1989. Control of Transducer Methylation Levels in Escherichia coli: Investigation of Components Essential for Modulation of Methylation

and

Demethylation

Reactions.

J. Bacteriol. 171:3609-3618.

Sakimura, K. (1963). Frankliniella fusca, an additional vector for the tomato spotted wilt virus, with notes on Thrips tabaci, a thrips vector. Phytopathology 53:412-415. Saldarelli, P.; Barbarossa, L.; Grieco, F.; and Gallitelli D. 1996. Digoxigenin- labelled riboprobes applied to phytosanitary certification of tomato in Italy. Plant Disease 80: 1343-1346. Salomone, A.; Masenga, V.; Minuto, G.; Parodi, C.; and Roggero P. 2003. First report of Tomato spotted wilt virus (Tospovirus, Bunyaviridae) infecting Euphorbia eritrea and Asclepias curassavica in Liguria, Italy. New Disease Report, (http://www.bspp. org.uk/ndr/july2003/2003-33.asp). Sambrook, J.; Fritsch, E. F.; and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press). San Millan, J. L.; Boyd, D.; Dalbey, R.; Wickner, W. and Beckwith, J. 1989. Use of phoA Fusions to Study the Topology of the Escherichia coli Inner Membrane Protein Leader Peptidase. J. Bacteriol. 171: 5536-5541. Satyanarayana, T.; Gowda, S.; Reddy, K.L.; Mitchell, S.E.; Dawson, W.O. and Reddy D.V. 1998. Peanut yellow spot virus is a member of a new serogroup of Tospovirus genus based on small (S) RNA sequence and organization. Arch. Virol. 143(2):353-64.

Schuster, G. L. and Halliwell, R. S. 1994. Six new hosts of tomato spotted wilt virus in Texas. Plant Disease vol. 78 (1): 100 Sether, D.M. and De Angelis, J.D. 1991. Tomato spotted wilt virus host list and bibliography. Agricultural Experimental Station, Special Report n° 888, pp. 16. Department of Entomology, Oregon State University, Corvallis. Sherwood, J. S.; Sanborn, M. R.; Keyser, G. C. and Myers, L. D. 1989. Use of monoclonal antibodies in detection of tomato spotted wilt virus. Phytopathology 79:61-64. Singh, H.S.; Krishnareddy and Kuo, G.G. 1996. Watermelon bud necrosis: a new tospovirus disease. Acta Horticulture 431: 68-77. Smith, K.M. 1932 . Studies on plant virus diseases. XI. Further experiments with ringspot virus: Its identifications with tomato spotted wilt of tomato. Ann. Appl. Biol. 19, 305–330. Snippea, M.; Goldbach, R.; and Kormelink, R. 2005. Tomato spotted wilt virus particle assembly and the prospects of fluorescence microscopy to study protein-protein interactions involved. Adv Virus Res.65:63-120. Southern, E.M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98:403-517. Takeda, A.; Sugiyama, K.; Nagano, H.; Mori, M.; Kaido, M.; Mise, K.; Tsuda, S. and Okuno, T. 2002. Identification of a novel RNA silencing suppressor, NSs protein of Tomato FEBS Lett.532 (1-2):75-9 (ISSN: 0014-5793) Thomas, J.E.; Schwinghamer, M.W.; Parry, J.N.; Sharman, M.; Schilg, M.A. and Dann, E.K. 2004. First report of Tomato spotted wilt virus in chickpea (Cicer arietinum) in Australia. Australas Plant Pathol. 33 (4):597-599. Ullman D.E. 1996. Thrips and Tospoviruses: Advances and Future Directions. Acta Horticulturae 431:310-324.

Ullman, D.E.; Cho, J.J.; Mau, R.F.L.; Westcot, D.M. and Cantone, D.M. 1992. A Midgut epithelial cells act as a barrier to tomato spotted wilt virus acquisition by adult western flower thrips. Phytopathology 85: 456-463. Urban, L. A.; Huang, P.Y.; and Moyer, J. W. 1991. Cytoplasmic inclusions in cells infected with isolates of L and I serogroups of tomato spotted wilt virus. Phytopathology 81:525-529. Vaira, A.M.; Vecchiati, M.; Masenga, V. and Accotto, G.P. 1996. A polyclonal antiserum against a recombinant viral protein combines specificity with versatility. J. Virol Methods. 56(2):209-219 . Vaira, A.M.; Semeria, L.; Crespi, S.; Lisa, V.; Allavena, A.; and Accotto, G.P. 1995. Resistance to tospoviruses in Nicotiana benthamiana transformed with the N gene of Tomato spotted wilt virus: correlation between transgene expression and protection in primary transformants. Molecular Plant Microbe Interactions 8 (1): 66-73. Van Os B.; Stancatelli G.; Mela L.; and Lisa V. 1993. Ruolo delle piante spontanee ed infestanti nell'epidemiologia di tospovirus in Liguria. Informatore Fitopatologico 43: 40-44. Vicchi, V. and Bellardi, M. G. 1996. Evaluation of ELISA technique in the diagnosis of tospoviruses in ornamental plants. Inform. Fitopatol. 46 (4): 60-64. Wang, M. and Gonsalves, D. 1990. ELISA detection of various tomato spotted wilt virus isolates using specific antisera to structural proteins of the virus. Plant Disease 75: 154-158. Weekes, R.; Barker, I; and Wood 1996. An RT-PCR test for the detection of tomato spotted wilt tospovirus incorporating immunocapture and colorimetric estimation. Journal of Phytopathology 144: 575-580. Whitfield, A. E.; Campbell, L. R.; and Ullman D. E. 2003. Tissue Blot Immunoassay for Detection of Tomato spotted wilt virus

in Ranunculus asiaticus and Other Ornamentals. Plant Dis. 87:618-622. Wijkamp, I.; Almarza, N.; Goldbach, R. and Peters, D. 1995. Distinct levels of specificity in thrips transmission of tospoviruses. Phytopathology 85: 1069-1074. Wijkamp, I.; Van Lent, J.; R., K.; Goldbach, R. and Peters, D. 1993. Multiplication of tomato spotted wilt virus in its vector, Frankliniella occidentalis. J. Gen. Virol. 74:341-349. Yeh, S.D. and Chang, T.F. 1995 . Nucleotide sequence of the N gene of watermelon silver mottle virus, a proposed new member of the genus tospovirus. Phytopathology 85 (1): 58-63. Zitter,T. A. and Daughtrey, M. L. 1989. Vegetable crops (Tomato Spotted Wilt Virus). Vegetable MD Online. Cooperative Extension, New York State, Cornell University. Department of Plant Pathology, Fact Sheet Page: 735-790.

‫يعتبر فير ا ذبرل ال ذبتبقعر فر ذبمار من ارف ذبسي‬ ‫حيث يصري‬

‫ر‬

‫عردةذ ببير ذ ارف ذباح صري ذبه ار اسربب سسر‬

‫تن عزل هلذ ذبسي ا اف ثا ر نب ت‬

‫ذذ‬

‫ذألهاير ذﻹقتصر ةي‬

‫ببير فر اصر‬

‫مار من عييهر رعر ذم اا ثير بضعر ذم ذبتر يحردثه‬

‫هلذ ذبسي ا تن تجايعه اف ازرع ذبتج رب بكيي ذبزرذع بج اع عيف شرا‬

‫هررلذ ذبسي ر ا بررد ارردل ع راذ ي‬ ‫ذبسير ا رهر‬ ‫اررف نب تر‬

‫ه ر‬

‫قررد ررهر‬

‫ذبما ر من تحررة ذبد ذر ر تررن عزبررد‬

‫ذبما ر من عير ر ر ر ر ا ذبد ذر ر‬

‫قرد‬

‫رد ر‬

‫ععررد عا ر عررد ل ايك نيكي ر أل رذق ذبما ر من ه ررلذ‬

‫ذ ر‬

‫رعر ذم احيير‬

‫ذبعر بن قرد‬

‫ذبد ذر ر ر ذبسير ا ذباعررز ل‬

‫نت ر‬

‫تع سررد عي ر رنررد فير ا ذبررل ال ذبتبقعر ف ر‬

‫ذببيابا ير ر ذالستبر ر ذر‬

‫ذبسرري با ي ذببيابا ير ر ذبجز ير ر‬

‫ذبت تاة عييد‬ ‫و يمكن تلخيص النتائج المتحصل عليها كاﻵتى‪:‬‬

‫الجزء اﻷول ‪ :‬الدراسات البيولوجية‪:‬‬ ‫‪ – 1‬عزل و تعريف الفيروس ‪.‬‬ ‫‪ -1 -1‬عزل الفيروس ‪.‬‬

‫ترن عررزل ذبسير ا تحررة ذبد ذر ر اررف نب تر‬

‫ذبتج ر رب‪ -‬بييهر ر ذبز ذرع ر ‪-‬‬

‫اعر ر عرريف ش ررا‬

‫ذبمار من ذباصر ب مبيعير اررف احمر‬

‫عييهر ر‬

‫‪ -‬اح فظ ر ذبقييابير ر ذبت ر رهر ر‬

‫رع ذم تش بد ذألع ذم ذبا ضي ذباص حب بالص بد بسي ا ذبل ال ذبتبقع ف ذبما من‬ ‫يسررب‬

‫ه ررلذ ذبسير ر ا اظه ر تظي رريق ذ رذق ذبمار ر من ذبصررظي‬

‫رحي ن اصحاب بحيق‬

‫ا بز‬

‫نز ذبيا ا حد ث ذنحع ءذ‬

‫ذباتقدا ر اررف ذبا ر م ت ررا عي ر هي ر ذصررس ذر تب ر ق‬

‫ذبثا ر ر رعي هي ر ر اس ر ر ح‬

‫ش ر ر حب ذبير ررا‬

‫تشررا‬

‫س صر ر ذبعر ر ق‬ ‫إب ذبدذس‬

‫ت ررا‬

‫ذألع ذم‬

‫تظه ر ذألع ر ذم عي ر‬

‫صر ررس ذء عي ر ر ذببش ر ر ذبمبيعي ر ر ذبحا ر ر ذء بثا ر ر ر‬

‫ذبما من ذبع ضج‬

‫قد تن تعقي هلذ ذبسي ا يابا ي اف سالل ذبعق ذباتسيس بيبق ذباحيي ذباعس ة‬

‫عي عاذ‬

‫تعمر بقر ااضرعيد اثر ذببيتانير ‪ Petunia hybrida‬ذعمرة بقعر ااضرعي‬

‫بعرد ‪ 4 – 2‬رير ب بعرد ذبعررد ل ريىر عير نب تر‬

‫‪Datura metel and Nicotiana‬‬

‫‪ tabacum cv Kentuckey.‬قررد تررن ذبعق ر ذبايك ر نيك عي ر نب ت ر‬ ‫‪ Money maker‬بتى ع‬

‫ذبسي ا‬

‫ذبما ر من ص ررع‬

‫‪ – 2 – 1‬تعريف الفيروس‬ ‫‪ – 2 - 1‬االنتقال‬ ‫ٲ – النقل الميكانيكى‬

‫تررن نقر فير ا ذبررل ال ذبتبقعر فر ذبمار من ذبعزبر ذباصر‬ ‫ييا تتب ‪ 16‬ع يد نب تي اعزرع ف‬

‫‪ 33‬نب ت‬

‫ذبصاب‬

‫ايك نيكير عير ب ر ة ذر‬

‫ذبك ب تخدذب احيال اعظن‬

‫ب ‪ -‬النقل بواسطة حشرة التربس‬

‫تررن نق ر في ر ا ذبررل ال ذبتبقع ر ف ر ذبما ر من عررف م ر حش ر ذبت ر ب‬

‫ذبعت‬

‫ذ‬

‫رره ر‬

‫ذبي ق ذبظي ب اي ه ذبت به ذبقردر عير ذكتسر ب ذبسير ا ذنتق برد بشرتال‬

‫ذبما من ذباص بد اذ م ذبحش ذب اير حيرث تظهر رعر ذم ذألصر ب فر ارد تتر ذ ن ارف‬ ‫مار من اصر ب بارد‬

‫‪ 21 -14‬ياار بعرد ذبتظلير عير نب تر‬

‫ف ر ر ‪ 14‬نب ت ر ر ار ررف ذ ا ر ر ب ‪ 20‬نب ت ر ر رل عسر ررب ‪%70‬‬

‫رع‬

‫قرد شراهد‬

‫ذألعر ذم‬

‫تر ررن ذبتأكر ررد ار ررف ايك نيكي ر ر ذبعق ر ر‬

‫ب تخدذب ذستب ذر ذﻹبي ذز ذبظي اب ش‬ ‫‪ 2 – 2 – 1‬دراسه المدى العوائلى‬ ‫رره ر ر‬

‫ذباصر ر‬

‫ة ذر ر ر ذبار رردل ذبع ر راذ ي بسي ر ر ا ذبر ررل ال ذبتبقع ر ر‬

‫رن ررد ذ ا رردل عر راذ ي‬

‫نب تي اختيس‬ ‫عي ر‬

‫قد تب يعة ذبعب ت‬

‫عسرريف يتبع ر‬

‫رع ذم ااضعي ر‬

‫ذ ر ر حي ررث‬

‫ذبع ي ر ذبعجييي ر را ر ب ر ق ذبعب ت ر‬

‫تب يعة مبق ﻹستالف ذبع‬

‫‪ 3 - 2 - 1‬درجة ثبات الفيروس‬ ‫رك ررد‬

‫ذبعتر ر‬

‫‪ 31‬نب تر ر تى ررن ‪ 14‬ع ير ر‬

‫ذباعدذ صع عي ق رة فعيه حيث بن تظه ري ﺇص ب‬

‫ذبع ي ر ذبق عي ر‬ ‫ه‬

‫ررد رن ررد يص رري‬

‫ف ر ر ذبما ر ر من بيعزب ر ر‬

‫ذباعدل‬

‫ر ذبسير ر ا تح ررة ذبد ذر ر ر يعتقر ر ب بعص رري ذبعبر ر ت‬

‫ذبح ر ر ذر ذباسقر ررد بعش ر ر مد ذبا ض ر ر ت ذ حر ررة ر رريف ‪ 50– 45‬ةر ر ر ا ا ر ر‬ ‫ذبعه ير ب نررة ‪10-3‬‬

‫فقررد ره ر عييه ر‬

‫فت ر ذبتعاي ر فر ذألن ي ر‬

‫ح ذر ذباعا‬

‫‪ 4 – 2 - 1‬الدراسات السيرولوجيه‪:‬‬

‫ا – االلي از الغير مباشره ‪.‬‬

‫ت ذ حررة ا ر‬

‫ب ن ررة ةر ر ر‬ ‫ةر ر ر ذبتخسي ر ر‬

‫رريف ‪ 6– 5‬ر ع‬

‫ف ر ةر ر‬

‫تن ذستب ر ذباردل ذبعراذ ي ذباعردل صرع عي بسير ا ذبرل ال ذبتبقعر فر ذبمار من‬ ‫عييد رع ذم ام بق بيت يحدثه هلذ ذبسي ا ب رتخدذب ت عيرك ذألبير ذز ذبظير‬

‫ذبت ره‬ ‫اب ش‬

‫ب – التنقيط المناعى‬ ‫رثبتة نت‬

‫ذبتعقيط ذباع ع ببعض ذبعب ت‬

‫رع ر ر ذم اش ر ر ه بيت ر ر يحر رردثه هر ررلذ ذبسي ر ر ا‬

‫ذبت ره‬

‫ذباص ب مبيعي‬

‫عييه‬

‫ر رراة ذص ر ر ب بسي ر ر ا ذبر ررل ال ذبتبقع ر ر ف ر ر‬

‫ذبما من‬ ‫‪ 5 – 2 – 1‬تفاعل البلمره المتسلسل‬ ‫ررهر ذ نتر‬

‫ذستبر ر تس عر ذببيار ذباتسيسر‬

‫ذبما من ف نسي ذبعب ت‬

‫عجر ن ذب شر‬

‫نرف فير ا ذبرل ال ذبتبقعر فر‬

‫ذباص ب‬

‫‪ 6 – 2 – 1‬الميكروسكوب االلكترونى‬

‫ر ر رركاب ذﻹب ت ن ر ر ر ب ر ر ررتخدذب م ق ر ر ر ذبصر ر ررب‬

‫رره ر ر ر ذبسحر ر ررس تحر ر ررة ذبايك‬

‫(يارذنير ر ريتية) بيسير ا ذباعقر‬ ‫قم ه ا يف ‪ 100 :80‬ن ناايت‬

‫ز ير ب باعار‬

‫راة ز ر‬

‫ب‬

‫ر‬

‫اىريع‬

‫ذبجزء ذبث ن ‪ :‬ذبد ذر‬

‫ذبس ر ر ر ب‬ ‫يت ر ذ ن‬

‫ذبجز ي‬

‫ر‪ -‬إ نت ج ر س ب اى ة احيي ب تخدذب ذببيابا ي ذبجز ي‬ ‫ا ‪ -‬استخالص الحامض النووى‬

‫تن ذ تخدذاهن م قتيف عج ن ف عزل ذبحر اض ذبعرا ل ذب ير ارف نسري ذبعب تر‬

‫ذباصر ب‬

‫ذبم قتر‬

‫ه ‪CTAB‬‬

‫هار ‪ :‬م قرد ‪ High Pure RNA Tissue Kit‬ذبم قرد ذبث نير‬

‫قرد ررهر‬

‫نتر‬

‫اف ذبك بقي ا ةر د ذبعق‬

‫ذبتس رد ذب ه بر‬

‫اذ مد ه‬

‫راة‬

‫نقر‬

‫‪ RNA‬ذباعرز ل ترن ذبتأكرد‬

‫بكت فاتاايت‬

‫أ‪ 2-‬تفاعل النسخ العكسى و البلمرة المتسلسل‬

‫بعرد ذ رتخدذب ذنرز ن ذبعسرع ذبعكسر بتحا ر ذبحر اض ذبعرا ل ذب برا ل ذباعرز ل ذبر‬

‫ذبخريط ذباكار ارف ذبحر اض ذبعرا ل ذبديربسر ر برا ل‬ ‫تس عر ذببيار ذباتسيس ر‬

‫ب ررتخدذب ذببر ة يف ذباتخصصرريف‪Tsw1‬‬

‫تصر ر رراياهن اذ ر ر ررمد ذبع با ر ر ر‬

‫ذبعيكيياب تيف‬

‫ب رتخدذب ذنرز ن ذببيار فر عايير‬

‫‪ Tsw2‬ذبيررلذ تررن‬

‫ب ر ر ر تا ه ن ر ر ر ةذ عر ر ر ر ب ‪ 2003‬تر ر ررن ذكث ر ر ر ر قمعر ر ررد ار ر ررف ر ر رريف‬

‫نه ذبجز ئ حراذب‬

‫‪ 780‬نيابيياتيرد ارف بر ذألنسرج ذباصر ب‬

‫ب رف بر‬

‫ن ت تس ع ذببيا ذباتسيس ذباتحص عييد اف نب تر‬ ‫ذبدس‬

‫نب‬

‫‪ N. rustica‬ذباصر ب ذقر فر ذبرا‬

‫ن ت تس ع ذببيا ذباتسيس حاذب‬ ‫تر ررن تأكير ررد ذبعت ر ر‬

‫ذبردذتار ذباصر ب ‪Datura metel‬‬

‫ذبجز ر ارف ذباتاقر حيرث بر‬

‫‪ 600‬نيابيياتيد‬

‫ذباتحص ر ر عييه ر ر اذ ر ررمد تهج ر رريف ذبح ر ر اض ذبعر ررا ل‬

‫حجرن‬

‫ذبتت بعر ر ر‬

‫ذبعيابيياتيدي‬ ‫‪ - 3‬االستنساخ‬

‫تاة عايي ذب يان عف م‬

‫ذبتح ب رزء ارف ريف ذبعيكيراب تيف بسير ا ذبرل ال‬

‫ذبتبقعر فر ذبمار من اب شر ةذسر ذببال ايررد ذببكتير ل ذباخصررس بعايير ذبتعبير ذبب تيعر‬ ‫‪pBAD-Topo‬‬

‫ذةس ر ل هررلذ ذببال ايررد ذباحررار رذثي ر ف ر سالي ر بكتي ر تسررا ريك راالل‬ ‫ذباحر ر ر ررار يعي ر ر ر ر ذبح اي ر ر ر ر بجر ر ر رريف‬

‫ر ر ر ررالب )‪ (BL21-DE3‬ثر ر ر ررن تر ر ر ررن عر ر ر ررزل ذببال اير ر ر رردذ‬

‫ذبح اي بجيف ذبعيابياب تيف‬

‫ذبعيابياب تيف رثع ء عا اسح بيتع ف عي ذببال ايدذ‬

‫‪ - 4‬التأكد من نجاح االستنساخ‬

‫ذبح ايررد بيعيكيرراب تيف رريف ذبررلل‬

‫تررن ذبتأكررد اررف نج ر ن عايي ر ذب يان ر بيبال ايرردذ‬

‫نرد ذبجز رئ ‪ 780‬ب رتخدذب بر ة يف اتخصصريف هار ‪tsw1‬‬ ‫ذببيا ذباتسيس‬ ‫ذستي ر ذببال ايدذ‬

‫‪ tsw2‬فر عايير تس عر‬

‫تن ت كيرد هرل ذبعتيجر ب رتخدذب م قر تهجريف ذبحر اض ذبعرا ل اعرد ترن‬ ‫اف تستخدب ف عايي ذبتعبي ذبب تيع‬

‫ذبت‬

‫‪ -5‬التعبير البروتينى لجين النيكلوبروتين داخل البكتيريا‬

‫ت ر ررن ذبتعبي ذبب تيعر ر ر بج ر رريف ذبعيكي ر رراب تيف ذب ر ررلل ت ر ررن بيانت ر ررد ةذسر ر ر ذبع قر ر ر ذب ر ررلل‬ ‫‪ pBAD – Topo‬تحرة ذبب ااترار (ذباحسرز) ‪ pBAD‬ذبارعظن ‪ AraC‬ةذسر‬

‫يسرا‬

‫ذبسررالبد ذببكتي ر ر )‪ (BL21-DE3‬بع ررد ذضر ر ف ذباعش ررط‬ ‫ذعير ت بيررز اررف ذبب ر تيف فر ح بر اتج نسر‬

‫قال ف تج بد ذبحقف ذباع عي ةذس ذألرذن‬

‫بكاي ر‬

‫‪ L-arabinose‬بيحص ررال عير ر‬

‫نير احررد ة ال ررتخدذاد بررأنتيجيف‬

‫النت ج ذ س ب اى ة عديد ذبتخصس‬

‫‪ - 6‬تنقيه النيكلوبروتين المهندس وراثيا تحت ظروف الدنترة‬ ‫ذبععت ذب‬ ‫ذ‬

‫تررن تعقي ر ذبعيابيرراب تيف ذباهعرردا رذثي ر ذباعررت ةذس ر ذببكتي ر ب ررتخدذب عا رراة‬

‫ر رري ذذ‬

‫ا تا ذف‬

‫ذألنتيسي ب ذأل عب‬

‫رره‬

‫نتر‬

‫ير ذألك الايرد‬

‫ز ر ررئ ‪ 28‬بيير ررا ةذبتر ررا‬

‫ت عيرك ذبا سرت‬

‫راة ر تيف ب نرد‬

‫قر ررد تس ع ر ر هر ررلذ ذبب ر ر تيف بكس ر ر ء ع بي ر ر ا ر ر‬

‫‪ - 7‬اختبار النيكلوبروتين المهندس وراثيا مع اﻷنتيسيرم اﻷجنبى‬

‫ذستبر ر ر ر ذر ذ بتعق ر ر رريط ذباع عير ر ر ر ق ر ر ررا ذبتسر ر ر ر عال‬

‫ر ض ر ر ررحة نتر ر ر ر‬

‫ذباع عير ر ر ر ر ر رريف‬

‫ذبعيابيياب تيف ذباهعدا رذثي ذباعق ارف ذببكتي ر ار ذأل سر ب ذباىر ة ذباتحصر عييهر‬ ‫اف ذبهعد تحة ذبظ ف ذبمبيعي‬ ‫‪ - 8‬إ نتاج اجسام مناعية مضاده لفيروس الذبول التبقعى فى الطماطم محليا‬ ‫تررن إنت ر ج ر س ر ب اع عي ر عديررد ذبتخصررس ضررد ذبعزبررد ذباص ر‬

‫بسي ر ا ذبررل ال‬

‫ذبتبقع ر ف ر ذبما ر من عررف م ر حقررف سيرريط اررف ذبعيكيرراب تيف ذباهعرردا رذثي ر‬ ‫ذبارردنت ف ر ذرن ر‬

‫تحررة ذبظ ر ف ذبمبيعي ر‬

‫ذباعق ر‬

‫نيا يعرردل قررد تررن ذستب ر ر ذأل س ر ب ذباى ر ة‬

‫ذباص د ذباعتج احيي ف ذبتج رب ذبسي با يد ب تخدذب ت عيك ذبتعقيط ذباع ع‬

‫ذبظي ر اب ش ر حيررث تس عيررة ا ر ب ر ا ررف نب ت ر‬

‫ما ر من اص ر بد بسي ر ا ذبررل ال ذبتبقعر ر‬

‫(ذبعيكيرراب تيف ذبمبيع ر ) بررلبك ا ر ذبعيابيرراب تيف ذباهعرردا رذثي ر‬ ‫(ذبا رردنت ) حتر ر تخسير ر‬

‫ذألبير ذز‬

‫ذباعق ر اررف ذببكتي ر‬

‫‪ 1:1000‬اار ر ي رردل عير ر ا رردل ذبتخصص رري‬

‫ذبع بي بيض س ب ذباى ة ذباعتج احيي‬

‫ذب سر ر ء ذباع عير ر‬

‫‪ - 9‬تقدير أفضل تخفيف لألجسام المضادة المنتجة محليا ضدد فيدروس الدذبول التبقعدى‬ ‫في الطماطم‬

‫بض سر ر ب ذباىر ر ة بيعيكي رراب تيف ذبخر ر‬

‫ررد ر رفىر ر تخسير ر‬

‫ذبتبقعر فر ذبما ر من ب ررتخدذب ت عيررك ذبتعقرريط ذباعر ع هررا تخسي ر‬ ‫ذبب تيف ذبمبيع ف ذبعيع‬

‫عف ذبب تيف ذبسي‬

‫‪ 1:1000‬بي شر‬

‫ذبادنت ب تخدذب ت عيك ذألبي ذز ذبظي اب ش حت تخسي‬

‫تررن تعقير ر ذبج ا ر‬

‫ةر ر ر ر نقر ر ر‬

‫ذاتص‬

‫عررف‬

‫ذباص بد ب بسي ا يعا ذ تم عة ذال س ب ذباىر ة ذب شر‬

‫‪ - 10‬تنقيه الجاما جلوبيولين‬ ‫ذبج ار ر ر‬

‫بسير ر ا ذبر ررل ال‬

‫يابي ررابيف ب ررتخدذب م ق ررد ذب ب يررك ذ رريد‬

‫يابي ر ررابيف‬

‫ععد ‪ 250‬ن ناايت‬

‫رعم ر ررة رعير ر ر ذاتصر ر ر‬

‫‪1:4000‬‬

‫قرردرره‬

‫عع ر ررد ‪ 280‬نر ر ر ناايت‬

‫ذبعتر ر‬ ‫رقر ر ر‬

‫‪ - 11‬تقدير نقطدة التخفيدف النهائيدة لألجسدام المضدادة المنتجدة باسدتخدام االليد از الغيدر‬ ‫مباشرة‬ ‫تررن تقرردي نقم ر ذبتخسي ر‬

‫ذبظي اب شر ر‬ ‫ذبخ‬

‫تخسي‬

‫ق ررد‬

‫ررد ذ‬

‫ذبعه ي ر‬

‫ذبج ار ر‬

‫بض س ر ب ذباى ر ة ذباعتج ر ب ررتخدذب ت عيررك ذالبي ر ذز‬

‫يابي ررابيف بهر ر ذبق رردر عير ر ذب شر ر‬

‫بسي ا ذبل ال ذبتبقع ف ذبما من ف عصر‬

‫‪4000 : 1‬‬

‫ذبعب تر‬

‫ع ررف ذبعيكي رراب تيف‬

‫ذباصر ب بر بسي ا حتر‬

‫‪ - 12‬تقدددير نقطددة التخفيددف النهائيددة فاالعصددير النبدداتى المصدداب بددالفيروس باسددتخدام‬ ‫االلي از الغير مباشرة‬

‫فر ر ذب شر ر‬

‫ت ررن ذ ررتخدذب ذأل سر ر ب ذباىر ر ة ذباعتجر ر‬

‫ع ررف رفىر ر تخسير ر‬

‫ذبعصي ذبعب ت ذباص ب ياكف ذ تخدذاد ف ت عيك ذالبي ذز ذبظير اب شر‬ ‫عف ذبسي ا دق‬

‫ذب ش‬

‫ف عصي ذبعب ت‬

‫قرد‬

‫ا ررف‬

‫رد رنرد ياكرف‬

‫ذباص بد ععدا تن تخسيسه ا يف & ‪10-2‬‬

‫‪ 10-3‬ذبك ذذذ تن ذ تخدب ذبج ا يابيابيف تخسي‬

‫‪1:1000‬‬

‫‪ - 13‬تقدديم اﻷجسدددام المضددادة المنتجدددة محليددا لفيدددروس الددذبول التبقعدددى فددى الطمددداطم‬ ‫باستخدام تكنيك التنقيط المناعى ‪.‬‬

‫عف في ا ذبل ال ذبتبقع ف بعض ذبعاذ‬

‫ذبعب تيد ذباص ب اذ مد‬

‫تن ذب ش‬

‫ت عيك ذبتعقيط ذباع ع ب تخدذب ذأل س ب ذباى ة ذباعتج رره‬

‫بس ء حس يد‬

‫ذبعت‬

‫ذأل سر ر ب ذباىر ر ة ذباعتجر ر احيير ر عع ررد اق رنتهر ر ار ر ذأل سر ر ب ذباىر ر ة ذأل عبير ر فر ر نسر ر‬

‫ذبت عيك‬ ‫ب – صفات الفيروس الجزيئيه‪.‬‬ ‫ب – ‪ 1‬التتابع النيكلوتيدى‬

‫ذبتتر ب ذبعيابيياتيردل ذباب شر بجزء ارف ريف ذبعيابييراب تيف (نر ت‬

‫ر ضحة ة ذر‬

‫ذببيار ذباتسيسر ) ب رتخدذب هر‬ ‫اعه ررد بح رراث ذار ر ذم ذبعب تر ر‬ ‫نر ا اتخصررس يسررا‬

‫ذبتت بعر‬

‫‪% 81‬‬

‫قررد راكررف ذ ررتعب‬

‫ركد نس‬

‫بع ررد ت ات ررد ذبر ر ذألحار ر م ذالايعير ر ذباش ررس ب ررد ب ررتخدذب‬

‫)‪(DNAMAN V 5.2.9 package‬‬

‫ذباسررجي فر‬

‫ذبعتيج ذبس بق‬

‫ذبتتر ب ذبعيكياتيردل ذباا رراة با برز ذببحراث ذبزرذعيررد –‬

‫عررك ذبجيعر‬

‫بربعض عرزال‬

‫اق رنتررد ار بعررض‬

‫‪ TSWV‬ب نررة نسررب ذبتمر‬

‫شررج ذبق ذبررد رريف هررل ذبتت بع ر‬

‫ب ررتخدذب نس ر‬

‫ح راذب‬

‫ذبب نر ا ذبررلل‬

‫ب‪ 2 -‬شجره القرابة‬ ‫رثبتة نت‬

‫تحيي شج ذبق ذب بضحا م ذألايعي بيتت ب ذبعيابيياتيدل بجيف‬

‫ذبعيابياب تيف ذباتحص عييد ر ةر‬

‫ذبق ذب يف ذبعزب ذباص‬

‫ف ذبما من تص ذب ‪ % 81‬ا بعض ذبعزال‬

‫ب تخدذب ن ا اتخصس ف ذبتحيي ذبارذث‬

‫ذباعشار ف‬

‫بسياا ذبل ال ذبتبقع‬

‫عك ذبجيع‬

‫ذبك‬

‫‪(DNAMAN V 5.2.9 package,‬‬

‫)‪Madison, Wisconsin, USA‬‬

‫الذي يصيب نباتات الطماطم‬

‫رسالة مقدمة من‬

‫جـماالت مـحمـد عالم‬ ‫بك بار اا عياب رذعي ‪ ،‬استكمال شعبة أمراض نبات ‪ ،‬كلية الزراعة ‪ ،‬جامعة عين شمس‬ ‫‪2002‬‬ ‫للحصول على‬

‫درجة املاجستري يف العلوم الزراعية‬ ‫(أمراض نبات)‬

‫قسم أمراض النبات‬ ‫كلية الزراعة ‪ -‬جامعة عين شمس‬

‫‪2006‬‬

‫صفحة الموافقة على الرسالة‬

‫دراسات مرضية و جزيئية لفيروس الذبول المتبقع الذي يصيب‬ ‫نباتات الطماطم‬ ‫رسالة مقدمة من‬

‫جماالت محمد عالم‬ ‫بك بار اا عياب رذعي ‪ ،‬استكمال شعبة أمراض نبات ‪ ،‬كلية الزراعة ‪ ،‬جامعة عين شمس‬ ‫‪2002‬‬ ‫للحصول على درجة‬

‫الماجستير في العلوم الزراعية‬ ‫(أمراض نبات)‬

‫وقد تمت مناقشة الرسالة و الموافقة عليها‬ ‫اللجنة‪:‬‬

‫أ‪.‬د‪ .‬محمد أحمد عوض‬

‫‪------------------------------------------‬‬

‫أستاذ أمراض النبات ‪ ،‬كلية الزراعة‪ ،‬جامعة المنوفية‬

‫أ‪.‬د‪ .‬فوزي مرسي أبو العباس ‪------------------------------------------‬‬ ‫أستاذ أمراض النبات‪ ،‬كلية الزراعة ‪ ،‬جامعة عين شمس‬

‫أ‪.‬د‪ .‬مصطفى حلمي الحمادي‬

‫‪-----------------------------------------‬‬

‫أستاذ أمراض النبات المتفرغ ‪ ،‬كلية الزراعة‪ ،‬جامعة عين شمس‬

‫تاريخ المناقشة ‪2006 / 5 / 22‬‬

‫جامعة عني مشس‬ ‫كلية الزراعة‬

‫رسالة ماجستري‬ ‫اسم الطالبة‬

‫‪ :‬جماالت محمد عالم‬

‫عنوان الرسالة ‪ :‬دراسات مرضية و جزيئية لفيروس الذبول المتبقع الذي‬ ‫يصيب نباتات الطماطم‬ ‫اسم الدرجة ‪ :‬ا ستي ف ذبعياب ذبزرذعي (را ذم نب )‬ ‫لجنة اإلشـراف‪:‬‬ ‫أ‪.‬د‪ .‬مصطفى حلمي الحمادي‬ ‫أستاذ أمراض النبات المتفرغ‪ ،‬قسم أمراض النباات‪ ،‬للياة الزراعاة‪ ،‬جامعاة عاي‬ ‫شمس‬ ‫( المشرف الرئيسي )‬

‫د‪ .‬طارق عبد الكريم مصطفى‬ ‫مادرس أمااراض النبااات ‪ ،‬قساام أمااراض النبااات ‪ ،‬لليااة الزراعااة ‪ ،‬جامعااة عااي‬ ‫شمس‬

‫د‪ .‬هيام سامي عبد القادر‬ ‫بـاحـث بقسـم بحـوث الفـيـروس و الفيتوبالزما ‪ ،‬معهـد بحـوث أمـراض الـنـبـاتـات ‪ ،‬مرلز‬

‫البحوث الزراعية‬ ‫تاريخ البحث‪5/2/2003 :‬‬

‫الدراسات العليا‬ ‫ختم اإلجازة‬

‫اجيزت الرسالة بتاريخ‬ ‫‪2006/5/22‬‬

‫موافقة مجلس الكلية‬ ‫‪2006 / /‬‬

‫موافقة مجلس الجامعة‬ ‫‪/‬‬

‫‪2006 /‬‬