Isolation and Identification of Bacteriophage(S) Infecting Bacteria Which Cause Potato Soft Rot Disease by Eman Abd Elhafeez Aldeeb Abd Elhamid

Faculty of Science Department of Botany and Microbiology MiMMMMicrobiology Isolation and identification of

bacteriophage(s) infecting bacteria which cause potato soft rot disease A Thesis Submitted to the Faculty of Science, Cairo University In Partial Fulfillment of the Requirements for the M.Sc.Degree In (Microbiology) By Eman Abd Elhafeez Aldeeb Abd Elhamid

B.Sc. (Microbiology-Chemistry) 2012

Department of Botany and Microbiology Faculty of Science Cairo University

2017

Faculty of Science Department of Botany and Microbiology Microbiology

Approval sheet for submission Thesis Entitled Isolation and identification of bacteriophage(s) infecting bacteria which cause potato soft rot disease By Eman Abd Elhafeez Aldeeb Abd Elhamid

This thesis has been approved for submission by supervisors: Prof. Dr. El-Sayed Tarek Abd El-Salam Professor of Virology Botany and Microbiology Department Faculty of Science- Cairo University Signature……………………………………

Dr/ Maha Mohammed Al Khazindar Assistant professor of Virology Botany and Microbiology Department Faculty of Science- Cairo University Signature……………………………………..

Prof. Dr. Neveen Saleh Geweely

Chairman of Botany and Microbiology Department

Faculty of Science-Cairo University Signature……………………………………..

Declaration

This thesis hasn’t been submitted previously for any degree at Cairo University or at any other University and it’s the original work of the author.

Eman Abd Elhafeez Aldeeb Abd Elhamid

Acknowledgment

Firstly, I thank “Allah”, who gave me the patience, strength and knowledge to accomplish this work.

I should express my deep respect and gratitude to Prof. Dr. El Sayed Tarek Abd ElSalam, Professor of Virology, Department of Botany and Microbiology, Faculty of Science, Cairo University. Who supported me throughout this work and taught me how to be patient and how to be a scientific researcher. I thank him for his supervision, continuous encouragement and consultant during this work.

I should express my grateful sincere thanks to Dr. Maha Mohamed AlKhazindar, Assistant Professor of Virology, Department of Botany and Microbiology, Faculty of Science, Cairo University. Who encouraged me to choose this research point. I thank her for her faithful supervision, continuous guidance and support throughout this work.

Finally, I devote all my thanks to my supervisors for their tremendous help during writing and revision of this thesis.

Dedication

This thesis is dedicated to the memory of my father. The man who gave me the key to the life. I’m proud to be his daughter.

I should thank my mother who was the backbone that supported me through all my hard times. She was like the wind beneath my wings.

I sincerely express all my gratitude and thanks to my faithful man and beloved husband, Mahmoud Hussein, who supported and encouraged me to accomplish this work from the beginning till the end.

Contents

Contents Page

List of Figures……………………………………………………...... I List of Tables……………………………………………………………..IV List of Abbreviation……………………………………………...……..VII Abstract…………………………………………………………………...IX Introduction and Aim of work…………………………………………...1 Review of Literature…………………………………………………...... 6 Part I Isolation of bacteria causing soft rot disease……………………………….6 Part II Isolation of bacteriopages………………………………………………….19

Materials and Methods…………………………………………………...35

A. Materials

1. Isolates………………………………………………………...... 35 2. Media………………………………………………………...... 35 3. Buffers………………………………………………………………….37 4. Chemicals………………………………………………………………38 5. Equipments……………………………………………………………..38

B. Methods Part I 1.Bacterial isolation from potato ……………………………...... 39 2. Pathogenicity test ……………………………………………………...... 39 3. Bacterial identification………………………………………………...... 40 3.1.Gram stain…………………………………………………………....40 3.2.Motility……………………………………………...... 41 3.3.Biochemical tests……………………………………………………..41 3.4. Molecular identification...... 42

Contents

Part II 1. Isolation of bacteriophages……………………………………………...44

2. Transmission Electron Microscope…………………………...... 46

3. Extraction of phage nucleic acid………………………………………....46 3.1. Detection of nucleic acid type…………...... 47 3.2. Agarose Gel Electrophoresis………………...... 47

4. Bacteriophage nomenclature……………………………...... 47 5. Host range………………………………………………………….…....48 6. Physical properties………………………………………...... 48 6.1. Dilution end point………………………………………………….48 6.2. Thermal inactivation point……………………………………….....48 6.3.Effect of pH……………………………………………………….....49 6.4 Aging………………………………………...... 50

7. Effect of different salts………………………….…………………….…..50 7.1. Effect of different salts on Kosakonia sacchari……………………....50 7.2. Effect of different salts on the isolated bacteriophages……………….51

8. Effect of different heavy metals……………...... 52 8.1. Effect of different heavy metals on Kosakonia sacchari…………….52 8.2. Effect of different heavy metals on the isolated bacteriophages….….52

9. Effect of different sugars……………………...... 53

9.1. Effect of different on Kosakonia sacchari……………………………53 9.2. Effect of different sugars on the isolated bacteriophages………….....54

10. Effect of some plant extracts…………………………………...... 54

10.1 Effect of some plant extracts on Kosakonia sacchari...... 54 Contents

10.2. Effect of some plant extracts on the isolated bacteriophages …..55 11. Effect of different essential oils………………………………...... 56

11.1. Effect of some essential oils on Kosakoniasacchari……………..56

11.2. Effect of different essential oils on the isolated bacteriophages…56

12. Application of phage therapy in the lab………………...... 57 13. Statistical analysis……………………………………………………...58

Results………………………………………………………….……...... 59 Part I 1. Bacterial isolation from potato…………………………………………..59 2.Pathogenicity test………………………………………...... 59 3. Bacterial identification………………………………………...... 60 3.1. Gram stain…………………………………………………………...60 3.2. Motility test………………………………………………………….60 3.3.Biochemical tests……………………………………………………..60

3.4.Molecular identification…………………………………...... 62 Part II 1. Isolation of bacteriophages………………………………………..……67 2. Transmission Electron Microscope…………………………...... 68 3. Detection of nucleic acid………………………………………………....70 4. Bacteriophage nomenclature……………………………………………...71 5. Host range……………………………………………………………...... 71 6. Physical properties……………………………………………...... 73 6.1. Dilution end point…………………………………………………...73 6.2 Thermal inactivation point…………………………………………...75 Contents

6.3. Effect of pH…………………………………………...... 77 6.4. Aging…………………………………………...... 80 7. Effect of different salts………………………………………………....81 7.1. Effect of different concentrations of salts on Kosakonia sacchari…81 7.2. Effect of different salts on the isolated bacteriophages …………....83 7.2.1. Effect of monovalent salts…………………………………...83 7.2.2. Effect of divalent salts……………………………………….88 7.2.3. Effect of trivalent salts……………………………………….93

8. Effect of different heavy metals salts…………………………………..97 8.1. Effect of different heavy metals on Kosakonia sacchari…………..97 8.2. Effect of different heavy metals salts on the isolated bacteriophages……………………………………………………..98

9. Effect of different sugars…………………………………………....….103 9.1. Effect of different sugars on Kosakonia sacchari…...... 103 9.2. Effect of different sugars on the isolated bacteriophages ……...... 104 9.2.1. Effect of monosaccharides…………………………………..104 9.2.2. Effect of disaccharides………………………………………108

10. Effect of some plant extracts………………………………...... 113 10.1. Effect of some plant extracts on Kosakonia sacchari…………...113 10.2. Effect of some plant extracts on the isolated bacteriophages…....114

11. Effect of different essential oils……………………………...... 120 11.1. Effect of different essential oils on Kosakonia sacchari………..,.120 11.2. Effect of different essential oils on the isolated bacteriophages....120

12. Application of phage therapy in the lab………………………………..126 Contents

12.1. Application of phage therapy in the lab using vB_KsaM-C1………...... 126

12.2 . Application of phage therapy in the lab using vB_KsaO-C2 …………………………………………………………127

Discussion………………………………………………………...... 128 Summary…………………………………………………………………...139 References……………………………………………………………….....143

List of Figures

List of Figures Page

Fig.(1): Showing potato soft rot symptoms ………………………...….…..59 Fig.(2):Pathogenicity test of three different bacterial isolates…………… ..60

Fig.(3): Simmon citrate test of the three bacterial isolates ………………..61 Fig.(4): Phylogenetic analysis of the bacterial isolate B1 according to Blastn……………………………………………………………..64 Fig.(5): Phylogenetic analysis of the bacterial isolate B2 according to Blastn………………………………………………………….....65 Fig.(6): Phylogenetic analysis of the bacterial isolate B3 according to Blastn……………………………………………………………...66

Fig.(7): Two different shaped plaques appeared in case of using Kosakonia sacchari as a host………………………………………………….67

Fig.(8): Double layer assay of Kosakonia sacchari as a host and Ph1 diluted to 10-7(A) and Ph2 diluted to 10-5 (B)…………………..…………68

Fig.(9): TEM of Ph1 negatively stained by 3% uranyl acetate, TEM magnification x150000 and bar 100 nm…………………………..69

Fig.(10): TEM of Ph2 negatively stained by 3% uranyl acetate, TEM magnification x200000 and bar 50 nm…………………………..69

Fig.(11): 0.8% Agarose gel electrophoresis of Ph1 nucleic acid.……….....70

Fig.(12): 0.8% Agarose gel electrophoresis of Ph2 nucleic acid…………..70

Fig.(13): The effect both isolated bacteriophages on a list of bacteria…….72

Fig.(14): Showing the thermal inactivation point for phage vB_KsaM-C1 at 65oC……………………………………………………………….76

I List of Figures

Fig.(15): Showing the thermal inactivation point for phage vB_KsaO-C1 at 65oC…………………………………………………………...... 77 Fig.(16): Showing the optimum activity for vB_KsaM-C1 at pH (7-8)………………...... 78 Fig.(17):Showing the optimum activity for vB_KsaM-C1 at pH 7………………………………………………………………..80 Fig.(18): Effect of different concentrations of salts on Kosakonia sacchari ……………………………...……………...82 Fig.(19) :Effect of different concentrations of NaCl on vB_KsaM- C1…………...... 84 Fig.(20): Effect of different concentrations of NaCl on vB_KsaO-C2…………………………………………………...... 85 Fig.(21): Effect of different concentrations of KCl on vB_KsaM-C1…………………………………………..……….....86 Fig.(22):Effect of different concentrations of KCl on vB_KsaOC2…………………...... 87

Fig.(23): Effect of different concentrations of CaCl2.2H2O on vB_KsaM-C1……………………………………………………..89

Fig.(24): Effect of different concentration of CaCl2.2H2O on vB_KsaO-C2…...………………………………………………...90

Fig.(25):Effect of different concentrations of MgCl2.6H2O on vB_KsaM-C1……………………………………………...... 91

Fig.(26):Effect of different concentrations of MgCl2.6H2O on vB_KsaO-C2…………...... 92

Fig.(27):Effect of different concentrations of anhydrous AlCl3 on vB_KsaM-C1……...... 94

Fig.(28):Effect of different concentration of anhydrous AlCl3 on vB_KsaO-C2……………………………………………………...95

Fig.(29):Effect of different concentration of FeCl3 on vB_KsaM-C1………96

Fig.(30): Effect of different concentration of FeCl3 on vB_KsaO-C2……...97

II List of Figures

Fig.(31): Effect of different concentrations of heavy metals salts on Kosakonia sacchari…………………………………………...... 98 Fig.(32): Effect of different heavy metals salts on vB_KsaM-C1………....100 Fig.(33): Effect of different heavy metals salts on vB_KsaO-C2 ………....102 Fig.(34): Effect of different sugars on Kosakonia sacchari …………….....103 Fig.(35): Effect of different concentration of glucose on vB_KsaM-C1…..105 Fig.(36): Effect of different concentration of glucose on vB_KsaO-C2….. 106

Fig.(37): Effect of different concentration of fructose on vB_KsaM-C1.....107

Fig.(38): Effect of different concentration of fructose on vB_KsaO-C2…..108

Fig.(39): Effect of different concentration of lactose on vB_KsaM-C1…...109 Fig.(40): Effect of different concentration of lactose on vB_KsaO-C2…....110 Fig.(41): Effect of different concentration of sucrose on vB_KsaM-C1…..111 Fig.(42): Effect of different concentration of sucrose on vB_KsaO-C2…...112 Fig.(43): Effect of different plant extracts on Kosakonia saccahri ………..113

Fig.(44): Effect of different plant extracts on vB_KsaM- C1………...…....116

Fig.(45): Effect of different plant extracts on vB_KsaO-C2 …………...... 119

Fig.(46): Effect of different essential oils on Kosakonia saccahri ………..120

Fig.(47): Effect of different essential oils on vB_KsaM-C1……………….122 Fig.(48): Effect of different essential oils on vB_KsaO-C2 ……………....125 Fig.(49):Application of phage therapy in the lab using vB_KsaM-C1 ……126 Fig.(50): Application of phage therapy in the lab using vB_KsaO-C2……127

III

List of Tables

List of Tables Page

Table (1): List of plant extracts used to study their effect on the isolated bacteriophages…………………………………………………55

Table (2): List of Essential oils used to study their effect on the isolated bacteriophages………………………………………………….57

Table (3): Biochemical tests of the three isolated bacteria (B1, B2, B3)…...... 62 Table (4): Host range of the two isolated bacteriophages vB_KsaM-C1 (Ph1) and vB_KsaO-C2 (Ph2) against different bacteria causing soft rot disease…………………………………………………………...73

Table (5): Tenfold dilution assay of vB_KsaM-C1 (Ph1)…………………74 Table (6): Tenfold dilution assay of vB_KsaO-C2 (Ph2)……………….....75 Table (7): The effect of different temperatures on vB_KsaM-C1(Ph1)…...75 Table (8): The effect of different temperatures on vB_KsaO-C2 (Ph2)……76 Table (9): Effect of different pH values on vB_KsaM-C1 (Ph1)………...... 78

Table (10): Effect of different pH values on vB_KsaO-C2 (Ph2)…………79 Table (11): Aging of both isolated bacteriophages vB_KsaM-C1 (Ph1) and vB_KsaO-C2 (Ph2)…………………………………………….81

Table (12): Effect of different concentrations of NaCl on vB_KsaM-C1...83 Table (13): Effect of different concentrations of NaCl on vB_KsaO-C2…84 Table (14): Effect of different concentrations of KCl on vB_KsaM-C1….86 Table (15): Effect of different concentrations of KCl vB_KsaO-C2……...87

IV List of Tables

Table (16): Effect of different concentrations of CaCl2.2H2O on

vB_KsaM-C1…………………………………………………..88

Table (17): Effect of different concentration of CaCl2.2H2O on vB_KsaO-C2 ………………………………...…………….90

Table (18): Effect of different concentrations of MgCl2. 6H2O on vB_KsaM-C1…………………………………………………..91

Table (19): Effect of different concentration of MgCl2.6H2O on vB_KsaO-C2..……………………………………...…………..92

Table (20): Effect of different concentration of anhydrous AlCl3 on vB_KsaM-C1……………………………………………………93

Table (21): Effect of different concentrations of anhydrous AlCl3 on vB_KsaO-C2…………………………………………………..94

Table (22): Effect of different concentration of FeCl3 on vB_KsaM-C1….95

Table (23): Effect of different concentration of FeCl3 on vB_KsaO-C2….96 Table (24): Effect of different heavy metals on vB_KsaM-C1…………..100 Table (25): Effect of different heavy metals salts on vB_KsaO-C2……..102 Table (26): Effect of different concentration of glucose on vB_KsaM-C1 ………………………………………………..104 Table (27): Effect of different concentration of glucose on vB_KsaO-C2…………………………………………………105 Table (28): Effect of different concentration of fructose on vB_KsaM-C1………………………………………………...107 Table (29): Effect of different concentration of fructose on vB_KsaO-C2…………………………………………………108 Table (30): Effect of different concentration of lactose on vB_KsaM-C1………………………………………………...109

V List of Tables

Table (31): Effect of different concentration of lactose on vB_KsaO-C2………………………………………………...110 Table (32): Effect of different concentration of sucrose on vB_KsaM-C1………………………………………………...111 Table (33): Effect of different concentration of sucrose on vB_KsaO-C2…………………………………………………112 Table (34) : Effect of different plant extracts on vB_KsaM-C1………….116 Table (35) : Effect of different plant extracts on vB_KsaO-C2…………..119 Table (36): Effect of different essential oils on vB_KsaM-C1…………...122 Table (37) : Effect of different essential oils on vB_KsaO-C2…………...124

List of Abbreviations

Abbreviations

Bacterial virus vB Crystal Violet Pectate CVP Crystal Violet Sodium PolyPectate CVPP DeoxyRiboNucleic Acid DNA Degree Celsius oC Dilution End Point DEP Enrichment-Enzyme-linked Immunosorbent Assay E-ELISA Field Asymmetric Ion Mobility Spectrometry FAIMS Gram g Hectar ha Immuno Fluorescence bacterial Colony staining IFC Intergenic Transcribed Spacer ITS ITS-Restriction Rragment Length Polymorphism ITS-RFLP Kosakonia sacchari Ksa Longevity In Vitro LIV Matrix Assisted Laser Desorption/Ionization–Time of Flight MALDI-TOF Microbiological Resource Center MIRCEN Multilocus Sequence Analysis MLSA Microviridae O Microlitre µL Milliliter mL

VII List of Abbreviations

Millimolar mM Myoviridae M Nanometer nm Nutrient Agar NA Nutrient Broth NB Nutrient Glycerol Manganese NGM Plant Tissue PT Poly Ethylene Glycol PEG Polymerase Chain Reaction PCR Phosphate Buffer Saline PBS Random Amplified Polymorphic DNA RAPD Restricted Fragment Length Polymorphism RFLP Ribosomal RNA rRNA RiboNucleic Acid RNA Sodium Dodecyl Sulfate SDS Species sp. Thermal Inactivation Point TIP Transmission Electron Microscope TEM Triphenyl Tetrazolium Chloride TTC Tris-Acetate-EDTA TAE Ultraviolet UV Volatile Organic Compounds VOCs

VIII Abstract

Candidate Name: Eman Abd Elhafeez Aldeeb Abd Elhamid

Thesis title: Isolation and identification of bacteriophage(s) infecting bacteria which cause potato soft rot disease

Degree: Master of Science

Abstract

Potato crop is one of the most economically important crops, and is considered as the fourth main food crop in the world. Potato plants are subjected to numerous pathogens and insects which cause considerable loss in the potato yield. One of the most important serious diseases of potato is bacterial soft rot disease. In our study, the main bacteria causing soft rot disease were isolated from two infected potato species (Solanum tuberosum L.cv. Spunta and Solanum tuberosum L.cv. Lady rosetta). The isolated bacterial samples were identified by biochemical tests (Gram stain, oxidase, catalase, citrate, starch hydrolysis and gelatin hydrolysis) and confirmed by molecular sequencing to be strains of Lelliottia amnigena, Kosakonia sacchari and Enterobacter cloaca. The bacterial isolates were recorded in the gene bank and were given accession numbers KY427021, KY235364 and KY235363, respectively. In our study, two bacteriophages infecting soft rot bacteria were isolated and identified. Soil samples were collected from potato rhizosphere from a potato cultivated area in Giza, Egypt. The two bacteriophages were isolated, purified and characterized using Transmission Electron Microscope (TEM), Dilution End Point (DEP), Longevity In Vitro (LIV), Thermal Inactivation Point (TIP), pH effect and host range. The two bacteriophages were named vB_EsaM-C1 and vB_EsaO-C2 belonging to Myoviridae and Microviridae, respectively. Bacteriophage vB_EsaM-C1 possessed icosahedral head (58.04 nm), neck (13.13nm(L) X 8.06nm(W)) and tail (96.42 nm(L)X 14.34nm(W)). DEP was 10-7, LIV was up to 20

IX Abstract months but with a significant decrease in phage titre, TIP was 65oC, optimum pH was 7-8. Bacteriophage vB_EsaO-C2 showed polyhedral heads (23.45nm). DEP was 10-9, LIV was up to 20 months but with a significant decrease in phage titre, TIP was 65oC, optimum pH was 7. Effect of different mono, di and trivalent salts, heavy metals, different types of monosaccharides and disaccharides and different types of plant extracts and essential oils were tested against the two isolated bacteriophages. A small scale application of the two isolated bacteriophages on infected potato tubers discs, in the lab, successfully inhibited bacterial soft rot disease.

Key words: Potato soft rot, Bacteriophages, Titre, Host range, Longevity in vitro, Myoviridae, Microviridae.

Supervisors:

Signature

1. Prof. Dr. Elsayed Tarek Abd El Salam………………………..

2. Dr. Maha Mohamed Al Khazindar…………………………….

Prof. Dr. Neveen Saleh Geweely

Head of Botany and Microbiology Department

Faculty of Science – Cairo University

Introduction and Aim of work

Tuber and root crops are the most important carbohydrate sources of food produced in many subtropical and tropical countries. Potato (Solanum tuberosum L.) is one of the most economically important tuber crop, and is considered as the fourth main food crop in the world after rice (Oryza sativa), maize (Zea mays) and wheat (Triticum aestivum). Potato does not require special growth conditions. It is the major field crop in temperate regions, and increasingly in warmer regions (Czajkowski et al., 2011). Recently, potato production has spread in both cool and hot dry areas, so, many varieties have become available in many countries (Kabiel et al., 2008).

In Egypt, potato is one of the most important and strategic vegetable crop for local consumption, export and industry, therefore, potato can be cultivated more than two times in a year (El Helaly, 2012). Approximately 20% of total area devoted for vegetable production is cultivated with potato (Kabiel et al., 2008), as 17800 ha of cultivated areas is responsible for the production of about 4800000 tons of potato (Abdalla et al., 2016). Thus, any disturbance in the production of this crop affects severely its local consumption and export impact. Potato crop is subjected to a great annual loss in the world due to viral, bacterial and fungal diseases and pests. In Egypt, those diseases affect greatly the quantitative and qualitative potato yield, working as a key barrier to the improvement of potato (Kabiel et al., 2008).

1 Introduction and Aim of work

One of the most important serious diseases of potato is bacterial soft rot which causes approximately one billion dollars of damage annually in potato worldwide (Perombelon, 2002 and Chung et al., 2013). Soft rot of potato is the second important economic disease after bacterial wilt disease (Czajkowski et al., 2011). Enterobacteriaceae is a large family of Gram-negative bacteria comprising about twenty nine phytopathogenic bacteria as clarified by Hauben et al., (1998). Enterobacteriaceae were familiarly known as enterobacteria or enteric bacteria.

Enterobacter is considered as an important genus in Enterobacteriaceae, found in all habitats as water, plants, soils, stool of animals and normal intestinal flora. Enterobacters were found to be phytopathogens causing many economically important plant diseases (Liu et al, 2016). From the most important examples that have been detected recently: Enterobacter cloacae which was isolated as the causative agent of soft rot disease for dragon fruit in Peninsular Malaysia (Masyahit et al., 2009), onion bulbs decay in Columbia Basin of Washington State, USA (Schroeder and du Toit, 2010), ginger rhizome rot in Brazil (Moreira et al., 2013) and bacterial wilt of mulberry in China (Wang et al., 2008). Enterobacter mori was isolated and identified to cause bacterial wilt of mulberry in China (Zhu et al., 2010). Enterobacter nimipressuralis was isolated from elm trees infected by bacterial wet wood in Rafsanjan, Iran (Khodaygan et al., 2012) and Lelliottia amnigena was isolated as new Enterobacter species causing soft rot decay of onion bulbs in China (Liu et al., 2016). . Enterobacter was known as one of the largest genera in Enterobacteriaceae family, characterized by its long and complicated history and comprising about 19 species, 50% of these species have been identified to be novel species recently. Several species were transferred to and from this genus depending

2 Introduction and Aim of work upon its polyphyletic nature and 16S rRNA gene sequences, where many species were reclassified from this genus into three proposed novel genera, known as: Lelliottia gen. nov., Pluralibacter gen. nov. and Kosakonia gen. nov (Brady et al., 2013).

Bacteriophages are viruses that specifically infect bacteria. They are effective against antibiotic or heavy metal-resistant bacteria, so can be used as alternatives of antimicrobial agents in plant protection (Soleimani et al., 2012 and Lim et al., 2013). Bacteriophages are natural components of the biosphere and are nontoxic to the eukaryotic cell (Lim et al., 2013). They are found where bacteria reside as in soil and sewage (Sheng et al., 2010). The infection cycle of a phage is an important consideration when choosing a phage for antibacterial application. Thus, they are commonly classified as either virulent or temperate according to their replication cycle. Temperate phage directs its replication towards lysogenic cycle giving chance for transference of virulence factors as antibiotic resistance, therefore, temperate phages are unsuitable for phage therapy while, virulent phages that use lytic cycles are ideal for phage therapy application (Keary et al., 2013). Recently, bacteriophages have been isolated, identified and proposed as biocontrol agents for bacterial diseases in plants. Examples of scientific contributions in controlling soft rot disease: bacteriophages belonging to Myoviridae were isolated by Ravensdale et al., (2007) from fertilizer solutions of green house, Adriaenssens et al., (2012) from potato soil samples, Lim et al., (2014) from Chinese cabbage field, Delfan et al., (2015) from Capsian sea water and Czajkowski et al., (2015) from potato samples collected from two different potato fields. Other phages belonging to Podaviridae were isolated by Lim et al., (2013) from soil samples and Hirata et al., (2016) from an infected Japanese horseradish

3 Introduction and Aim of work rhizome by soft rot. Other bacteriophages belonging to Siphoviridae were isolated by Ravensdale et al., (2007) from fertilizer solutions of green house and Delfan et al., (2015) from Capsian sea water. Phage therapy used to control phytopathogens is associated with some factors: a) the rapid destruction of phage by UV radiation surmounted by the application of phage in the evening, b) the location of susceptible bacteria, c) the moisture levels in the environment and on the leaf, d) the presence of chemicals which may compromise the viability of phage population (Keary et al., 2013). The aim of our study is to: 1) isolate and identify bacteria causing potato soft rot from infected potato tubers, 2) isolate and characterize bacteriophages infecting those bacteria causing potato soft rot that can be used as biocontrol agents.

❖ Objectives: 1. Collection of infected potato samples from different potato cultivated areas in Egypt infected by soft rot bacteria. 2. Isolation of the infecting bacteria from the infected potato tubers. 3. Identification of the bacteria causing soft rot disease using biochemical tests and molecular sequencing using 16S rRNA. 4. Isolation of the specific bacteriophages from potato tubers rhizosphere. 5. Purification of the isolated bacteriophages. 6. Studying the morphology of the isolated bacteriophages by transmission electron microscope.

4 Introduction and Aim of work

7. Studying the physical properties (dilution end point, longevity in vitro, thermal inactivation point and effect of different pHs) of the isolated bacteriophages. 8. Studying the effect of different chemicals, sugars, some natural plant extracts and essential oils. 9. Small scale application of the isolated phages on potato tubers to control soft rot diseese.

Review of Literature

Part I

Isolation of bacteria causing soft rot disease

Jones (1901) was the first to record soft rot disease in carrot, caused by a motile, peritrichously flagellated bacterium called Bacillus carotovorus.

Rahn (1937) suggested the introduction of Enterobacteriaceae as a new family comprising the two genera of peritrichously flagellated bacteria, Enterobacter of animal enterics and the plant pathogenic bacteria, Erwinia.

Burkholder (1939) found that Escherichia coli and Aerobacter spp. were the causative agents for some rot diseases.

Waldee (1945) separated the soft rot pectolytic group of bacteria from the nonpectinolytic one into two separate genera, Pectobacterium for pectolytic soft rot bacteria and Erwinia for the nonpectinolytic group.

Malcolmson (1959) isolated three Erwinia isolates from infected potato tubers by soft rot and blackleg. The bacterial isolates were identified as Erwinia carotovora, Erwinia atroseptica and Erwinia aroideae, using some biochemical tests such as utilization of nitrogen, ethyl alcohol, glycerol and maltose.

6 Review of Literature

Perombelon (1971) isolated soft rot bacterial species of Erwinia carotovora var carotovora and Erwinia carotovora var atroseptica from soil, using modified semi-selective Stewart’s medium to enumerate all soft rot Erwinia in soil.

Cuppels and Kelman (1974) isolated soft rot bacteria from soil and decaying plant tissue on Crystal Violet Pectate (CVP) medium as a selective medium containing NaNO3, sodium polypectate and crystal violet. Pectolytic Erwinia spp. and Pseudomonas spp. were identified by the colonial morphology and shape of depression formed on the selective CVP medium.

Graham (1976) suggested the reason of re-infection of healthy propagated potato tubers by soft rot bacteria was due to the spread of bacteria as Erwinia carotovora var carotovora by insects or by aerosols droplets.

Burr and Schroth (1977) isolated bacteria causing soft rot disease from the rhizosphere of weeds and crop plants on Plant Tissue (PT) medium as a differential medium, using an anaerobic enrichment method. The isolated soft rot bacteria were identified as Erwinia carotovora var. carotovora and Erwinia carotovora var. atroseptica.

Perombelon and Kelman (1980) found that the causative agents of potato soft rot are Erwinia carotovora and Erwinia atrosepticum while black leg is caused by Erwinia crysanthemi. Soft rot was described as slimy, wet, black rot lesions spreading from the mother tuber up the stems, under wet conditions, due to the presence of pectate lyase enzyme. Other symptoms were stunting, yellowing, wilting and desiccation of stems and leaves under dry conditions. On the other

7 Review of Literature hand, extensive stem rotting from top and progress downwards was recorded in late summer and rainy conditions, which could be confused with those symptoms of black leg.

Quinn et al., (1980) isolated Erwinia carotovora var carotovora and Erwinia carotovora var atroseptica from the atmospheric air during rain fall around potato crops. This proved that the contamination of healthy potato crops by soft rot Erwinia might have occurred during rain fall carrying bacterial aerosols.

Liao (1987) reported pectolytic bacterial genera causing soft rot disease other than the known Erwinia species. Some strains that caused rotting of potato slices were identified as Aeromonas liquefaciens and Flavobacterium sp. which are common inhabitants of soil and plant surface. Furthermore, he isolated Klebsiella oxytoca and Yersinia enterocolitica with pectolytic activity which are known for their pathogenicity to animals but not plants.

Leifert et al., (1989) investigated the causative agents of contamination of micropropagated plant cultures by isolating different bacterial strains from infected plants. Bacteriological identification of these isolated strains reported species of Bacillus, Enterobacter, Micrococcus, Staphylococcus, Pseudomonas and Lactobacillus.

Perombelon and Burnett (1991) isolated the three soft rot Erwinia by using two modified selective media of the known selective Crystal Violet Pectate (CVP) media. One medium was a single layer and the other was a double layer medium consisting of an agar base and a pectate over layer. Both media contained novobiocin instead of

8 Review of Literature sodium lauryl sulphate of the original CVP. This modification showed higher recovery rate and selectivity than in the original CVP one.

Barras et al., (1994) mentioned that soft rot Erwinia produce a group of cell wall degrading enzymes upon infection of its host to allow its penetration and maceration of host plant tissue to obtain its nutrients.

Parent et al., (1996) isolated 40 bacterial strains from infected plants cultivated in Quebec province of Canada to screen the Random Amplified Polymorphic DNA (RAPD) primers. Two primers were selected to differentiate between soft rot bacterial strains as differentiation between Erwinia carotovora subsp. carotovora and Erwinia carotovora subsp. atroseptica, also differentiation between Erwinia carotovora and pectolytic Pseudomonas spp..

Hauben et al., (1998) clarified the phylogentic position of the twenty nine phytopathogenic bacteria in Enterobacteriaceae, including genera of Erwinia, Pantoea and Enterobacter, using 16s rDNA. Erwinia species were divided into three groups: the first group represented Erwinia species as Erwinia amylovora, Erwinia maliotivora, Erwinia persicinus, Erwinia psidii, Erwinia rhapontici and Erwinia tracheiphila. The second group represented the merging of Erwinia carotovora subsp. (atroseptica, betavasculorum, carotovora, odorifera and wasabiae), Erwinia cacticida, Erwinia chrysanthemi and Erwinia cypripedii in the genus Pectobacterium, to gain new nomenclatures to be Pectobacterium carotovorum subsp. (atrosepticum, betavasculorum, carotovorum, odoriferum and wasabiae), Pectobacterium cacticidum, Pectobacterium chrysanthemi

9 Review of Literature

and P. cypripedii, respectively. The third group that comprised Erwinia alni, Erwinia nigrifluens, Erwinia paradisiaca, Erwinia quercina, Erwinia rubrifaciens and Erwinia salicis, was placed in a new genus Brenneria to gain new names of Brenneria sp.. On the other hand, Pantoea spp. were placed in monophyletic unit related to Erwinia, while the phytopathogenic Enterobacter spp. were spreading among Citrobacter and Klebsiella phylogenetic distribution.

Kushalappa and Zulfiqar (2001) studied the effect of wet incubation time and temperature on the infection of potato tubers by soft rot disease caused by Erwinia cartovora subsp. carotovora. The effect of storage time and temperature on the expansion of soft rot lesions in the infected potato tubers were also studied. The recorded infection by soft rot at wet conditions was at 20oC after 12h, and lesions expansion after 60 days at 16oC.

Toth et al., (2001) used the 16S-23S rRNA Intergenic Transcribed Spacer (ITS) and ITS-Restriction Fragment Length Polymorphism (ITS-RFLP) as a simple, precise and rapid method for identification of soft rot Erwinia. In contrast to other traditional molecular and phenotypic identification tools which were characterized by the imprecision and time consumption.

Perombelon and van der Wolf (2002) developed and evaluated four methods for the detection of Pectobacterium carotovorum subsp. atrosepticum from contaminated potato seed tubers. Three of which depended on Pectobacterium carotovorum subsp. atrosepticum specific antibodies for its detection. Immunomagnetic separation of bacterial

10 Review of Literature cells followed by selective growth at differential temperatures on crystal violet pectate media (IMS-CVP), immunofluorescence bacterial colony staining (IFC) and bacterial enrichment before ELISA detection (E-ELISA). The fourth method depends on the bacterial DNA amplification using Pectobacterium carotovorum subsp. atrosepticum specific primer by Polymerase Chain Reaction (PCR).

De Boer (2003) characterized Pectobacterium spp. as sophisticated group of bacteria related to their pathogenicity and virulence genes as hrp gene cluster in association with type III secretion system and virulence global regulators which control motility and production of cell wall extracellular enzymes that were activated by the diffusible signal molecule N-acyl-homoserine lactone.

Bdliya and Langerfeld (2005) isolated the phytopathogenic bacteria, Erwinia carotovora subsp. carotovora using a modified semi-selective media of Crystal Violet Sodium PolyPectate (CVPP) double layer media, that could be used for both selection and differentiation between the three types of soft rot Erwinia, where Erwinia carotovora subsp. carotovora appeared as red to pink colonies with cavity formation.

Charkowsky (2006) characterized soft rot Erwinia as pectinolytic, Gram-negative, nonspore forming, facultative anaerobic, motile rods with peritrichous flagella and belonging to ɣ-protobacteria in Enterobacteriaceae.

Lee and Yu (2006) isolated and identified Erwinia chrysanthemi from infected Phalaenopsis orchids in Taiwan, using nutrient glycerol

11 Review of Literature manganese (NGM) medium as a differential medium on which Erwinia chrysanthemi strains could grow in the form of dark brownish to blue colonies that could be differentiated from other soft rot Erwinia spp.. PCR amplification for indC oligonucleotide primer was specific for Erwinia chrysanthemi strains only.

Mills et al., (2006) studied the effect of several chemical compounds on the inhibition of Erwinia carotovora subsp. carotovora and Erwinia carotovora subsp. atroseptica. Sodium metabisulfite, propyl-paraben, alum, potassium sorbate, calcium propionate and copper sulfate pentahydrate showed inhibitory action at the lowest concentration of 0.002M, which proved their role in soft rot disease control.

Mahmoudi, et al., (2007) isolated and purified 8 strains of pectolytic Erwinia spp. from infected bulbs and stems of crown imperial showing brown spot and soft rot symptoms. They were identified phenotypically, biochemically and by PCR amplification, in which a pair of specific primers of (EXPCCF/EXPCCR) and (INPCCF/INPCCR) was used, to identify these isolated bacterial strains to be strains of Pectobacterium carotovorum subsp. carotovorum.

Laurila et al., (2008) isolated soft rot Pectobacterium strains from infected potato stems and tubers that were identified as Pectobacterium carotovorum and Pectobacterium atrosepticum using PCR tests. Dickeya strains were isolated from potato samples and river waters in Finland. The identification was done using phylogenetic analysis with

12 Review of Literature

16S-23S rDNA intergenic spacer sequences which divided them into three different groups causing diseases in potato.

Masyahit et al., (2009) isolated the pathogenic bacteria causing dragon soft rot and identified the isolated bacteria by in vitro pathogenicity test and biolog analysis to be Enterobacter cloaca. This was the first report of soft rot caused by Enterobacter cloaca on dragon fruit in Peninsular Malaysia.

Hammed and Alyaa (2010) isolated 7 strains of Erwinia carotovora from 20 samples of infected potato in Iraq local markets. The isolated bacterial strains were subjected for obtaining pectin lyase. The enzyme activity increased in the presence of Ca2+ and Mg2+ ions, and was inhibited in the presence of Co2+, Hg2+, Ni2+, Zn2+ and Sn2+ ions.

Schroeder and du Toit (2010) reported the causative bacteria of bulb decay of stored onion. It was identified as Enterobacter cloaca.

Zhu et al., (2010) isolated thirty two strains of Pectobacterium carotovorum ssp. from Chinese cabbage and other vegetables. The isolated bacterial strains were identified by morphological, biochemical, physiological and molecular tests using 16S rDNA sequence analysis. Five strains of the identified bacteria were found to be related to novel strains of Pectobacterium carotovorum ssp. as indicated by the phylogenetic analysis of 16S rDNA sequence.

Czajkowski et al., (2011) stated that tuber soft rot is initiated at lenticels, the stolon end or wounds under wet conditions. This results in

13 Review of Literature macerated tissue sites in the form of lesions that spread to the whole tuber turning into macerated mass, that spreads to the neighboring tubers in storage. This rotting is accompanied by blackening of macerated tissue in the presence of air developing foul odour.

Ismail et al., (2012) isolated soft rot Erwinia carotovora subsp. carotovora, from infected girasole tubers (Helianthus tuberosus L. cv. Balady). The isolated bacterial strains were rod shaped and Gram negative bacteria. Biochemical tests showed positive results in α- methyl-d-glucoside medium, sucrose reducer, phosphatase activity and formed deep cavities on pectate medium. The isolated bacterial strain showed broad host range infecting potato tubers, sweet potato, egg plant, cloves of bulbs, garlic, onion, radish root and some fruits as, apricot, apple, olive and lemon.

Ngadze et al., (2012) isolated strains of bacteria causing potato soft rot and black leg from infected potato tubers and stems. The isolated bacteria were identified as 12 isolates of Pectobacterium carotovorum subsp. brasiliensis, 20 isolates of Dickeya dadantii subsp. dadantii, 16 isolates of Pectobacterium carotovorum subsp. carotovorum and 4 isolates of Pectobacterium atrosepticum. The identification was based on biochemical tests, rep-PCR, Restricted Fragment Length Polymorphism (RFLP) and sequences of gyrB and recA genes.

Al-Zomor et al., (2013) isolated 46 bacterial strains of Erwinia carotovora subsp. atroseptica causing potato black leg disease, in Jordan. The isolated bacterial strains were identified using different

14 Review of Literature biochemical, physiological and nutritional tests. The identification was confirmed by molecular studies.

Chung et al., (2013) studied the opportunity of potato infection by soft rot disease during long term storage and reported that some resistant potato cultivars as Freedom Russet, Anett and Alaska Red Eye, sustained its resistance against soft rot up to six months. On the other hand, other susceptible potato cultivars lost their resistance for infection by soft rot disease during storage.

Moreira et al., (2013) isolated Pseudomonas fluorescens and Enterobacter cloacae and reported them to cause ginger rhizome rot in Brazil.

Nykyri (2013) identified and characterized novel virulence genes of Pectobacterium wasabiae SCC3193 and Pectobacterium atrosepticum SCRI1043 by genomic studies including genome sequencing and post genomic studies.

Rashid et al., (2013) isolated soft rot bacterium from infected potato tuber that was identified as Erwinia carotovora subsp. carotovora using morphological, pathological and biochemical studies. In vitro screening of five different chemicals and five biocontrol agents against the isolated soft rot bacterium was done by well diffusion method. Copper oxychloride was the most effective chemical and Bacillus subtilis was the most effective biocontrol agent against Erwinia carotovora subsp. carotovora.

Anajjar et al., (2014) isolated Pectobacterium strains from symptomless potato tubers and soil on Crystal Violet Pectate (CVP)

15 Review of Literature medium after enrichment step. The strains were identified by biochemical and pathogenicity tests and confirmed by PCR using Y1 and Y2 primers.

Nunes et al., (2014) reported the first incidence of potato black leg caused by Pectobacterium carotovorum subsp. brailiensis in Netherlands.

Potrykus et al., (2014) introduced multiplex PCR as a fast and reliable assay for the identification of soft rot and black leg bacteria isolated from infected potato tubers in Poland. The three pectinolytic bacteria causing soft rot, were identified as Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. carotovorum and Pectobacterium wasabiae.

Rutolo et al., (2014) introduced a new tool for the detection of potato soft rot bacteria using a new technology via gas/vapour analysis known as Field Asymmetric Ion Mobility Spectrometry (FIAMS).

Van Der Wolf et al., (2014) isolated a novel bacterial strain of Dickeya solani from infected potato tubers in different European countries and Israel. Polymerase chain reaction using pelADE primers and bacterial gene sequencing using 16S rRNA placed the novel bacterial strain in a distinct genetic clade. In addition, the characterization by whole cell MALDI-TOF mass spectroscopy, pulsed field gel electrophoresis after cutting DNA by rare restriction enzymes, average nucleotide identity analysis and DNA-DNA hybridization studies, showed the isolation of a new Dickeya strain known as Dickeya solani sp. nov. .

Delfan et al., (2015) isolated two phytopathogenic strains of Dickeya daddanti, causing potato soft rot. Both strains were identified using

16 Review of Literature

16S rRNA gene sequencing and submitted to the gene bank with the accession numbers HQ423668 and HQ423669.

Panda et al., (2015) reported a draft genome sequence [ICMP 5702 (ATCC 15713)] of the soft rot enterobacteria and Pectobacterium carotovorum subsp. carotovorum. This is considered as an important reference for any future phylogenomic and taxonomic studies concerning the phytopathogenic Enterobacteriaceae.

Ansermet et al., (2016) studied the symptoms developed on potato tubers infected by Dickeya spp. either soil borne infection or seed tuber infection. It was found that both induced wilting and black leg symptoms in addition to decrease in the plant transpiration rate due to the colonization of xylem vessels by the bacterial cells.

Liu et al., (2016) reported the first incidence of onion bulbs decay in China, caused by a new Enterobacter species, Lelliottia amnigena.

Mohamed et al., (2016) isolated two bacterial strains causing potato soft rot diseases from infected potato tubers and potato cultivated soil in Saudi Arabia. The isolated bacterial strains were identified using bacterial gene sequencing using 16S rRNA. The phylogenetic tree showed that the two bacterial isolates belong to strains of Pseudomonas stutzeri and Bacillus pumilus.

Umunna and Austin (2016) reported the great destructive loss in wide range of vegetables caused by Erwinia spp.. The pathogenic bacteria enter its host through wounds or natural openings as lenticels, stomata or hydrathodes releasing cell wall degrading enzymes as pectin methyl estrases, cellulases, zylanases and polygalacturanases causing degradation of the middle lamella followed by cell death. Soft rot

17 Review of Literature infection spreads at high temperature and humidity and is transmitted by water or insects from infected plant to healthy one. On the other hand, the infection at storage might occur by touching cell exudates of infected plant.

Van Der Wolf et al., (2017) compared Pectobacterium carotovorum subsp. brasiliense with each of Pectobacterium carotovorum subsp. atrosepticum , Pectobacterium carotovorum subsp. wasabiae, Dickeya solani and Dickeya dianthicola. It was found that Pectobacterium carotovorum subsp. brasiliense was the most virulent bacteria causing soft rot disease.

Sinha et al., (2017) introduced a new technology for the early detection of potato soft rot disease caused by Pectobacterium carotovorum subsp. carotovorum. Field Asymmetric Ion Mobility Spectroscopy (FAIMS) senses the mobility of ions of Volatile Organic Compounds (VOCs) released as a result of tuber rotting.

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Part II

Isolation of bacteriophages

Hankin (1896) reported that drinking water from Ganges and Jumna rivers in India showed antibacterial action against Vibrio cholerae preventing spread of cholera epidemics. He suggested that an unidentified, filterable and heat labile substance caused this phenomenon.

Towrt (1915) described a “glassy transformation” as an ultramicroscopic virus that infected Micrococci bacteria that was isolated from calf vaccinia lymph.

D’Herelle (1917) described the effective agent against dysentery pathogen as a filterable virus that was parasitic on bacteria. It was obtained from feces of people recovering from this disease. The agents were named bacteria eaters. This led him to be the first discoverer of bacteriophage.

Mallman and Hemstreet (1924) isolated a phage from the filtrate of rotted cabbage tissues, that proved to inhibit the growth of Xanthomonas campestris pv. campestris that causes soft rot of cabbage.

Coons and Kotila (1925) proved that phages play an important role in the soil by limiting plant pathogenic bacteria. Moist carrot discs were inoculated by Erwinia carotovora subsp. carotovora. in combination with the specific isolated phage and compared with control discs. The phage protected the carrot discs from rotting.

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Anderson (1928) isolated a phage infecting Xanthornonas pruni from the soil of infected peach trees.

Massey (1934) isolated phages infecting Xanthomonas axonopodis pv. malvacearum from the soil of infected cotton crops and water of Nile river to control bacterial blight disease.

Thomas (1935) isolated a phage from diseased corn seeds to control Pantoea stewartii that causes Stewart’s wilt of corn. The phage caused the reduction of the disease from 18% to 1.4%.

Kent (1937) isolated a phage infecting Agrobacterium tumefaciens from the healthy part of tomato stem, six inches away from the formed gall.

Ruska (1940) showed the first electron micrograph photos of bacteriophages. The electron micrographs identified different phage types according to their morphology as enterobacteria phages T4, T1, T7, T5, 7–11, VII and pseudomonas phage PB1.

Rosberg and Parrack (1955) isolated a phage infecting Xanthomonas axonopodis pv. malvacearum from two-years old dry leaves of cotton plant.

Holmes (1956) studied the physico-chemical agents effect on Staphylococcus phage. The physical study comprised thermal inactivation, effect of temperature on adsorption rate, ultrasonic vibration, long term storage, lypholization and photoreactivation to UV. On the other hand, the chemical study targeted the evaluation of some types of salts and pH values effect on phage infectivity.

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Brenner and Horne (1959) introduced negative stain to visualize phage particles under electron microscope with unpredictable clear images.

Clark (1962) tried different methods for preserving bacteriophages under different conditions for long period of time. He compared preserving bacteriophage as phage lysates, phage lysates in 50% glycerol and freeze dried at room temperature (24oC-28oC) and at 4oC. After phage titre determination at different periods of time, it was found that the best method for preserving bacteriophage with least damaging effect and highest titer could be obtained by storing it as phage lysate at 4oC. Freeze drying method was unsuitable damaging method and storing phage lysate with glycerol had less damaging effect. The phage titre decreases with time independently on the preservation method.

Okabe and Goto (1963) stated that bacteriophages of plant pathogen were divided into two groups, virulent and temperate. Bacteriophages are also divided according to host range (some have wide host range and others have narrow host range), plaque morphology, biological and serological properties. phages are found where plant pathogenic bacteria reside in soil or intestines.

Eisenstark (1967) published the first phage survey listing 111 phages, 99 of which were tailed, three were isometric and nine were filamentous.

Bradley (1967) defined six basic morphotypes depending on gross morphology and nucleic acid.

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Tan and Reanney (1976) studied the interaction between bacteriophages and bacterial cells in soil. They demonstrated that a sensitive equilibrium was found between them that could be altered by altering any external factor.

Ritchie and Klos (1977) isolated three types of bacteriophages infecting Erwinia amylovora, in the form of clear-centered plaques with a spreading translucent halo or small plaques without any halo. Bacteriophages were isolated from the diseased aerial parts of apple trees at different geographical locations without the need of enrichment step due to high population of Erwinia amylovora bacteriophages at these plant parts. The isolated phages displayed thermal stability at - 20oC and 4oC but lost their activity at 24oC.

Smith and Huggins (1982) studied phage therapy on animals. They found that phages were effective against E. coli and calves diarrhea.

Ackermann and Du Bow (1987) stated that phages are the most abundant viruses infecting bacteria and archaebacteria, their sizes ranges from 20 nm to 200 nm.

Pirhonen et al., (1988) used Escherichia coli phage T4 as a selective agent to isolate resistant mutant strains of Erwinia carotovora subsp. carotovora and Erwinia carotovora subsp. atroseptica.

Gross et al., (1991) used the isolated bacteriophages controlling soft rot to study the distribution of pectobacterium spp. in the potato rhizosphere and stem, this was known as phage typing.

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Goto (1992) stated that the practical application of phages for the control of phytopathogens in the field wasn’t successful for some reasons as their narrow spectrum and broad spectrum of antibiotics.

Eayre, et al., (1995) isolated bacteriophages infecting Erwinia carotovora subsp. carotovora and Erwinia ananas from fresh water lakes in Florida and Texas.

Ackermann (1996) stated that among 4400 phages, 96% are tailed while only 4% range from cubic, filamentous or pleomorphic. He listed phages according to their morphology and host genera into 12 families, 60 % of them belong to Siphoviridae.

Lederberg (1996) isolated various phages from spleen cells and suggested that phages could be used as therapeutic viruses against antibiotic resistant organisms and could be a good target against agricultural diseases.

Toth et al., (1997) isolated M1 bacteriophage which proved to be able to infect over 25% Erwinia carotovora strains and showed transduction ability in Erwinia carotovora subsp. Atroseptica.

Barrow et al., (1998 ) isolated a lytic bacteriophage from sewage to control septicemia and meningitis disease in chickens and calves, caused by K1+ strain of Escherichia coli. In an attempt of using this isolated phage in phage therapy, phage multiplication in blood was recorded and hence delayed appearance of the host bacterium in blood was noticed.

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Gill (2000) isolated six phage types (from soil surrounding infected trees by fire blight disease and from aerial plant tissue), to control fire blight disease caused by Erwinia amylovora. Some types proved to infect the epiphytic bacteria Pantoea agglomerans. In vitro study bioassay was made by using immature pear fruit disc to evaluate the role of the isolated phages in disease control. The phages decreased the bacterial population by about 97%, but didn’t show complete elimination at the disc surface.

Keel et al., (2002) isolated a lytic bacteriophage GP100 from soil, that infected pseudomonas strains. Pseudomonas fluorescens CHA0 was used as a biocontrol agent controlling cucumber root infection caused by Pythium ultimum. Consequently, the biocontrol of this disease ceased and as a result, this isolated lytic virulent phage affected the beneficial bacteria in soil.

Tovkach (2002a) studied a new species of temperate bacteriophage (ZF40), belonging to Myoviridae and infecting Erwinia carotovora. The isolated bacteriophage was characterized by isometric head 58.3 nm in diameter and a contractile tail of 86.3 nm in length with a complex basal plate and short tail fibers of 31.5 nm in length.

Tovkach (2002b) studied the resistance of the pectinolytic Erwinia carotovora to bacteriophages using the temperate bacteriophage (ZF40). He found that the reason was probably due to blocking of bacteriophage adsorption to the surface of the bacterial cell at the level of phage-cell interaction.

24 Review of Literature

Balogh (2006) isolated several identical bacteriophages from citrus canker lesions in Florida and Argentina to control citrus canker disease caused by Xanthomonas axonopodis pv. citri and X. axonopodis pv.aurantifolii. The isolated phages proved their efficacy to eradicate citrus canker disease both in green house and field trials.

Revensdale et al., (2007) isolated fourteen bacteriophages from fertilizer solution samples from greenhouse producing calla lilly tubers, to control soft rot disease caused by Erwinia carotovora subsp. carotovora. The isolated phages belong to Caudovirales of Myoviridae and Siphoviridae. They proved to have the ability to reduce the soft rot disease by more than 50%.

Panshchina et al., (2007) characterized the temperate ZF40 bacteriophage as P2-like phages belonging to order Caudovirales of Myoviridae, and proved the ability to infect Pectobacterium carotovorum subsp. carotovorum that causes soft rot disease.

Kropinski et al., (2009) introduced a rational scheme for the nomenclature of bacteriophages depending on their shape under electron microscope and nucleic acid type. The name is preceded by vB (bacterial virus) followed by: the host abbreviation, the abbreviated virus family and ended by the research laboratory designation or common name.

Tan et al., (2009) isolated 132 phages from sewage samples. Three phages (P45, P71, P631) showed lysis effect against Ralstonia solanacearum and one phage (P621) showed lysis effect against

25 Review of Literature

Erwinia chrysanthemi, while the other isolated phages hadn’t any lysis effect.

Evans et al., (2010) isolated a novel flagellatropic bacteriophage AT1 to control potato soft rot disease caused by Erwinia carotovora. This was characterized by electron microscopy and restriction analysis and was found to have an icosahedral head and a long contractile tail. The isolated phage belongs to Myoviridae. In vivo study assay revealed that the isolated phage (AT1) was able to attenuate the virulence of mutant strains, as Erwinia carotovora flagellum act as a receptor for AT1 phage, thus affecting the bacterium motility.

Sheng et al., (2010) isolated and genetically characterized a T4-like bacteriophage IME08, depending on gene cloning and sequencing technique, it was found that the bacteriophage IME08 was highly similar to T4-like Enterobacteria phage T4 by 94%, JS98 by 95%, JS10 by 95% and RB14 by 94%.

Kim et al., (2011) isolated twenty one virulent bacteriophages from sewage at different locations to control brown blotch disease of mushrooms caused by Pseudomonas tolaasii. Biocontrol trials to study the toxicity of these isolated phages to host bacteria revealed good results to be applied for the control of brown blotch disease.

Müller et al., (2011) isolated and characterized phages from North America and Germany for controlling fire blight of apple and pear trees caused by Erwinia amylovora. The isolated phages, Ea1h and Ea100 belong to Podaviridae while Ea104 and Ea116 belong to

26 Review of Literature

Myoviridae. Biocontrol experiments showed that phages belonging to Myoviridae possessed better lysis activity upon Erwinia amylovora and hence protected apple flowers significantly more than those belonging to Podaviridae. 272 Adriaenssens et al., (2012) isolated two closely related and specific bacteriophages, vB_DsoM_LIMEstone1 and vB_DsoM_LIMEstone2, to control Dickeya solani and Dickeya dianthicola (the causative agents of potato rotting and black leg disease). The isolated phages were able to control potato infection at laboratory and field trails. The study recommended the use of phage therapy instead of antibiotics.

Comeau et al., (2012) reported a new group of dwarf bacteriophages belonging to Myoviridae family. It possesses short contractile tails of (65-85 nm), isomeric head of (60-70 nm) and genome size of 45kb. This group infect various strains of bacteria, including ZF40 phage infecting Pectobacterium carotovorum, in addition to Aeromonas salmonicida phage 56, Vibrio cholerae phages 138 and CP-T1 and Bdellovibrio phage 1422.

Jee et al., (2012) isolated bacteriophages from 6 different soil samples cultivated by Chinese cabbage in Korea, belonging to Myoviridae, Siphoviridae and Podaviridae of Caudovirales. The phages were able to control soft rot disease caused by different strains of Pectobacterium carotovorum subsp. carotovorum.

Lee et al., (2012) isolated a bacteriophage PP1, to control soft rot disease, caused by Pectobacterium carotovorum subsp. carotovorum. It

27 Review of Literature was identified by complete genome sequencing of the 44,400-base pairs. No lysogen genes were encoded, which enhanced the target of using this isolated phage as a biocontrol agent.

Mishra et al., (2012) isolated an aquatic T1-like phage named as F20 belonging to Siphoviridae. It was found to be virulent against two strains of Enterobacter aerogenes which were ATCC 13048 and the multi-drug-resistant strain K113.

Paunikar et al., (2012) studied the effect of different concentration of different metal ions, heavy metals and chemical solvents on the infectivity of bacteriophages to their hosts. They found that some metal ions at special concentration enhanced higher plaque count by supporting the phage adsorption to its host. On the other hand, some other heavy metals and chemical solvents showed negative effect on the phages infectivity.

Radhakrishnan and Ananthasubramanian (2012) isolated lytic bacteriophages from sewage that showed antibacterial effect against Pseudomonas fluorescens strains isolated from sewage. There was no effect against those isolated from rhizosphere regions of cultivated soils.

Soleimani et al., (2012) isolated a novel bacteriophage belonging to Cystoviridae family from soil and wastewater to control potato soft rot disease caused by Pseudomonas putida.

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Yamada (2012) isolated and characterized various kinds of bacteriophages belonging to Inoviridae and Myoviridae for controlling bacterial wilt disease caused by Ralstonia solanacearum. This helped to eliminate the disease from contaminated soils, and is used as a diagnostic tool for discovering the pathogen present in growing crops.

Cannon et al., (2013) studied the effect of different water parameters such as salinity, pH, dissolved oxygen and temperature on PCR sensitivity. These parameters were used as a detection tool for bacteriophages as T4 in environmental samples. Hence, PCR identification tool for bacteriophages was used as an indicator of contamination by pathogenic coliform bacteria.

Czajkowski et al., (2013) isolated nine bacteriophages from soil samples collected in Poland in an attempt to control soft rot and black leg diseases of potato caused by Pectobacterium and Dickeya spp.. The isolated phages belong to order Caudovirales of Myoviridae. Phage (D5) showed a wide host range while phage (D7) showed a narrow host range. Both phages proved to have an antibacterial effect against strains of Dickeya dadantii and eradicated the growth of Dickeya Solani. Potato tubers treated with phages in vitro were protected from displaying disease symptoms. On the other hand, no antibacterial effect was recorded against Pectobacterium spp.

Lim et al., (2013) isolated a bacteriophage PP1, belonging to Caudovirales of Podaviridae, from soil to control soft rot disease caused by Pectobacterium carotovorum subsp. carotovorum. The

29 Review of Literature isolated phage showed a significant lytic effect against Pectobacterium carotovorum subsp. carotovorum.

Marie (2013) isolated a virulent, tailed isometric shape bacteriophage from soil cultivated with liquorices root in Matruh governorate, Cairo, Egypt, that infect Bacillus subtilis.

Kalbage and Costa (2014) isolated a mixture of 6 bacteriophages from soil, cultivated by vegetables and soils rich with organic matter, to control tomato wilt disease caused by Ralstonia solanacearum. The mixture of 6 phages was applied to the plant rhizosphere as a soil drench and tested against two isolates of Ralstonia solanacearum (isolate 6 and AB3). The percentage of disease incidence for isolate 6 was reduced by 10% while, for AB3 was reduced by 20%. As a result, this proved the role of phage potential in controlling bacterial wilt disease.

Cormier and Janes (2014) introduced a double layer plaque assay of 10ml bottom layer of 1% agar and 10ml soft upper layer of 0.45% agar, using spread plate method in an attempt to improve phage count and visibility of MS2 bacteriophage. In addition, the use of some supplements as glucose, CaCl2 and thiamine aided the improvement of phage visibility and count.

Lim et al., (2014) isolated PM1 bacteriophage from Chinese cabbage field. The phage belongs to Myoviridae. It infected both Pectobacterium carotovorum subsp. carotovorum and Pectobacterium carotovorum subsp. brasiliense strains.

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Czajkowski, et al., (2015a) isolated and characterized two novel broad host range lytic bacteriophages belonging to Myoviridae family. Samples were isolated from potato samples collected from two different potato fields in central Poland, infecting three main types of bacteria causing soft rot disease (Dickeya solani, Pectobacterium wasabiae and Pectobacterium carotovorum subsp. carotovorum). The isolated bacteriophages PD10.3 and PD23.1 were characterized by head diameter of 85 and 86 nm and tail length of 117 and 121 nm, respectively.

Czajkowski, et al., (2015b) isolated infectious lytic bacteriophages that infect Pectobacterium spp. and Dickeya spp. from environmental samples using zinc chloride precipitation method. Soft agar overlay assay was used to isolate phage plaques, without the application of neither the enrichment step nor precipitation steps by Poly Ethylene Glycol (PEG) or ultrafiltration or ultracentrifugation.

Delfan et al., (2015) isolated bacteriophages belonging to Myoviridae and Siphoviridae, from Capsian sea water in order to control soft rot disease caused by Dickeya dadantii. The isolated bacteriophages proved to control antibiotic resistant strains of Dickeya dadantii.

Addy et al., (2016) isolated two bacteriophages, 휙RSSKD1 and 휙RSSKD2, from the soil of infected banana trees to control bacterial wilt disease caused by Ralstonia solanacearum. The isolated bacteriophages were able to infect nine isolates of Ralstonia solanacearum.

31 Review of Literature

Al Khazindar et al., (2016) studied the effect of different chemicals and some plant extracts on the isolated phages and recorded the enhancement or inhibitory effect of some of these chemicals and plant extracts on the phage activity.

Hirata et al., (2016) isolated a novel lytic bacteriophage PPWS1 from an infected Japanese horseradish rhizome by soft rot in order to control soft rot disease caused by Pectobacterium carotovorum. Nuclear sequencing showed the high similarity between PPWS1 and Peat1phage that infect Pectobacterium atrosepticum, both belonging to Podoviridae.

Mohamed et al., (2016) isolated virulent bacteriophages infecting two bacterial strains of Pseudomonas stutzuri and Bacillus Pumilus causing potato soft rot, from infected potato tubers and rhizosphere soil of potato and other plant species.

Pereira et al., (2016) used a phage cocktail of phT4A/ECA2 as an application of phage therapy to control the contamination of cockles disease caused by Escherichia coli.

Rombouts et al., (2016) isolated five novel bacteriophages from Flanders infected fields to control fire blight of leek (Allium porrum) caused by Pseudomonas syringae pv. Porri. The five isolated phages, (vB_PsyM_KIL1, vB_PsyM_KIL2, vB_PsyM_KIL3, vB_PsyM_KIL4 and vB_PsyM_KIL5) were used in phage therapy complemented by the

32 Review of Literature host range mutant phage vB_PsyM_KIL3b, and was proved to attenuate disease symptoms development in the tested plants.

Czajkowski et al., (2017) studied the viability of the lytic bacteriophage ΦD5 in tuber extract, on tuber surface, in potting compost, in rainwater and on leaf surface, in addition to, the effect of copper sulphate. The interaction of lytic bacteriophage ΦD5 with Dickeya solani was evaluated to reduce the bacterial infection by 50% in tissue culture and compost grown potato. The bacteriophage infectivity remained up to 72h in potato tuber extract. However, it was inactivated in presence of copper sulphate. Furthermore, the phage population remained stable up to 28 days on potato tuber surface and potting compost. .

Lee et al., (2017) isolated bacteriophages from potato tubers rhizosphere to control soft rot disease caused by different Pectobacterium spp.. These isolated bacteriophages showed stability at 16oC-40oC and pH 6-7 and were characterized to belong to Podoviridae, Myoviridae, and Siphoviridae. .

Lim et al., (2017) isolated a novel bacteriophage vB_PcaP_PP2 (PP2) to control soft rot disease caused by Pectobacterium carotovorum subsp. carotovorum. This bacteriophage was genetically characterized to be related to subfamily Autographivirinae in Podoviridae.

Smolarska et al., (2017) isolated two bacteriophages ϕA38 and ϕA41 to control potato soft rot caused by Pectobacterium parmentieri

33 Review of Literature

(former Pectobacterium wasabiae) strain. The two bacteriophages were characterized to be related to Caudovirales of Podoviridae.

Zahran (2017) isolated two phages belonging to Podaviridae and Siphoviridae to control potato scab disease in Egypt. Physical properties (dilution end point, thermal inactivation point, pH and host range) were studied. Phage infectivity was studied in presence of different chemicals salts (Na+, K+, Ca++, Cu++, Mg++, Zn++ and Al+++ ions) and some plant extracts (Camellia sinensis L., Cinnamomum cassia, Cuminum cyminum L., Ellicium verum Hook L, Maracaria chemomilla L., Menthea Sativa L., Nigella sativa L., Origanum marjoranum L., Psidium guajava L., Tilia Americana L. and Zingiber officinal Roscoe) to record the enhancement or inhibitory effect.

Materials and Methods

A. Materials 1. Isolates Bacterial isolates used in our study were isolated from naturally infected potato tubers (Solanum tuberosum L. cv. Spunta and Solanum tuberosum L. cv. Lady Rosetta) showing soft rot symptoms. The pure cultures obtained were maintained on nutrient agar media to be identified. Five strains were brought from Microbiological Resource Center (MIRCEN), Ain Shams University, Cairo, Egypt: Bacillus pumilus Escherichia coli Pectobacterium carotovorum subsp carotovorum Pseudomonas flourecens Pseudomonas putida 2. Media Nutrient agar (NA) Each 1000 mL distilled water contain the following constituents: 5 g peptone 5 g Sodium chloride 1.5 g Beef extract 1.5 g Yeast extract 15 g Agar Final pH (at 25°C) 7.4±0.2

35 Materials and Methods

Nutrient broth (NB) Each 1000 mL distilled water contain the following constituents: 5 g peptone 5 g Sodium chloride 1.5 g Beef extract 1.5 g Yeast extract 0.2 g Magnesium chloride Final pH (at 25°C) 7.4±0.2

Nutrient gelatin Each 1000 mL distilled water contain the following constituents: 5g Peptone 3g Beef extract 120g Gelatin Final pH (at 25°C) 6.8±0.2

Simmon citrate media Each 1000 mL distilled water contain the following constituents: 0.2g Magnesium sulphate 1.0g Ammonium dihydrogen phosphate 1.0g Dipotassium hydrogen phosphate 2.0g Sodium citrate 5.0g Sodium chloride 0.08g Bromothymol blue 15g Agar Final pH (at 25°C) 6.8±0.2

36 Materials and Methods

Starch agar Each 1000 mL distilled water contain the following constituents: 3g Beef Extract 5g Peptone 2g Starch 15g Agar Final pH (at 25°C) 7.2±0.1

3. Buffers Gel loading buffer dye (6x) 0.05% w/v Bromophenol blue 0.05% w/v Xylene cyanol FF 30% v/v Glycerol

Phosphate buffer saline (PBS) tablets, Sigma, USA.

Sodium Dodecyl Sulfate (SDS) sample buffer

62.5mM Tris-HCL 2% SDS 700mM β-mercaptoethanol 10% v/v Glycerol 0.01% w/v Bromophenol blue

Tris-Acetate-EDTA (TAE) electrophoresis buffer (50x), AppliChem, Germany.

37 Materials and Methods

4. Chemicals

MicroSeq 500, 16S rDNA Bacterial Identification Kits, Applied

Biosystems, USA.

Oxidase reagent ampoule, BioMerieuxR, France.

PrepMan Ultra Sample Preparation Reagent, Applied

Biosystems, USA.

Proteinase K, Sigma, USA.

Triphenyl Tetrazolium Chloride (TTC), (0.75% w/v), Roth, Germany.

5. Equipments

Centrifuge, 2-16PK, Sigma, Germany.

DNA electrophoresis sub-cells, Cleaver Scientific Ltd,

(nanoPAC-300), France.

3500 genetic analyzer, Applied Biosystems, USA.

JEOL-JEM.1010 TEM, Japan.

Photo-documentation system (microDOC), camera using Canon

instant image films, Cleaver Scientific Ltd, France.

38 Materials and Methods

B. Methods

Part I

1. Bacterial isolation from potato Naturally infected potato tubers (Solanum tuberosum L. cv. Spunta and Solanum tuberosum L. cv. Lady Rosetta) showing soft rot symptoms were collected from local markets. The bacterial isolation was done according to Gao et al., (2014) with some modifications. Four infected tubers, from each type, were selected, washed thoroughly by running tap water, dried and surface sterilized by 75% ethyl alcohol. The tubers were peeled off using sterile sharp blade and the infected tissue of each tuber was separately macerated in sterile mortar and pestle using sterile distilled water and incubated at room temperature for 30 min to liberate the bacteria from potato tissues. Streak plate method was done on nutrient agar to obtain pure single colonies. Duplicates of each single colony were done by subculturing on nutrient agar and all samples were incubated in an incubator at 37oC for 48h. Subculturing step was repeated several times until pure single colonies were obtained.

2. Pathogenicity test Pure single colonies of the isolated bacterial samples were subjected to pathogenicity test according to Moreira et al., (2013). Two healthy potato tubers were chosen, washed thoroughly by tap water then dried by tissue paper and surface sterilized by 75% ethyl alcohol. The tubers were cut into slices (0.5 cm thick) by a suitable sterile cork borer and placed in sterilized petri plates (12cm)

39 Materials and Methods

containing wet Whatmann No.1 filter papers. A small hole was done using sterile sharp blade at the center of each slice. An inoculum of bacterial culture (24h old) suspended in 250 µL of PBS was placed at the center of each potato slice. Duplicates were done for each bacterial sample. A control plate was done using PBS at the center of the potato slice. All plates were incubated at 37oC for 48h.

3. Bacterial identification Each bacterial sample causing soft rot was subjected to the following tests: (Duplicates were done for each sample)

3.1. Gram stain A smear was prepared for each sample by placing a drop of distilled water on the middle of a clean glass slide and then the organism was transferred to the drop of water by sterile cooled loop. The smear was air dried then heat fixed and left to cool for few seconds. Crystal violet was added to the fixed smear as primary stain and left for 1min followed by iodine solution as a mordant and left for 1min. The excess of the stain was washed by 95% ethyl alcohol as decolorizing agent. Safranin was added as a counter stain for 45s. Then all samples were washed by tap water and blotted to dry by filter paper to be examined under oil immersion lens (Mitruka, 1976).

40 Materials and Methods

3.2 . Motility Motility for each of the isolated bacterial samples was detected using the Hanging Drop Method. A drop of 24h old nutrient broth culture for each bacterial sample was placed on a clean cover slip, and was turned with a quick movement onto the hollow ground part of the slide avoiding the drop from touching the hollow part of the slide. The slide was examined under light microscope using the 40x lens (Aygan and Arikan, 2007).

3.3. Biochemical tests

The following tests were done according to Mitruka, (1976) with some modifications:

Catalase test A loopful of bacterial colony (24h old culture) was transferred to the surface of a clean dry glass slide.

A drop of 3% H2O2 was added and mixed with the bacterial cells then left for 5-10 s to notice the evolution of air bubbles.

Oxidase test Bacterial smear of 24 h old culture was taken on a sterile cotton swab wetted by oxidase reagent. The colour change was recorded after 1 min.

Citrate test Citrate slants were prepared, and inoculated by 24 h old cultures. All samples were incubated for 24 h at 37oC and the change of media colour was recorded.

41 Materials and Methods

Gelatin hydrolysis test Nutrient gelatin tubes were prepared and left to harden in the refrigerator before inoculation. Each nutrient gelatin tube was inoculated by heavy inoculum of 24 h old culture. All samples of inoculated and uninoculated control tubes were incubated at 37oC for 7 days. Gelatin liquefaction was checked every day by transferring all incubated tubes to the refrigerator and comparing all inoculated tubes by the uninoculated ones.

Starch hydrolysis test Starch agar media was prepared and poured in into sterile petri plates (9 cm) and each starch agar plate was inoculated by 24 h old bacterial culture as a small streak at the center. All plates were incubated at 37oC for 24 h, all plates were flooded by 10 % iodine solution to illustrate the zone around the bacterial growth.

3.4. Molecular identification Molecular sequencing was carried out for each bacterial sample causing soft rot symptoms at Colours for Research Laboratories, Cairo, Egypt.

DNA extraction DNA extraction was performed using PrepMan™ Ultra Sample Preparation Reagent (PN 4322547). A small loopful of bacterial culture was suspended in 200μL of PrepMan Ultra Sample Preparation Reagent in a 2 mL microcentifuge tube, followed by vortexing for 10 to 30 s. The sample was heated at

42 Materials and Methods

100oC in a water bath for 10 min then spinned at 16000xg for 3 min. The supernatant was transferred to a new microcentrifuge tube and kept at -20oC.

PCR amplification and Molecular sequencing

In a microcentrifuge tube (1.5 mL) (Ambion PN 12450), 5 µL of the supernatant, 495 µL nuclease free water (Ambion PN 9937) was added, and mixed by vortexing. PCR was performed by pipetting 15 µL of the prepared sample stock to 15 µL PCR Module, in addition to, preparation of negative control (15 µL PCR Module and 15 µL nuclease free water) and positive control (15 µL PCR Module and 15 µL E. coli positive-control DNA). PCR product was determined by agarose gel electrophoresis, to detect the amplified DNA comparing with the positive and negative controls. The PCR product was purified by Montage PCR Filter Unit (Millipore PN UFC7 PCR50), and prepared for molecular sequencing using Sequencing kit (PN 4346479). The purified PCR (7 µL) was pipetted into 0.2 mL microcentrifuge tube containing 13 µL Sequencing Module and entered sequencing cycle that sequence the first 500 base pairs of the 16S ribosomal RNA (16S rDNA) bacterial gene, by using 3500 genetic analyzer - applied biosystems, using forward and reverse primers. The obtained sequencing product was purified using DyeEx™ 2.0 Spin Kit (Qiagen PN 63204). The resulted DNA sequence was analyzed and compared to a library of 16S rDNA bacterial gene sequences using Microseq ID Analysis Software.

43 Materials and Methods

Part II

1. Isolation of bacteriophages

Duplicates of three soil samples were collected at wintertime (15oC- 20oC), in polyethylene bags from the soil surface, the rhizosphere and rhizoplane of potato tubers, from potato cultivated land, of pH 7.8, in Giza, Egypt. The soil samples were sieved to remove chunks, gravel and twigs. Phage enrichment was done according to Revensdale et al., (2007) with slight modification. Triplicates of 5g from each soil sample was added to 250 mL Erlenmeyer flasks containing 50 mL nutrient broth and inoculated by 200 µL of 24 h old culture. The same method was repeated to each of the following isolates; Kosakonia sacchari, Lelliottia amnigena and Enterobacter cloaca. These flasks were incubated on a horizontal shaking incubator at 37oC for 24 h. One mL of chloroform was added to each flask and incubated on an orbital shaker for 30 min at room temperature. The suspension in each flask was decanted and centrifuged at 9700xg for 10 min to get rid of all bacterial cells and soil debris. The resulting supernatant was decanted and filter sterilized through nitrocellulose membrane bacterial filters of 0.22 nm pore size. The enriched samples were stored in dark at 4oC until use.

Bacteriophage purification and enumeration

Each bacteriophage filtrate was serially tenfold diluted (10-1-10-10) using PBS and used in double layer assay described by Łos´et al., (2008). Triplicates of sterile plates (9cm) were done for each dilution for each bacteriophage filtrate. Bacteriophage purification and

44 Materials and Methods enumeration was done according to Revensdale et al., (2007) with some modification as following; in each plate, an approximate volume of 15 mL of bottom layer of nutrient agar medium containing 12g/L bacteriological agar was poured and left to harden. 10mL soft nutrient agar medium containing 7g/L bacteriological agar and supplemented by 0.2 g/L magnesium chloride was overlaid over the bottom layer. The soft upper layer (10mL) contains 8mL soft nutrient agar, 750 µL of an overnight bacterial culture of one of the following bacterial isolates (Kosakonia sacchari, Lelliottia amnigena, Enterobacter cloaca), 750 µL bacteriophage filtrate and 500 µL of 0.75% triphenyl tetrazolium chloride (TTC) dye to visualize the bacteriophage plaques. After hardening of the soft upper layer, all plates were put in an incubator at 37oC and checked for plaque formation after 24 h. After displaying plaques, single plaque was isolated using sterile pipette tips into a sterile eppendorf tube containing 1mL sterile PBS. The eppendorf tube was vortexed and centrifuged at 11092xg at 10oC for 15 min to remove bacterial cells debris as described by Tovkach et al., (2012). The supernatant was filter sterilized through nitrocellulose bacterial filter of 0.2nm pore size. The obtained filtrate was serially diluted as described previously and the process of double layer assay was repeated several times to ensure obtaining pure single plaques of the isolated bacteriophage(s). The isolated purified bacteriophage(s) were stored in PBS at 4oC in dark.

Kosakonia sacchari was chosen as the main host to complete the rest of experiments, since it showed two different-shaped plaques.

45 Materials and Methods

2. Transmission electron microscope

A drop of the phage lysate was adsorbed on a carbon coated copper grid, left to dry, then negatively stained by (3%) uranyl acetate and examined under transmission electron microscope and photographed at Regional Center for Mycology and Biotechnology, Azhar University, Cairo, Egypt.

3. Extraction of phage nucleic acid A highly concentrated phage lysate was prepared for each isolated bacteriophage by picking 100 clear single plaques in a sterile eppendorf tube containing 1mL PBS then vortexed and centrifuged to remove bacterial cell debris, as described previously in section (1). The supernatant was filter sterilized through nitrocellulose bacterial filters of 0.2nm pore size. Phage lysate (500µL) was transferred to a clean sterile eppendorf tube then 20µL of 10% SDS and 1µL of 20mg/mL proteinase K were added and incubated at 56oC for 1 h. Equal volume of phenol was added and mixed. The mixture was centrifuged at 6050xg for 5 min at room temperature (20oC-30oC). The supernatant was transferred to a fresh eppendorf tube and an equal volume of chloroform was added, inverted and spinned. The supernatant was transferred to a fresh tube, then two volumes of ice cold ethanol were added, mixed and kept overnight at -20oC. The mixture was spinned at maximum speed for 20 min, the supernatant was discarded and 70% ethanol was added, spinned at maximum speed for 2 min. The excess ethanol was removed by leaving the tube opened on bench for 15-30 min and the pellet was dissolved in 20 µL TAE buffer.

46 Materials and Methods

3.1. Detection of nucleic acid type

Two eppendorf tubes containing 5 µL of the extracted nucleic acid were tested to detect the type of nucleic acid of the isolated bacteriophages. One tube was tested using 1µL of DNase and the other was tested using 1µL RNase. The mixtures were incubated at 37oC for 2 h.

3.2. Agarose Gel Electrophoresis

Agarose gel electrophoresis was done using DNA electrophoresis system. Agarose concentration was adjusted to be 0.8%, stained by ethidium bromide (0.5µg/mL). Electrophoresis was performed in 1x TAE buffer. The extracted nucleic acid from each isolated bacteriophage was mixed with gel loading dye. The extracted nucleic acid was visualized on a UV transilluminator (λ = 254 nm) and photographed by digital camera (Canon).

4. Bacteriophage nomenclature

The two isolated bacteriophages were named according to Kropiniski et al., (2009). The nomenclature of bacteriophages was designed depending upon three main criteria preceded by vB which referred to bacterial virus: The first part is the host bacteria abbreviation name, followed by the viral family abbreviation which is indicated by the viral morphology illustrated by electron microscope and the nucleic acid type. The last part is a simple abbreviation of common name in letter/number designated specifically for the laboratory depending upon the working researchers.

47 Materials and Methods

5. Host range

The host range of the isolated bacteriophages was determined using seven bacterial isolates. Two isolates: Lelliottia amnigena and Enterobacter cloaca were isolated and identified in our lab. Five strains: Bacillus pumilus, Escherichia coli, Pectobacterium carotovorum subsp carotovorum, Pseudomonas flourecens, Pseudomonas putida, were purchased from the Microbiological Resource Center (MIRCEN), Ain Shams University, Cairo, Egypt. Fresh overnight bacterial cultures were prepared. The isolated bacteriophages were used and plated with the bacteria under test using the double layer assay as described previously in section (1). The plates were incubated at 37oC and checked for plaque formation after 24h.

6. Physical properties

6.1. Dilution end point

A fresh overnight culture of Kosakonia sacchari was prepared. For each isolated bacteriophage, row of 9 sterile eppendorf tubes were serially tenfold diluted (10-1 to 10-10) using 900µL of sterile PBS in each tube and transferring 100 µL from each tube to the next. The double layer method was done as described previously in section (1). The dilution end point was determined as last dilution of phage displayed clear plaques.

6.2. Thermal inactivation point

Thermal inactivation point was determined according to Holmes (1956) with some modifications. A fresh overnight culture of Kosakonia sacchari was prepared. One single clear plaque of the

48 Materials and Methods

isolated phage was picked by sterile pipette tip into a sterile eppendorf tube containing 1mL PBS. Each eppendorf tube was subjected to different temperature degrees (5, 15, 25, 35, 45, 55, 65oC) for 1h by gentle vortexing in intervals then each eppendorf tube was cooled and centrifuged to remove bacterial cell debris as described previously. The supernatant was filter sterilized through nitrocellulose bacterial filters of 0.2nm pore size. The obtained filtrate was diluted and used in the double layer assay as described previously in section (1). The number of plaques produced was counted after 24 to determine the temperature at which the isolated bacteriophage lost its activity. .

6.3. Effect of pH

A fresh overnight culture of Kosakonia sacchari was prepared . Clear single plaque of the isolated bacteriophage was picked by sterile pipette tip into a sterile eppendorf tubes containing 1 mL sterile PBS of different pH values (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) and stored at 5oC for 24h. Then vortexed and centrifuged at 11092xg for 15 min at 10oC to remove bacterial cell debris as described previously. The supernatant was filter sterilized through nitrocellulose bacterial filters of 0.2nm pore size and used in the double layer method as described previously and incubated for 24 h. The number of plaques was counted.

49 Materials and Methods

6.4. Aging The effect of aging on the isolated bacteriophages was determined at room temperature (20oC-30oC) and in the refrigerator at 5oC as following: Single clear plaques of the isolated bacteriophage were picked by sterile pipette tip into sterile eppendorf tubes containing 1mL PBS. These tubes were vortexed and centrifuged to remove bacterial cell debris as described previously. The supernatant was filter sterilized through nitrocellulose bacterial filters of 0.2 nm pore size. The obtained filtrate from each eppendorf tube was collected in a sterile 15mL falcon tube and stored in dark conditions at room temperature and another one was stored at refrigerator. Both were tested for the phage infectivity using double layer assay as described previously, after 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 weeks and up to 20 months.

7. Effect of different salts

7.1. Effect of different salts on Kosakonia sacchari

Stock solutions of 100mM concentration were prepared for sodium chloride (NaCl) and potassium chloride (KCl) as

monovalent chloride salts, calcium chloride (CaCl2) and

magnesium chloride (MgCl2 .6H2O) as divalent chloride salts and

aluminum chloride (AlCl3) and ferric chloride (FeCl3) as trivalent chloride salts. The stock solution for each salt was used for preparation of 3 different concentrations (10mM, 1mM, 0.1mM). The effect of the selected salts with the 3 different concentrations

50 Materials and Methods

was tested on the host bacteria Kosakonia sacchari. This was done using agar well diffusion method as described by Irshad et al., (2012) with slight modification. An overnight culture of Kosakonia sacchari was prepared. In a 250mL conical flask, 1mL of the prepared bacterial culture was added to 100 mL of molten nutrient agar, cooled to 45oC, in addition, 1mL of 0.75% triphenyl tetrazolium chloride (TTC) was added to visualize the inhibition zones by staining the bacterial cells. The agar was poured into 9 cm sterile petri plates, left to harden. Wells were done in each petri plate of the tested chemical type. Each well was filled by the tested concentration of each tested salt type. All plates were incubated at 37oC and tested for inhibition zone formation after 24h. Triplicates of plates were done for each tested salt type.

7.2. Effect of different salts on the isolated bacteriophages Fresh phage lysate was prepared by picking a clear single plaque using sterile pipette tip into sterile eppendorf tube, containing 1 mL sterile PBS. The eppendorf was vortexed, centrifuged and the supernatant was filter sterilized as described previously. Studying the effect of different salts was done according to Paunikar et al., (2012) with some modifications. Duplicates of soft agar tubes were mixed with each salt to obtain final concentrations (10mM, 1mM, 0.1mM). Phage filtrate (750µL) was added to each soft agar tube plus 750µL of an overnight bacterial culture of Kosakonia sacchari and 50µL of 0.75% triphenyl tetrazolium chloride (TTC). The mixture was poured over a fresh previously hardened bottom nutrient agar in a 9cm sterile petri plate. All plates were incubated at 37oC for 24 h, the number of plaques

51 Materials and Methods

was recorded and compared with a control plate, without any salt treatment.

8. Effect of different heavy metals

8.1. Effect of different heavy metals on Kosakonia sacchari

Stock solutions of 100mM concentration were prepared for

cadmium chloride (CdCl2), cobalt chloride (CoCl2), copper

chloride (CuCl2), mercuric chloride (HgCl2), silver nitrate

(AgNO3), and zinc sulphate (ZnSO4). The stock solution for each heavy metal was used for preparation of 4 different concentrations (10mM, 1mM, 0.1mM, 0.01mM). The effect of the selected heavy metals with the 4 different concentrations was tested on the host bacteria Kosakonia sacchari. This was done using agar well diffusion method as described by Irshad et al., (2012) with slight modification as described previously in section (7.1). The agar was poured into 9cm sterile petri plates, left to harden. Wells were done in each petri plate of the tested heavy metal type. Each well was filled by the tested concentration of each tested heavy metal type. All plates were incubated at 37oC and tested for inhibition zone formation after 24h. Triplicates of plates were done for each tested heavy metal type.

8.2. Effect of different heavy metals on the isolated bacteriophages Freshly prepared phage lysate was prepared as described previously in section (7.2) to study the effect of different heavy metals according to Paunikar et al., (2012) with some modifications. Duplicates of soft agar tubes were mixed with each heavy metal to obtain final concentrations (10mM, 1mM, 0.1mM,

52 Materials and Methods

0.01). Phage filtrate (750µL) was added to each soft agar tube plus 750µL of an overnight bacterial culture of Kosakonia sacchari and 50µL of 0.75% triphenyl tetrazolium chloride (TTC). The mixture was poured over a fresh previously hardened bottom nutrient agar in a 9 cm sterile petri plate. All plates were incubated at 37oC for 24 h, the number of plaques was recorded and compared with a control plate, without any heavy metal treatment.

9. Effect of different sugars

9.1. Effect of different sugars on Kosakonia sacchari Stock solutions of 100mM concentration were prepared for fructose and glucose as monosaccharides and for lactose and sucrose as disaccharides. The stock solution for each sugar was used for preparation of 3 different concentrations (10mM, 1mM, 0.1mM). The effect of the selected sugar with the 3 different concentrations was tested on the host bacteria Kosakonia sacchari. This was done using agar well diffusion method as described by Irshad et al., (2012) with slight modification as described previously in section (7.1). The agar was poured into 9 cm sterile petri plates, left to harden. Wells were done in each petri plate of the tested sugar type. Each well was filled by the tested concentration of each tested sugar type. All plates were incubated at 37oC and tested for inhibition zone formation after 24h. Triplicates of plates were done for each tested sugar type.

53 Materials and Methods

9.2. Effect of different sugars on the isolated bacteriophages

Freshly prepared phage lysate was prepared as described previously, to study the effect of different sugars according to Paunikar et al., (2012) with some modifications. Duplicates of soft agar tubes were mixed with each sugar to obtain final concentrations (10mM, 1mM, 0.1mM). Phage filtrate (750µL) was added to each soft agar tube plus 750µL of an overnight bacterial culture of Kosakonia sacchari and 50µL of 0.75% triphenyl tetrazolium chloride (TTC). The mixture was poured over a fresh previously hardened bottom nutrient agar in a 9 cm sterile petri plate. All plates were incubated at 37oC for 24 h, the number of plaques was recorded and compared with a control plate, without any sugar treatment.

10. Effect of some plant extracts

10.1. Effect of some plant extracts on Kosakonia sacchari

Stock solutions of different plant extracts listed in Table (1) were prepared by boiling 1.5g of the dried plant organ in 100mL distilled water for 10 min. The extract was left to cool and filtered using whatman No.1 filter paper. The obtained filtrate was filter sterilized through nitrocellulose bacterial filters of 0.2nm pore size. The extract was used at 100% concentration. The effect of the selected plant extract was tested on the host bacteria Kosakonia sacchari. This was done using agar well diffusion method as described by Irshad et al., (2012).

54 Materials and Methods

10.2. Effect of some plant extracts on the isolated bacteriophages A single plaque was picked by a sterile pipette into a sterile eppendorf tube containing 1mL of the plant extract filtrate. The mixture was incubated at 5oC for 1h followed by centrifugation and filtration as described previously. Double layer assay was done as described previously, the number of plaques was recorded and compared with a control plate without any treatment by plant extract.

Table (1): Plant extracts used to study their effect on the isolated bacteriophages Latin name Family Common name Plant organ used Camellia Sinensis L. Theaceae Tea Leaves Cinnamomum Lauraceae Cinnamon Bark cassia L. Hibiscus rosa- Malvaceae Hibiscus Flower sinensis L. Matricaria Asteraceae Chamomile Flower chamomilla L. Mentha piperita L. Lamiaceae Peppermint Flower Organum Marjoram Leaves majorana L. Pimpinella Apiaceae Anise Fruit anisum L. Zingiber officinal Zingiberaceae Ginger Rhizome Roscoe

55 Materials and Methods

11. Effect of different essential oils

11.1. Effect of some essential oils on Kosakonia sacchari

Essential oils listed in Table (2) were used. The effect of the selected essential oil was tested on the host bacteria Kosakonia sacchari. This was done using agar well diffusion method as described by Irshad et al., (2012).

11.2. Effect of different essential oils on the isolated bacteriophages

A single plaque was picked by a sterile pipette into a sterile eppendorf tube containing 1mL of the essential oil. The mixture was incubated at 5oC for 1h followed by centrifugation and filtration as described previously. Double layer assay was done as described previously. The number of plaques was recorded and compared with a control plate without any treatment by essential oil.

56 Materials and Methods

Table (2): Essential oils used to study their effect on the isolated bacteriophages Latin name Family Common name Plant organ used Acacia sp. Fabaceae Gum Stem Allium sativum L. Amaryllidaceae Garlic Bulb Lactuca sativa L. Asteraceae Lettuce Leaves Lavandula Lamiaceae Lavendar Flower angustifolia Mill. Pimpinella Apiaceae Anise Fruit anisum L. Zingiber officinale Zingiberaceae Ginger Rhizome Rosocoe

12. Application of phage therapy on small scale

The ability of the isolated bacteriophages to control potato soft rot by preventing rotting of potato slices was evaluated on small scale as described by Czajkowski et al., (2013) with slight modification. Concentrated phage lysate (100 plaques/mL) was prepared as previously described in section (3). Healthy potato tubers were selected from local markets, washed thoroughly by tap water, dried by tissue paper and surface sterilized by ethanol (70%). The tuber was cut into slices (0.5cm thick) by sterile sharp blade and placed in sterilized petri plates (12 cm) containing wet Whatman no.1 filter papers. Small hole was done by a suitable sterile cork borer at the center of each slice. Phage lysate (250 µL) was added to the hole at

57 Materials and Methods

the center of the potato slice and left for 30 min before adding an equal volume of bacterial inoculum of 24h old culture of Kosakonia sacchari suspended in PBS. Positive control was done by adding 500 µL of the bacterial suspension only to the hole. Negative control was done by adding 500 µL of PBS only to the hole. All plates were incubated at 37oC for (48h-72h). The treated plates were compared by both the positive control plates and negative control ones to investigate the ability of the isolated bacteriophages in protecting potato slices from soft rotting. Replicates of plates were done for each case and the experiment was repeated three times.

13. Statistical analysis The results obtained from experimental methods were analyzed using one way ANOVA.

Results

Part I

1. Bacterial isolation from potato

The soft rot symptoms in potato showed creamy consistency, degraded tissue and water soaked appearance with bacterial ooze as shown in Fig. (1).

Fig.(1): Showing potato soft rot symptoms

2. Pathogenicity test

Pure single colonies were selected, purified and tested for their pathogenicity on potato tuber discs. Three bacterial isolates (B1, B2, B3) caused soft rotting to the potato tuber discs, compared to the control (C) as shown in Fig. (2).

59 Results

C B1 B2 B3

Fig.(2):Pathogenicity test of three different bacterial isolates (B1, B2, B3) causing soft rot on potato tuber discs, compared to the control (C).

3. Bacterial identification

3.1 Gram stain

Microscopial examination using oil immersion lens for the three bacterial isolates, showed Gram negative rods.

3.2 Motility test

Microscopial examination by high power lens (40x), showed rotating, non polar movement of the bacterial cells in case of the three bacterial isolates (B1, B2, B3). Hanging Drop Method showed peritrichously flagellated motile bacteria.

3.3 Biochemical tests

All the following biochemical tests are summarized in Table (3)

Catalase test

The three bacterial isolates (B1, B2, B3) showed air bubbles after the addition of H2O2. They possess catalase enzyme.

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Oxidase test The treatment of the three bacterial isolates (B1, B2, B3) with cotton swabs wetted by oxidase reagent, showed no colour change. This indicate their lacking of cytochrome oxidase.

Simmon citrate test The three bacterial isolates (B1, B2, B3) showed change in the colour of the media from green to blue as shown in Fig. (3)

C B1 B2 B3

Fig. (3): Simmon citrate test of the three bacterial isolates (B1, B2, B3) showing change in colour from green to blue, compared with the control (C), from left to right.

Gelatin hydrolysis test

The three bacterial isolates (B1, B2, B3) showed positive gelatin hydrolysis. B1 showed gelatin liquefaction after 72 h, B2 showed gelatin liquefaction after 96 h, while B3 showed delayed gelatin liquefaction after 7 days.

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Starch hydrolysis test

The three bacterial isolates (B1, B2, B3) showed negative result for starch hydrolysis.

Table (3): Biochemical tests of the three bacterial isolates (B1, B2, B3).

Test B1 B2 B3 Catalase +ve +ve +ve Oxidase -ve -ve -ve Simmon citrate +ve +ve +ve Gelatinase +ve +ve +ve Starch hydrolysis -ve -ve -ve

3.4 Molecular identification

Phylogenetic analysis of 16S rRNA gene sequencing identified the three bacterial isolates (B1, B2, B3) to be strains closely related to Enterobacteriaceae family. Neighbor-joining approach illustrated that the isolate B1 was clustered with Lelliottia amnigena, Lelliottia nimipressuralis and Enterobacter soli as shown in Fig. (4). Blastn alignment closely related B1 to be a strain of Lelliottia amnigena by 99%.

Neighbor-joining approach illustrated that the isolate B2 was clustered with Kosakonia sacchari, Kosakonia sp. and Enterobacter sp. as shown in Fig. (5). Blastn alignment closely related B2 to be a strain of Kosakonia sacchari by 99%.

Neighbor-joining approach illustrated that the isolate B3 was related to Enterobacteriaceae family as shown in Fig.(6). Blastn alignment closely related B3 to be a strain of Enterobacter cloaca by 97%.

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The three bacteria isolates were deposited in the gene bank and given the following accession numbers.

B1: KY427021

B2: KY235364

B3: KY235363

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Fig. (4): Phylogenetic analysis of the bacterial isolate B1 according to Blastn according B1 of the bacterial (4): Fig. analysis isolate Phylogenetic

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according to Blastn according

): Phylogenetic analysis of the bacterial analysis ): isolate Phylogenetic Fig. (5 Fig.

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according to Blastn according

f the the fbacterial isolate

): Phylogenetic analysis o analysis ): Phylogenetic

Fig. (6 Fig.

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Part II

1. Isolation of bacteriophages

Two different shaped plaques appeared in case of isolating bacteriophages from potato cultivated soil, using Kosakonia sacchari as a host. However, in case of using Lelliottia amnigena or Enterobacter cloaca as a host, only one shape of plaques was observed. Therefore, Kosakonia sacchari was chosen as the phage host to complete the rest of experiments. The two plaques were clearly different in both size and morphology, indicating the presence of two different bacteriophages as shown in Fig. (7).

Fig.(7): Two different shaped plaques appeared in case of using Kosakonia sacchari as a host. Arrows show different plaques size and morphology.

Bacteriophage purification and enumeration

The two isolated bacteriophages were designated as Ph1 which was characterized by halo zone like plaques of (0.2 cm) diameter and Ph2 which was characterized by pin point like plaques as shown in Fig. (8). Each plaque was successfully isolated using double layer assay.

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Ph1 was diluted tenfold to 10-7 to show countable plaques. Ph2 was tenfold diluted to10-5 to show countable plaques.

A B

Fig. (8): Double layer assay of Kosakonia sacchari as a host. Ph1 diluted to 10-7(A) and Ph2 diluted to 10-5 (B).

2.Transmission Electron Microscope (TEM)

The examination of bacteriophages negatively stained by 3% uranyl acetate under TEM showed that ph1 was characterized by icosahedral head of 58.04nm diameter, short neck of 13.13nm length and 8.06nm width and long flexible tail of 96.42nm length and 14.34nm width as shown in Fig. (9). On the other hand, examination of Ph2 showed that it was characterized by small polyhedral heads of 23.45nm diameter as shown in Fig. (10).

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Fig. (9): TEM of Ph1 negatively stained by 3% uranyl acetate, TEM magnification x 150000 and bar 100 nm.

Fig. (10): TEM of Ph2 negatively stained by 3% uranyl acetate, TEM magnification x 200000 and bar 50 nm.

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3. Detection of nucleic acid

Nucleic acid of both bacteriophages was determined using enzymatic digestion by DNase and RNase. Both phages nucleic acids were digested by DNase and were not affected by RNase as shown in Fig. (11) and Fig. (12).

Fig.(11): 0.8% Agarose gel electrophoresis of Ph1 nucleic acid. Lane1: total nucleic acid, Lane2: RNase digestion of genomic DNA and lane 3: DNase digestion.

Fig.(12): 0.8% Agarose gel electrophoresis of Ph2 nucleic acid. Lane 1: total nucleic acid, Lane2: DNase digestion and lane 3: RNase digestion of genomic DNA

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4. Bacteriophage nomenclature

Nomenclature of both bacteriophages was based upon the nucleic acid type and phage morphology under TEM. Both phages possess DNA genome. Ph1 is tailed with icosahedral head and short neck, therefore, it belongs to family Myoviridae (M). Ph2 is characterized by small polyhedral head, therefore, it belongs to family Microviridae (O). Accordingly, both phages were named bacterial viruses (vB), followed by the first two letters of host genus and species according to restriction enzyme data base (Ksa), then by single letter referring to the viral family and ended by symbol designed for the institute were the work took place which was Cairo University (C). So Ph1 was named vB_KsaM-C1 and Ph2 was named vB_KsaO-C2 (Kropinski et al., 2009).

5. Host range

Both isolated bacteriophages showed lytic effect on Enterobacter cloaca isolate, Pectobacterium carotovorum subsp. carotovorum and Pseudomonas flourecens. In addition, both isolated phages showed no lytic effect on Escherichia coli and Pseudomonas putida. vB_KsaM-C1 (Ph1) showed lytic effect on Bacillus pumilus, however, it had no lytic effect on Lelliottia amnigena. On the other hand, vB_KsaO-C2 (Ph2) showed lytic effect on Lelliottia amnigena and showed no lytic effect on Bacillus pumilus. All these results are shown in Fig. (13) and Table (4).

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A1 B1 A2 B2

C1 C2 D1 D2

E1 E2 F1 F2

G1 G2

Fig. (13): The effect of both isolated bacteriophages on different bacterial strains causing soft rot disease. vB_KsaM-C1 (Ph1) showed lytic effect on Bacillus pumilus (A1), Enterobacter cloaca (B1), Pectobacterium carotovorum subsp. carotovorum (E1) and Pseudomonas flourecens (F1), on the other hand, it showed no lytic effect on Escherichia coli (C1), Lelliottia amnigena (D1) and Pseudomonas putida (G1). vB_KsaO-C2 (Ph2) showed lytic effect on Enterobacter cloaca (B2), Lelliottia amnigena (D2), Pectobacterium carotovorum subsp. carotovorum (E2) and Pseudomonas flourecens (F2), on the other hand, it showed no lytic effect on Bacillus pumilus (A2), Escherichia coli (C2) and Pseudomonas putida (G2).

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Table (4): Host range of the two isolated bacteriophages vB_KsaM-C1 (Ph1) and vB_KsaO-C2 (Ph2) against different bacterial strains causing soft rot disease.

The bacterial strain vB_KsaM-C1 vB_KsaO-C2 Bacillus pumilus +ve -ve Enterobacter cloaca +ve +ve isolate Escherichia coli -ve -ve Kosakonia sacchari +ve +ve Lelliottia amnigena -ve +ve Pectobacterium carotovorum subsp. +ve +ve carotovorum Pseudomonas flourecens +ve +ve Pseudomonas putida -ve -ve

6. Physical properties

6.1. Dilution end point

Dilution end point of phage vB_KsaM-C1

The tenfold dilutions (10-1 to 10-6) showed uncountable number of plaques. While, 150 plaques were recorded at dilution of 10-7 which decreased gradually upon dilution upto 10-9 which recorded 4 plaques as shown in Table (5). Therefore, the dilution end point of bacteriophage vB_KsaM-C1 (Ph1) is 10-9.

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Table (5): Tenfold dilution assay of vB_KsaM-C1 (Ph1).

Phage dilution Number of plaques 10-1 Uncountable 10-2 Uncountable 10-3 Uncountable 10-4 Uncountable 10-5 Uncountable 10-6 Uncountable 10-7 150 10-8 45 10-9 4 10-10 0

Dilution end point of phage vB_KsaO-C2

The tenfold dilutions (10-1 to 10-3) showed uncountable number of plaques. While 674 plaques were recorded at dilution of 10-4 and 200 plaques were recorded at 10-5 which decreased gradually upon dilution upto 10-7 which recorded 3 plaques as shown in Table (6). Therefore, the dilution end point of bacteriophage vB_KsaO-C2 (Ph2) is 10-7.

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Table (6): Tenfold dilution assay of vB_KsaO-C2 (Ph2).

Phage dilution Number of plaques 10-1 Uncountable 10-2 Uncountable 10-3 Uncountable 10-4 674 10-5 200 10-6 17 10-7 3 10-8 0

6.2. Thermal inactivation point

Thermal inactivation point of phage vB_KsaM-C1

Studying the effect of different temperatures by incubating the bacteriophage vB_KsaM-C1 (Ph1) in different temperatures of 5, 15, 25, 35, 45, 55, 65oC for 1h. The thermal inactivation point was at 65oC as shown in Table (7) and Fig. (14).

Table (7): The effect of different temperatures on vB_KsaM-C1(Ph1)

Temperature (oC) No. of plaques 5 111 15 135 25 89 35 73 45 48 55 12 65 0

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250 200

150

100

50 Mean No No plaques of Mean 0 5 15 25 35 45 55 65

Temperature (oc)

Fig. (14): Showing the thermal inactivation point for phage vB_KsaM-C1at 65oC.

Thermal inactivation point of phage vB_KsaO-C2

Studying the effect of different temperatures by incubating the bacteriophage vB_KsaO-C2 (Ph2) in different temperatures of 5, 15, 25, 35, 45, 55, 65oC for 1h. The thermal inactivation point was at 65oC as shown in Table (8) and Fig. (15).

Table (8): The effect of different temperatures on vB_KsaO-C2 (Ph2)

Temperature (oC) No. of plaques 5 197 15 189 25 180 35 150 45 46 55 35 65 0

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250 200 150

100

50 Mean No. of plaques of MeanNo. 0 5 15 25 35 45 55 65 Temperature(oc)

Fig. (15): Showing the thermal inactivation point for phage vB_KsaO-C1 at 65oC.

6.3. Effect of pH

Effect of pH on vB_KsaM-C1

Studying the effect of different pH values on vB_KsaM-C1 (Ph1) by incubating the bacteriophage in buffer of different pH values (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) at 5oC for 24 h. Optimum activity for phage vB_KsaM-C1 was determined at pH (7-8), while phage infectivity decreased towards acidic and alkaline conditions, as shown in Table (9) and Fig. (16).

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Table (9): Effect of different pH values on vB_KsaM-C1(Ph1)

pH value No. of plaques 1 0 2 0 3 0 4 35 5 55 6 66 7 104 8 102 9 76 10 34 11 0 12 0

250 200

150

100

MeanNo. plaques of 0 1 2 3 4 5 6 7 8 9 10 11 12

pH value

Fig. (16): Showing the optimum activity for vB_KsaM-C1 at pH (7-8).

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Effect of pH on vB_KsaO-C2

Studying the effect of different pH values on vB_KsaO-C2 (Ph2) by incubating the bacteriophage in buffer of different pH values (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) at 5oC for 24 h. The optimum activity for phage vB_KsaO-C2 was determined at pH 7, phage infectivity decreased in high acidic and alkaline conditions. Phage vB_KsaO-C2 can tolerate the alkaline conditions up to pH 9 as shown in Table (10) and Fig. (17).

Table (10): Effect of different pH values on vB_KsaO-C2 (Ph2)

pH value No. of plaques 1 0 2 0 3 0 4 45 5 78 6 120 7 195 8 137 9 125 10 43 11 3 12 0

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250 200 150

100

50 MeanNo. plaques of 0 1 2 3 4 5 6 7 8 9 10 11 12

pH value

Fig. (17): Showing the optimum activity for vB_KsaM-C1 at pH 7.

6.4. Aging

Both isolated bacteriophages sustained their lysis ability more than 20 months, either stored at room temperature (25-35oC) or at refrigerator (5oC). However, a significant decrease in phage infectivity expressed in a decrease in the plaque count by the time was recorded as shown in Table (11).

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Table (11): Aging of both isolated bacteriophages vB_KsaM-C1 (Ph1) and vB_KsaO-C2 (Ph2)

No of plaques Time (Months) vB_KsaM-C1 vB_KsaO-C2 (25-35oC) 5oC (25-35oC) 5oC 0 150 150 200 200 4 120 145 190 197 8 100 133 152 188 12 78 120 134 175 16 55 100 100 158 20 32 94 85 142

7. Effect of different salts

7.1. Effect of different salts on Kosakonia sacchari

Effect of different concentrations (10mM, 1mM and 0.1mM) of sodium chloride (NaCl), potassium chloride (KCl), calcium chloride

(CaCl2), magnesium chloride (MgCl2), aluminum chloride (AlCl3) and

ferric chloride (FeCl3) on Kosakonia sacchari was studied. No

significant antimicrobial effect was observed for all concentrations of

NaCl, KCl, CaCl2 and MgCl2. However, AlCl3 and FeCl3 showed a significant cidal effect on the host bacteria at high concentration of 10mM as shown in Fig. (18).

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10mM 10mM C 10mM C C

NaCl KCl CaCl2 1mM 0.1mM 1mM 0.1mM 1mM 0.1mM

A B C C

10mM 10mM C 1mM

MgCl2 C AlCl3 0.1mM 1mM

0.1mM D E F F

Fig. (18): Effect of different concentrations of salts on Kosakonia sacchari.

A: NaCl, B: KCl, C: CaCl2, D: MgCl2, E: AlCl3 and F: FeCl3

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7.2. Effect of salts on the isolated bacteriophages

7.2.1. Effect of monovalent salts

Effect of NaCl

Effect of NaCl on vB_KsaM-C1

Studying the effect of different concentrations of NaCl on vB_KsaM-C1 (Ph1) showed significant increase in the plaques count percentage. The optimum plaque count percentage was observed at salt concentration of 1mM as shown in Fig. (19). Statistical analysis showed significant differences (p < 0.05) between the different concentrations of NaCl compared with the control (0% NaCl). There is a high significant difference (p < 0.001) at 1mM NaCl compared with other concentrations and the control as shown in Table (12).

Table (12): Effect of different concentrations of NaCl on vB_KsaM-C1

NaCl Plaque count percentage concentrations (mM) (%)

0 100 0.1 108* 1 125** 10 110* *Significant difference at p< 0.05

**High significant difference at p< 0.001

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140 120 100 80 60 40 20 0 Plaque count percentage (%)percentage count Plaque 0 0.1mM 1mM 10mM

NaCl concentration in mM

Fig. (19): Effect of different concentrations of NaCl on vB_KsaM-C1 Effect of NaCl on vB_KsaO-C2

Studying the effect of different concentrations of NaCl on vB_KsaO- C2 (Ph2) showed high significant decrease in the plaques count percentage as shown in Fig. (20). Statistical analysis showed high significant differences (p<0.001) between the different concentrations of NaCl compared with the control (0% NaCl) as shown in Table (13).

Table (13): Effect of different concentrations of NaCl on vB_KsaO-C2

NaCl Plaque count percentage concentrations (mM) (%)

0 100 0.1 86** 1 67** 10 92** ** High significant difference at p < 0.001

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(%) 120 100 80 60 40 20 0 Plaque count percentage percentage count Plaque 0 0.1mM 1mM 10mM

NaCl concentration in mM

Fig. (20): Effect of different concentrations of NaCl on vB_KsaO-C2

Effect of KCl

Effect of KCl on vB_KsaM-C1

Studying the effect of different concentrations of KCl on vB_KsaM- C1 (Ph1) showed high significant increase in the plaques count percentage at concentrations of 1mM and 10mM of KCl and no significant change was observed at low concentration of 0.1mM of KCl. However, the optimum plaque count percentage was observed at salt concentration of 1mM as shown in Fig. (21). Statistical analysis showed significant differences (P < 0.05) between 1mM and 10mM concentrations of KCl compared with the control (0% KCl), however there is a highly significant difference (p < 0.001) at 1mM KCl compared with other concentrations and the control ( 0% KCl) as shown in Table (14).

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Table (14): Effect of different concentrations of KCl on vB_KsaM-C1

KCl concentrations (mM) Plaque count percentage (%)

0 100 0.1 102 1 371** 10 347* * Significant difference at p< 0.05 ** High significant difference at p< 0.001

400 (%) 350 300 250 200 150

count percentage count percentage 100 50

Plaque 0 0 0.1mM 1mM 10mM

KCl concentration in mM Fig. (21): Effect of different concentrations of KCl on vB_KsaM-C1.

Effect of KCl on vB_KsaO-C2

Studying the effect of different concentrations of KCl on vB_KsaO- C2 (Ph2) showed a high significant increase in plaques count percentage by increasing KCl concentration from 1mM to 10mM. However, a high significant decrease in plaque count percentage was observed at 0.1 mM as shown in Fig. (22). Statistical analysis showed highly significant differences (p < 0.001) between the different

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concentrations of KCl compared with the control (0% KCl) as shown in Table (15).

Table (15): Effect of different concentrations of KCl vB_KsaO-C2

KCl Plaque count percentage concentrations (mM) (%) 0 100 0.1 84** 1 117** 10 153** ** High significant difference at p < 0.001

180 160 140 120 100 80 60 40 20

Plaque count percentage (%)percentage count Plaque 0 0 0.1mM 1mM 10mM

KCl concentration in mM

Fig. (22): Effect of different concentrations of KCl on vB_KsaO-C2.

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7.2.2. Effect of divalent salts

Effect of CaCl2.2H2O

Effect of CaCl2.2H2O on vB_KsaM-C1

Studying the effect of different concentrations of CaCl2.2H2O on vB_KsaM-C1 (Ph1) showed a significant increase in plaque count

percentage at concentrations of 1mM and 10mM of CaCl2.2H2O. However, no significant change was observed at low concentration of 0.1mM as shown in Fig. (23). Statistical analysis showed high significant differences (p < 0.001) between the 1mM and 10mM concentrations of

CaCl2.2H2O compared with the control (0% CaCl2.2H2O). On the other hand, no significant difference was observed at concentration of 0.1mM as shown in Table (16).

Table (16): Effect of different concentrations of CaCl2.2H2O on vB_KsaM-C1

CaCl2.2H2O Plaque count percentage concentrations (mM) (%)

0 100 0.1 102 1 110** 10 128** ** High significant difference at p < 0.001

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140 120 100 80 60 40 20

0 Plaque count percentage (%)percentage count Plaque 0 0.1mM 1mM 10mM

CaCl2.H2O concentration (mM)

Fig. (23): Effect of different concentrations of CaCl2.2H2O on vB_KsaM-C1

Effect of CaCl2.2H2O on vB_KsaO-C2

Studying the effect of different concentrations of CaCl2.2H2O on vB_KsaO-C2 (Ph2) showed a significant increase in plaque count percentage especially at concentration of 0.1mM and high significant increase in plaque count percentage at concentrations of 1mM and 10mM as shown in Fig. (24). Statistical analysis showed a significant difference

(p < 0.05) between 0.1mM concentration of CaCl2.2H2O compared with the control (0% CaCl2.2H2O), however, there is a high significant difference (p < 0.001) at 1mM and 10mM concentrations of CaCl2.2H2O compared with the control ( 0% CaCl2.2H2O ) as shown in Table (17).

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Table (17): Effect of different concentration of CaCl2.2H2O on vB_KsaO-C2

CaCl2.2H2O Plaque count percentage concentrations (mM) (%)

0 100 0.1 103* 1 105** 10 118** *Significant difference at p < 0.05

** High significant difference at p< 0.001

120

115

110

105

100

Plaque count percentage (%)percentage count Plaque 90 0 0.1mM 1mM 10mM CaCl .H O concentration (mM) 2 2

Fig. (24): Effect of different concentration of CaCl2.2H2O on vB_KsaO-C2

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Effect of MgCl2.6H2O

Effect of MgCl2.6H2O on vB_KsaM-C1

Studying the effect of different concentrations of MgCl2.6H2O on vB_KsaM-C1 (Ph1) showed no significant effect on plaque count percentage of vB_KsaM-C1 as shown in Fig. (25). Statistical analysis showed no significant difference between different concentrations of

MgCl2.6H2O and the control (0% MgCl2.6H2O) as shown in Table (18).

Table (18): Effect of different concentrations of MgCl2.6H2O on vB_KsaM-C1

MgCl2.6H2O Plaque count percentage concentrations (mM) (%)

0 100 0.1 100 1 99 10 99

100 99 98 97 96 95 94

93 count percentage (%)percentage count 92 91

Plaque 90 0 0.1mM 1mM 10mM MgCl .6H O concentration (mM) 2 2

Fig. (25): Effect of different concentrations of MgCl2.6H2O on vB_KsaM-C1

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Effect of MgCl2.6H2O on vB_KsaO-C2

Studying the effect of different concentration of MgCl2.6H2O on vB_KsaO-C2 (Ph2) showed a high significant increase in plaque count

percentage by increasing the concentration of MgCl2.6H2O as shown in Fig. (26). Statistical analysis showed high significant differences

(p<0.001) between the different concentrations of MgCl2.6H2O

compared with the control (0% MgCl2.6H2O) as shown in Table (19).

Table (19): Effect of different concentration of MgCl2.6H2O on vB_KsaO-C2

MgCl2.6H2O Plaque count percentage concentrations (mM) (%)

0 100 0.1 112** 1 118** 10 139** ** High significant difference at p < 0.001

160 140 120 100 80 60

count percentage percentage (%) count 40 20

plaque 0 0 0.1mM 1mM 10mM

MgCl2.6H2O concentration (mM)

Fig. (26): Effect of different concentrations of MgCl2.6H2O on vB_KsaO-C2

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7.2.3. Effect of trivalent salts

Effect of AlCl3

Effect of AlCl3 on vB_KsaM-C1

Studying the effect of different concentration of anhydrous AlCl3 on vB_KsaM-C1 (Ph1) showed a significant increase in plaque count

percentage at low concentration (0.1mM) of AlCl3. However, a significant decrease in plaque count percentage at high concentration of

AlCl3 (1mM) was observed as shown in Fig. (27). Statistical analysis showed significant differences (p < 0.05) between different concentration

of AlCl3 compared with control (0% AlCl3 ) as shown in Table (20).

Table (20): Effect of different concentration of anhydrous AlCl3 on vB_KsaM-C1

AlCl3 Plaque count percentage concentrations (mM) (%)

0 100 0.1 118* 1 80* * Significant difference at p < 0.05

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140 120 100 80 60 40 20

0 Plaque count percentage (%)percentage count Plaque 0 0.1mM 1mM

Anhydrous AlCl concentration (mM) 3

Fig. (27): Effect of different concentrations of anhydrous AlCl3 on vB_KsaM-C1

Effect of AlCl3 on vB_KsaO-C2

Studying the effect of different concentrations of anhydrous AlCl3 on vB_KsaO-C2 (Ph2) showed a significant decrease in plaque count percentage at salt concentration of 1mM. However, no significant change

was observed at low concentration of AlCl3 (0.1mM) as shown in Fig. (28). Statistical analysis showed a significant difference between the two

concentrations (1mM and 0.1mM) compared with control (0% AlCl3 ) as shown in Table (21).

Table (21): Effect of different concentrations of anhydrous AlCl3 on vB_KsaO-C2.

AlCl3 Plaque count percentage concentrations (mM) (%)

0 100 0.1 100 1 96* * Significant difference at p < 0.05

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101 100 99 98 97 96 95 94 Plaque count percentage (%)percentage count Plaque 0 0.1mM 1mM Anhydrous AlCl3 concentration (mM)

Fig. (28): Effect of different concentration of anhydrous AlCl3 on vB_KsaO-C2.

Effect of FeCl3

Effect of FeCl3 on vB_KsaM-C1

Studying the effect of different concentration of FeCl3 on vB_KsaM-C1 (Ph1) showed a high significant decrease in plaque count percentage by increasing the concentration of Fe3+ as shown in Fig (29). Statistical analysis showed high significant differences between different

concentrations of FeCl3, compared with control (0% FeCl3) as shown in Table (22).

Table (22): Effect of different concentration of FeCl3 on vB_KsaM-C1

FeCl3 Plaque count percentage concentrations (mM) (%)

0 100 0.1 85** 1 60** ** High significant difference at p < 0.001

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120 100 80 60 40 20

Plaque count percentage (%) percentage count Plaque 0 0 0.1mM 1mM FeCl3 concentration (mM)

Fig. (29): Effect of different concentration of FeCl3 on vB_KsaM-C1

Effect of FeCl3 on vB_KsaO-C2

Studying the effect of different concentration of FeCl3 on vB_KsaO-C2 (Ph2) showed a high significant decrease in plaque count percentage by increasing the concentration of Fe3+ as shown in Fig (30). Statistical analysis showed highly significant differences between different

concentrations of FeCl3, compared with control (0% FeCl3) as shown in Table (23).

Table (23): Effect of different concentration of FeCl3 on vB_KsaO-C2

FeCl3 Plaque count percentage concentrations (mM) (%)

0 100 0.1 81** 1 67** ** High significant difference at p < 0.001

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120 100 80 60 40 20

Plaque count percentage (%)percentage count Plaque 0 0 0.1mM 1mM FeCl3 concentration (mM)

Fig. (30): Effect of different concentration of FeCl3 on vB_KsaO-C2

8. Effect of different heavy metals salts

8.1. Effect of different heavy metals on Kosakonia sacchari

Effect of different types of heavy metals salts as: cadmium chloride

(CdCl2), cobalt chloride (CoCl2), copper chloride (CuCl2), mercuric

chloride (HgCl2), silver nitrate (AgNO3) and zinc sulphate (ZnSO4) on Kosakonia sacchari was studied. All the tested heavy metals salts showed significant antibacterial effect against the host bacteria at

concentrations of 10mM and 1mM, except CoCl2 that had no antibacterial effect at concentration of 1mM. Low concentration of

0.1mM for CdCl2, CoCl2, CuCl2 and ZnSO4 showed no significant

antibacterial effect on the host bacteria. However, HgCl2and AgNO3 showed high significant antimicrobial effect at concentrations greater than 0.01mM as shown in Fig. (31).

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10mM 10mM 1mM 10mM 1mM 1mM

CoCl2 CuCl2 CdCl2 C C 0.1mM 0.1mM C 0.1mM

A B C

0.01mM C 10mM 0.1mM 0.1mM 0.01mM C 1mM HgCl 2 AgNO3 10mM C ZnCl2 10mM 1mM 1mM 0.1mM

D E F

Fig. (31): Effect of different concentrations of heavy metals salts on Kosakonia sacchari. A: CdCl2, B: CoCl2, C: CuCl2, D: HgCl2, E: AgNO3 and F: ZnCl2.

8.2. Effect of different heavy metals on the isolated bacteriophages

Effect of different heavy metals on vB_KsaM-C1 The effect of different heavy metals salts on vB_KsaM-C1 (Ph1) was summarized in Fig. (32). Statistical analysis showed high significant

differences between CdCl2, CoCl2, CuCl2 and ZnSO4 concentrations, compared with the control (0% of any heavy metal salts) as shown in Fig. (32) and Table (24).

Effect of CdCl2

Studying the effect of CdCl2 at concentration of 0.1mM showed a high significant decrease in plaque count percentage of the bacteriophage.

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Effect of CoCl2

Studying the effect of CoCl2 at concentrations of (0.1mM and 1mM) showed a high significant increase in plaque count percentage of the bacteriophage.

Effect of CuCl2

Studying the effect of CuCl2 at concentration of 0.1mM showed a high significant decrease in plaque count percentage of the bacteriophage.

Effect of HgCl2

Studying the effect of HgCl2 at concentration of 0.01mM showed no significant change in plaque count percentage of the bacteriophage.

Effect of AgNO3

Studying the effect of AgNO3 at concentration of 0.01mM showed no significant change in plaque count percentage of the bacteriophage.

Effect of ZnSO4

Studying the effect of ZnSO4 at concentration of 0.1mM showed a high significant decrease in plaque count percentage of the bacteriophage.

99 Results

Table (24): Effect of different heavy metals on vB_KsaM-C1

Type of heavy Concentration Control plaque Plaque count metals salts (mM) count percentage (%) percentage (%)

** CdCl2 0.1 48 ** CoCl2 1 207 0.1 159** ** CuCl2 0.1 100 55

HgCl2 0.01 103

AgNO3 0.01 103 ** ZnSO4 0.1 82 ** High significant difference at p< 0.001

250 200 150 100 50

Plaque count percentage (%)

1mM

0.1mM 0.1mM 0.1mM 0.1mM

0.01

0.001mM 0.001mM Control CdCl2 CoCl2 CuCl2 HgCl2 AgNO3 ZnSO4 Control CdCl2 CoCl2 CuCl2 HgCl2 AgNO3 ZnSO4

Fig. (32): Effect of different heavy metals on vB_KsaM-C1

100 Results

Effect of different heavy metals on vB_KsaO-C2

The effect of different heavy metals on vB_KsaM-C2 (Ph2) was summarized in Fig. (33). Statistical analysis showed high significant differences between CdCl2, CoCl2, CuCl2, HgCl2, AgNO3 and ZnSO4 concentrations, compared with the control (0% of any heavy metal salts) as shown in Fig.(33) and Table (25)

Effect of CdCl2

Studying the effect of CdCl2 at concentration of 0.1mM showed a high significant decrease in plaque count percentage of the bacteriophage.

Effect of CoCl2

Studying the effect of CoCl2 at concentrations of (0.1mM and 1mM) showed a high significant decrease in plaque count percentage of the bacteriophage.

Effect of CuCl2

Studying the effect of CuCl2 at concentration of 0.1mM showed a high significant decrease in plaque count percentage of the bacteriophage.

Effect of HgCl2

Studying the effect of HgCl2 at concentration of 0.01mM showed a high significant decrease in plaque count percentage of the bacteriophage.

Effect of AgNO3

Studying the effect of AgNO3 at concentration of 0.01mM showed a high significant decrease in plaque count percentage of the bacteriophage.

101 Results

Effect of ZnSO4

Studying the effect of ZnSO4 at concentration of 0.1mM showed a high significant decrease in plaque count percentage of the bacteriophage.

Table (25): Effect of different heavy metals on vB_KsaO-C2

Type of heavy Concentration Control plaque Plaque count metals salts (mM) count percentage (%) percentage (%)

** CdCl2 0.1 66 ** CoCl2 1 91 0.1 97** ** CuCl2 0.1 100 84 ** HgCl2 0.01 81 ** AgNO3 0.01 90 ** ZnSO4 0.1 63 ** High significant difference at p< 0.001

120 100 80 60 40 20

Plaquecount percentage(%)

1mM

0.1mM 0.1mM 0.1mM 0.1mM

0.01

0.001mM 0.001mM Control CdCl2 CoCl2 CuCl2 HgCl2 AgNO3 ZnSO4

Fig. (33): Effect of different heavy metals on vB_KsaO-C2

102 Results

9. Effect of different sugars

9.1. Effect of different sugars on Kosakonia sacchari

The effect of different sugars (fructose, glucose, lactose and sucrose) at concentrations of (10mM, 1mM and 0.1mM) on Kosakonia sacchari was studied. No antibacterial effect was observed as shown in Fig. (34).

10mM C C 10mM

Fructose Glucose 1mM 1mM 0.1mM 0.1mM

A B

10mM 10mM C C

Lactose Sucrose 1mM 1mM 0.1mM 0.1mM

C D

Fig. (34): Effect of different sugars on Kosakonia sacchari. A: fructose, B: glucose, C: lactose and D: sucrose.

103 Results

9.2 Effect of different sugars on the isolated bacteriophages

9.2.1. Effect of monosaccharides

Effect of glucose

Effect of glucose on vB_KsaM-C1

Studying the effect of different concentration of glucose on vB_KsaM- C1 (Ph1) showed high significant increase in plaque count percentage by increasing the sugar concentration as shown in Fig. (35). Statistical analysis showed highly significant differences (p < 0.001) between the different concentrations of glucose compared with the control (0% glucose) as shown in Table (26).

Table (26): Effect of different concentration of glucose on vB_KsaM-C1

Glucose Plaque count percentage concentrations (mM) (%)

0 100 0.1 110** 1 115** 10 140** ** High significant difference at p< 0.001

104 Results

160 140 120 100 80 60 40 20

Plaque count percentage count Plaque (%)percentage 0 0 0.1mM 1mM 10mM Glucose concentration (mM) Fig. (35): Effect of different concentration of glucose on vB_KsaM-C1

Effect of glucose on vB_KsaO-C2

Studying the effect of different concentration of glucose on vB_KsaO-C2 (Ph2) showed high significant increase in plaque count percentage by increasing the sugar concentration as shown in Fig. (36). Statistical analysis showed high significant differences (p < 0.001) between the different concentrations of glucose compared with the control (0% glucose) as shown in Table (27).

Table (27): Effect of different concentration of glucose on vB_KsaO-C2

Glucose Plaque count percentage concentrations (mM) (%)

0 100 0.1 108** 1 115** 10 144** ** High significant difference at p < 0.001

105 Results

160 140 120 100 80 60 40

20 Plaque count percentage percentage count Plaque (%) 0 0 0.1mM 1mM 10mM

Glucose concenteration (mM) Fig.(36): Effect of different concentration of glucose on vB_KsaO-C2

Effect of fructose

Effect of fructose on vB_KsaM-C1

Studying the effect of different concentration of fructose on vB_KsaM- C1 (Ph1) showed a high significant increase in plaque count percentage at fructose concentrations of 1mM and 10mM as shown in Fig. (37). However, no significant change was observed at low fructose concentration of 0.1mM. Statistical analysis showed high significant differences (p < 0.001) between the different concentrations of fructose compared with the control (0% fructose) as shown in Table (28).

106 Results

Table (28): Effect of different concentration of fructose on vB_KsaM-C1

Fructose Plaque count percentage concentrations (mM) (%)

0 100 0.1 102 1 122** 10 162** ** High significant difference at p < 0.001

180 160 140 120 100 80 60 40 20

Plaque count percentage (%)percentage count Plaque 0 0 0.1mM 1mM 10mM

Fructose concentration (mM) Fig. (37): Effect of different concentration of fructose on vB_KsaM-C1

Effect of fructose on vB_KsaO-C2

Studying the effect of different concentration of fructose on vB_KsaO- C2 (Ph2) showed a high significant increase in plaque count percentage at sugar concentrations of 1mM and 10mM as shown in Fig. (38). However, no significant change was observed at low sugar concentration of 0.1mM. Statistical analysis showed high significant differences (p < 0.001) between the different concentrations of fructose compared with the control (0% fructose) as shown in Table (29).

107 Results

Table (29): Effect of different concentration of fructose on vB_KsaO-C2

Fructose Plaque count percentage concentrations (mM) (%)

0 100 0.1 100 1 105** 10 108** ** High significant difference at p< 0.001

110 108 106 104 102 100 98

Plaque count percentage (%)percentage count Plaque 96 0 0.1mM 1mM 10mM Fructose concentration (mM) Fig. (38): Effect of different concentration of fructose on vB_KsaO-C2

9.2.2. Effect of disaccharides

Effect of Lactose

Effect of Lactose on vB_KsaM-C1

Studying the effect of different concentration of lactose on vB_EsaM-C1 (Ph1) showed a high significant increase in plaque count percentage by increasing the sugar concentration as shown in Fig. (39). Statistical

108 Results analysis showed high significant differences (p < 0.001) between the different concentrations of lactose compared with the control (0% lactose) as shown in Table (30).

Table (30): Effect of different concentration of lactose on vB_KsaM-C1

Lactose Plaque count percentage concentrations (mM) (%)

0 100 0.1 176** 1 311** 10 359** ** High significant difference at p< 0.001

400 350 300 250 200 150 100 50

0 Plaque count percentage (%)percentage count Plaque 0 0.1mM 1mM 10mM

Lactose concentration (mM) Fig. (39): Effect of different concentration of lactose on vB_KsaM-C1

109 Results

Effect of Lactose on vB_KsaO-C2

Studying the effect of different concentration of lactose on vB_KsaO-C2 (Ph2) showed a high significant increase in plaque count percentage by increasing the sugar concentration as shown in Fig. (40). Statistical analysis showed high significant differences (p < 0.001) between the different concentrations of lactose compared with the control (0% lactose) as shown in Table (31).

Table (31): Effect of different concentration of lactose on vB_KsaO-C2

Lactose Plaque count percentage concentrations (mM) (%)

0 100 0.1 113** 1 172** 10 Complete lysis ** high significant difference at p< 0.001

200

150

100

50 count percentage (%)percentage count

0 Plaque 0 0.1mM 1mM Lactose concentration (mM) Fig. (40): Effect of different concentration of lactose on vB_KsaO-C2

110 Results

Effect of Sucrose

Effect of Sucrose on vB_KsaM-C1

Studying the effect of different concentration of sucrose on vB_KsaM- C1 (Ph1) showed a high significant increase in plaque count percentage by increasing the sugar concentration as shown in Fig. (41). Statistical analysis showed high significant differences (p < 0.001) between the different concentrations of sucrose compared with the control (0% sucrose) as shown in Table (32).

Table (32): Effect of different concentration of sucrose on vB_KsaM-C1

Sucrose Plaque count percentage concentrations (mM) (%)

0 100 0.1 112** 1 115** 10 135** ** High significant difference at p< 0.001

160 140 120 100 80 60 40 20

0 Plaque count percentage (%)percentage count Plaque 0 0.1mM 1mM 10mM

Sucrose concentration (mM)

Fig. (41): Effect of different concentration of sucrose on vB_KsaM-C1

111 Results

Effect of Sucrose on vB_EsaO-C2

Studying the effect of different concentration of sucrose on vB_KsaO-C2 (Ph2) showed a high significant increase in plaque count percentage by increasing the sugar concentration as shown in Fig. (42). Statistical analysis showed high significant differences (p < 0.001) between the different concentrations of sucrose compared with the control (0% sucrose) as shown in Table (33).

Table (33): Effect of different concentration of sucrose on vB_KsaO-C2

Sucrose Plaque count percentage concentrations (mM) (%)

0 100 0.1 111** 1 132** 10 139** ** High significant difference at p< 0.001

160 140 120 100 80 60 40

20 Plaque count percentage count Plaque (%)percentage 0 0 0.1mM 1mM 10mM Sucrose concentration (mM) Fig. (42): Effect of different concentration of sucrose on vB_KsaO-C2

112 Results

10. Effect of some plant extracts

10.1. Effect of some plant extracts on Kosakonia sacchari

The effect of some plant extracts (Camellia sinensis L., Cinnamon cassia L., Hibiscus rosa-sinensis L., Matricaria camomilla L., Mentha piperta L., Organum majorana L., Pimpinella anisum L. and Zingiber officinale Roscoe) on Kosakonia sacchari was studied. No antibacterial effect was observed as shown in Fig. (43).

b c a

Control d h

e g f

Fig. (43): Effect of different plant extracts on Kosakonia saccahri. a: Camellia sinensis L. b: Cinnamon cassia L, c: Hibiscus rosa- sinensis L., d: Matricaria camomilla L., e: Mentha piperta L., f: Organum majorana L., g: Pimpinella anisum L. and h: Zingiber officinale Rosocoe.

113 Results

10.2. Effect of some plant extracts on the isolated bacteriophages Effect of some plant extracts on vB_KsaM-C1 The effect of some plant extracts on vB_KsaM-C1 (Ph1) was summarized in Fig. (44). Statistical analysis showed high significant differences (p < 0.001) between the different types of plant extract compared with the control (0% of any plant extract) as shown in Fig. (44) and Table (34).

Effect of Camellia sinensis L. Studying the effect of Camellia sinensis L. at concentration of 100% showed no significant change in plaque count percentage of the bacteriophage.

Effect of Cinnamomum cassia L. Studying the effect of Cinnamon cassia L. at concentration of 100% showed a high significant increase in plaque count percentage of the bacteriophage.

Effect of Hibiscus rosa-sinensis L. Studying the effect of Hibiscus rosa-sinensis L. at concentration of 100% showed a high significant decrease in plaque count percentage of the bacteriophage.

Effect of Matricaria camomilla L. Studying the effect of Matricaria camomilla L. at concentration of 100% showed a significant decrease in plaque count percentage of the bacteriophage.

114 Results

Effect of Mentha piperta L. Studying the effect of Mentha piperta L. at concentration of 100% showed a high significant increase in plaque count percentage of the bacteriophage.

Effect of Organum majorana L. Studying the effect of Organum majorana L. at concentration of 100% showed a high significant decrease in plaque count percentage of the bacteriophage.

Effect of Pimpinella anisum L. Studying the effect of Pimpinella anisum L. at concentration of 100% showed a high significant increase in plaque count percentage of the bacteriophage.

Effect of Zingiber officinale Roscoe Studying the effect of Zingiber officinale Roscoe at concentration of 100% showed a high significant increase in plaque count percentage of the bacteriophage.

115 Results

Table (34) : Effect of different plant extracts on vB_KsaM-C1

Type of plant extract Control plaque count Plaque count percentage (%) percentage (%) Camellia sinensis L. 100 Cinnamomum cassia L. 272** Hibiscus rosa- sinensis L. 1** Matricaria camomilla L. 96* Mentha Piperita L. 100 500** Organum majorana L. 63** Pimpinella anisum L. 163** Zingiber officinale 164** Roscoe * Significant difference at p < 0.05

** High significant difference at p < 0.001

600 500 400 300 200 100

Plaque count percentage (%)percentage count Plaque 0

Fig. (44): Effect of different plant extracts on vB_KsaM-C1

116 Results

Effect of some plant extracts on vB_KsaO-C2

The effect of some plant extracts on vB_KsaM-C2 (Ph2) was summarized in Fig. (45). Statistical analysis showed high significant differences (p < 0.001) between the different types of plant extract compared with the control (0% of any plant extract) as shown in Fig. (45) and Table (35).

Effect of Camellia sinensis L. Studying the effect of Camellia sinensis L. at concentration of 100% showed a high significant decrease in plaque count percentage of the bacteriophage.

Effect of Cinnamomum cassia L. Studying the effect of Cinnamon cassia L. at concentration of 100% showed a high significant decrease in plaque count percentage of the bacteriophage.

Effect of Hibiscus rosa-sinensis L. Studying the effect of Hibiscus rosa-sinensis L. at concentration of 100% showed a high significant decrease in plaque count percentage of the bacteriophage.

Effect of Matricaria camomilla L. Studying the effect of Matricaria camomilla L. at concentration of 100% showed a high significant decrease in plaque count percentage of the bacteriophage.

117 Results

Effect of Mentha piperta L. Studying the effect of Mentha piperta L. at concentration of 100% showed a high significant decrease in plaque count percentage of the bacteriophage.

Effect of Organum majorana L. Studying the effect of Organum majorana L. at concentration of 100% showed a high significant decrease in plaque count percentage of the bacteriophage.

Effect of Pimpinella anisum L. Studying the effect of Pimpinella anisum L. at concentration of 100% showed a high significant decrease in plaque count percentage of the bacteriophage.

Effect of Zingiber officinale Roscoe Studying the effect of Zingiber officinale Roscoe at concentration of 100% showed a high significant decrease in plaque count percentage of the bacteriophage.

118 Results

Table (35) : Effect of different plant extracts on vB_KsaO-C2

Type of plant extract Control plaque count Plaque count percentage (%) percentage (%) Camellia sinensis L. 50** Cinnamomum cassia L. 3** Hibiscus rosa- 31** sinensis L. Matricaria camomilla L. 100 90** Mentha piperita L. 25** Organum majorana L. 25** Pimpinella anisum L. 38** Zingiberofficinale 62** Roscoe ** High significant difference at p < 0.001

120 (%) 100 80 60 40 20

Plaque percentage count Plaque 0

Fig. (45): Effect of different plant extracts on vB_KsaO-C2

119 Results

11. Effect of different essential oils

11.1. Effect of different essential oils on Kosakonia sacchari

Studying the effect of some essential oils (Acacia sp., Allium sativum L., Lactuca sativa L., Lavandula angustifolia Mill., Pimpinella anisum L. and Zingiber officinale Roscoe) on Kosakonia sacchari. No antibacterial effect was observed as shown in Fig. (46).

b c

Control d f e

Fig. (46): Effect of different essential oils on Kosakonia saccahri. a: Acacia sp., b: Allium sativum L., c: Lactuca sativa L., d: Lavandula angustifolia Mill., e: Pimpinella anisum L. and f: Zingiber officinale Rosocoe

11.2. Effect of different essential oils on the isolated bacteriophages

Effect of different essential oils on vB_KsaM-C1

The effect of some essential oils on vB_KsaM-C1 (Ph1) was summarized in Fig. (47). Statistical analysis showed high significant differences (p < 0.001) between the different types of

120 Results essential oils compared with the control (0% of any essential) as shown in Fig. (47) and Table (36).

Effect of Acacia sp. Studying the effect of Acacia sp. at concentration of 100% showed a high significant increase in plaque count percentage of the bacteriophage.

Effect of Allium sativum L. Studying the effect of Allium sativum L. at concentration of 100% showed a high significant decrease in plaque count percentage of the bacteriophage.

Effect of Lactuca sativa L. Studying the effect of Lactuca sativa L. at concentration of 100% showed a high significant decrease in plaque count percentage of the bacteriophage.

Effect of Lavandula angustifolia Mill. Studying the effect of Lavandula angustifolia Mill. at concentration of 100% showed a high significant decrease in plaque count percentage of the bacteriophage.

Effect of Pimpinella anisum L. Studying the effect of Pimpinella anisum L. at concentration of 100% showed a high significant increase in plaque count percentage of the bacteriophage.

Effect of Zingiber officinale Roscoe Studying the effect of Zingiber officinale Roscoe at concentration

121 Results

of 100% showed a high significant increase in plaque count percentage of the bacteriophage.

Table (36): Effect of different essential oils on vB_KsaM-C1

Type of essential oils Control plaque count Plaque count percentage (%) percentage (%) Acacia sp. 162**

Allium sativum L. 25**

Lactuca sativa L. 38** 100 Lavandula angustifolia 83**

Mill. Pimpinella anisum L. 186** Zingiber officinale 140** Roscoe ** High significant difference at p< 0.001

200 180 160 140 120 100 80 60 40

Plaquecount oercentage (%) 20 0

Fig. (47): Effect of different essential oils on vB_KsaM-C1

122 Results

Effect of different essential oils on vB_KsaO-C2

The effect of some essential oils on vB_KsaO-C2 (Ph2) was summarized in Fig. (48). Statistical analysis showed high significant differences (p < 0.001) between the different types of essential oils compared with the control (0% of any essential) as shown in Fig. (48) and Table (37).

Effect of Acacia sp. Studying the effect of Acacia sp. at concentration of 100% showed a high significant decrease in plaque count percentage of the bacteriophage.

Effect of Allium sativum L. Studying the effect of Allium sativum L. at concentration of 100% showed a high significant decrease in plaque count percentage of the bacteriophage.

Effect of Lactuca sativa L. Studying the effect of Lactuca sativa L. at concentration of 100% showed a high significant decrease in plaque count percentage of the bacteriophage.

Effect of Lavandula angustifolia Mill. Studying the effect of Lavandula angustifolia Mill. at concentration of 100% showed a high significant decrease in plaque count percentage of the bacteriophage.

Effect of Pimpinella anisum L. Studying the effect of Pimpinella anisum L. at concentration of

123 Results

100% showed a high significant decrease in plaque count percentage of the bacteriophage.

Effect of Zingiber officinale Roscoe Studying the effect of Zingiber officinale Roscoe at concentration of 100% showed a high significant decrease in plaque count percentage of the bacteriophage.

Table (37) : Effect of different essential oils on vB_KsaO-C2

Type of essential oils Control plaque count Plaque count percentage (%) percentage (%) Acacia sp. 38** Allium sativum L. 60** Lactuca sativa L. 1** Lavadula angustifolia 45** Mill. 100 Pimpinella anisum L. 85**

Zingiber officinale 7** Roscoe ** High significant difference at p < 0.001

124 Results

120

100

20 Plaquecount percentage (%) 0

Fig. (48): Effect of different essential oils on vB_KsaO-C2

125 Results

12. Application of phage therapy in the lab

12.1 Application of phage therapy in the lab using vB_KsaM-C1

The isolated bacteriophage vB_KsaM-C1 (Ph1) successfully protected the potato slice from soft rot, that should be caused by the inoculated host bacteria Kosakonia sacchari, compared by the infected one as shown in Fig.(49).

B C

Fig. (49): Application of phage therapy in the lab using vB_KsaM-C1. Potato slice to which PBS was added (-ve control), showed normal intact tissue [A], while potato slice to which bacterial suspension only was added (+ve control), showed macerated soft tissue, indicating soft rot disease [B]. However, the potato slice to which the phage lysate of vB_KsaM-C1 was added, in addition to, the bacterial suspension, showed normal intact tissue [C].

126 Results

12.2. Application of phage therapy on small scale using vB_EsaO-C2

The isolated bacteriophage vB_KsaO-C2 successfully protected the potato slice from soft rot, that should be caused by the inoculated host bacteria Kosakonia sacchari, compared by the infected one as shown in Fig. (50).

B C

Fig.(50): Application of phage therapy in the lab using vB_KsaO-C2. Potato slice to which PBS was added B(- ve control), showed normal intact tissue [A], while potato slice to which bacterial suspension only was added (+ve control), showed macerated soft tissue, indicating soft rot disease [B]. However, the potato slices to which the phage lysate of vB_KsaO-C2 was added, in addition to, the bacterial suspension, showed normal intact tissue [C]

127

Discussion

Potato soft rot is a serious economic disease, caused by Enterobacteriacae (Czajkowski et al.,2011). Members of Enterobacter were recently detected to be phytopathogens causing soft rot (Liu et al., 2016).

In our study three Enterobacter species were isolated and identified to cause potato soft rot as indicated by pathogenicity test, biochemical tests and molecular identification by 16S rRNA gene sequencing. These isolates were identified to be strains of Lelliottia amnigena, Kosakonia sacchari and Enterobacter cloaca. These bacterial isolates were recorded in the gene bank by the following accession numbers: KY427021, KY235364 and KY235363 respectively.

Lelliottia amnigena is a new proposed genus, formerly known as Enterobacter amnigena, as proposed by Brady et al., (2013) depending on Multilocus Sequence Analysis (MLSA). In our study, it was isolated as a phytopathogenic bacteria from infected potato tubers and proposed to cause potato soft rot disease in Egypt. In agreement to our results, Lelliottia amnigena had been recently isolated from infected onion bulbs showing soft rot symptoms in China, that was considered as the first report of soft rot disease caused by this genus (Liu et al., 2016). However, Lelliottia amnigena was isolated from soil and identified to play an important role in nitrate reduction to ammonia (Fazzolari et al., 1990).

Kosakonia sacchari is a novel species of a new proposed genus, previously known as Enterobacter sacchari (Brady et al., 2013). In our study, this

128 Discussion bacterial strain was isolated from infected potato tubers and proposed to cause potato soft rot disease, that is considered as the first report in soft rot disease to be caused by this genus in Egypt. On the other hand, Kosakonia sacchari was isolated as a nitrogen fixing bacteria colonizing sugarcane promoting its growth (Chen et al., 2014).

Enterobacter cloaca is a part of intestinal microflora, however, it is an opportunistic, multidrug resistant member of Enterobacteriacae, causing many nosocomial infections in the last decades (Keller et al., 1998 and Davin-Regli and Pages 2015). On the other hand, it should be mentioned that Enterobacter cloaca species is a large group comprising diverse strains that spread in different environments (plants, soils and human) and show different reactions according to its host (Liu et al., 2013). In our study, a strain of Enterobacter cloaca was isolated as a phytopathogenic bacteria from infected potato tubers and was identified to cause potato soft rot disease. In agreement to our study, strains of Enterobacter cloaca were isolated and found to be phytopathogenic causing serious post harvest plant diseases, causing soft rot to dragon fruit in Malaysia and onion bulbs decay in USA, ginger rhizome rot in Brazil, and bacterial wilt of mulberry in China (Masyahit et al., 2009; Schroeder and du Toit 2010; Moreira et al., 2013 and Wang et al., 2008). In contrary to our results, strains of Enterobacter cloaca were isolated as endophytic bacteria, promoting plant growth as in case of isolation of Enterobacter cloaca mutants from cucumber roots (Liu et al., 2007). Furthermore, Enterobacter cloaca strains were used in biocontrolling plant diseases, as those strains that were found to colonize corn seedling roots and controlling corn kernels infection by Fusarium sp. (Hinton and Bacon 1995) and those used for biocontrolling of cotton seed rot and damping off caused by Pythium spp. (Nelson 1988).

129 Discussion

In our study, three Enterobacter species (Lelliottia amnigena, Kosakonia sacchari, Enterobacter cloaca) were found to cause soft rot disease to potato crop resulting in great economic loss.

In our study, two bacteriophages were isolated from potato tubers rhizosphere. The two isolated phages showed different plaques, in both shape and size, that facilitated their purification. The isolation and purification steps were done using double layer assay, to obtain clear visualized plaques. This was in agreement with Lillehaug (1997), who suggested the improvement of phage diffusion rate using double layer plates. The two isolated bacteriophages were preliminary named as Ph1 and Ph2. Transmission Electron Microscope photographing showed that, Ph1 possessed icosahedral head, short neck and long flexible tail. On the other hand, Ph2 possessed small polyhedral heads without tails. The genome of both phages was proved to be DNA. According to Ackermann (2007), the phages were classified depending on phage morphology and genome type. Ph1 was related to Myoviridae, while, Ph2 was related to Microviridae. Therefore, according to Kropiniski et al., (2009), Ph1 was named as vB_KsaM-C1and Ph2 was named as vB_KsaO-C2. In agreement to our results, previous studies reported the isolation of bacteriophages belonging to Myoviridae to control soft rot disease: Tovkach (2002a) isolated a new species of temperate bacteriophage ZF40, Revensdale et al., (2007) isolated bacteriophages from fertilizer solution samples, Evans et al., (2010) isolated a novel flagellatropic bacteriophage AT1, Jee et al., (2012) isolated bacteriophages from 6 different soil samples cultivated by Chinese cabbage in Korea, Czajkowski et al.,(2013)

130 Discussion isolated nine bacteriopages from soil samples collected in Poland, Lim et al., (2014) isolated PM1 bacteriophage from Chinese cabbage field, Czajkowski, et al.,(2015) isolated and characterized two novel broad host range lytic bacteriophages, PD10.3 and PD23.1, from potato samples collected from two different potato fields in central Poland, Delfan et al., (2015) isolated bacteriophages, from Caspian sea water and Lee et al.,(2017) isolated bacteriophages from potato tubers rhizospheres. On the other hand, bacteriophages belonging to Microviridae were reported to infect Enterobacteriacae members (Fane et al., 2011 and Roux et al., 2012). Contrary to our results, bacteriophages belonging to other families were isolated to control soft rot disease: Revensdale et al., (2007), Mishra et al., (2012) and Delfan et al., (2015) isolated bacteriophages belonging to Siphoviridae. Furthermore, Hirata et al., (2016), Lim et al., (2017) and Smolarska et al., (2017) isolated lytic bacteriophages, belonging to Podoviridae. In addition, Soleimani et al., (2012) isolated a novel bacteriophage belonging to Cystoviridae.

Our isolated bacteriophages infected different bacterial strains causing soft rot disease. Phage vB_KsaM-C1 showed lytic effect against; Pectobacterium carotovorum subsp. carotovorum, Pseudomonas flourecens, Enterobacter cloaca isolate and Bacillus pumilus. However, it showed no lytic effect against Lelliottia amnigena, Pseudomonas putida and Escherichia coli. On the contrary, Czajkowski et al., (2013) isolated bacteriophages belonging to Myoviridae to control soft disease and studied their host range. The isolated bacteriophages showed specific lytic effect against Dickeya spp., however, they showed no lytic effect against any Pectobacterium spp.. In our study, phage vB_KsaO-C2 showed lytic effect against; Pectobacterium carotovorum subsp. carotovorum, Pseudomonas flourecens,

131 Discussion

Enterobacter cloaca isolate and Lelliottia amnigena. However, it showed no lytic effect on Bacillus pumilus, Pseudomonas putida and Escherichia coli. On the other hand, Labrie et al., (2014) isolated a Microviridae phage and studied its host range. It showed specific lytic effect against Escherichia coli. Escherichia coli was used as a model for the normal microflora of human and animal intestines. The two isolated bacteriophages showed no lytic effect against it, suggesting the safe use of the two isolated bacteriophages in phage therapy for controlling potato soft rot disease, without any disturbance in the normal intestinal microflora.

In our study, the dilution end point for vB_KsaM-C1 was at 10-9, while the dilution end point for vB_KsaO-C2 was at 10-7. Furthermore, the two isolated phages were found to be thermo labile phages, as both showed thermal stability up to 55oC. Phage vB_KsaM-C1 showed optimum activity at 15oC, with a noticeable decrease in plaque count at temperature degrees greater or less than 15oC. In agreement to our results, Czajkowski et al., (2013) isolated bacteriophages belonging to Myoviridae showing maximum stability at temperature ranging from 4oC to 37oC with decreasing infectivity by increasing temperature. Phage vB_KsaO-C2 showed optimum activity at 5oC, with a noticeable decrease in phage plaque count at temperature greater than 35oC. Tsutsaeva et al., (1981) studied the surviving ability of Microviridae phages at freezing temperatures and found that over 60% survived the freezing temperature without change in protein and DNA biosynthesis.

In our study, both bacteriophages sustained their optimum activity at neutral pH values, with a decrease in plaque count at acidic or alkaline conditions and total inhibition of phages infectivity at acidic (less than pH 4) and

132 Discussion alkaline (more than pH 10) values. Our results are in agreement with Krasowska et al., (2015) who stated that the phage optimum activity should be at neutral pH values. This is attributed to the adsorption of the phage on the host bacterial cell which is affected by pH value, due to charge alteration of protein capsid (Wunsche 1989). Bacteriophages infectivity is affected by temperature and pH, as those factors cause lipid dissolving, DNA and protein denaturation, leading to phage structure damage (Wunsche 1989).

Studying the longevity in vitro showed that both isolated bacteriophages sustained their lytic activity more than 20 months, either stored at room temperature (25oC-35oC) or at refrigerator (5oC), but with a significant decrease in phage titre. Our results are in agreement with Ackermann et al., (2004) who proved the ability of the two phages T4 and C16 belonging to Myoviridae to sustain long -term storage up to years.

In our study, the effect of mono, di and trivalent salts (sodium chloride, potassium chloride, calcium chloride, magnesium chloride, aluminum chloride and ferric chloride) on the activity of both isolated phages was tested. The of monovalent salts of sodium chloride and potassium chloride on both isolated phages was studied. K+ ions showed a significant increase in the activity of both isolated bacteriophages at concentration greater than 1mM. Na+ ions showed a high significant increase in the activity of phage vB_KsaM-C1. This could be attributed to the increase of phage adsorption to host bacterial cells in the presence of monovalent salts as Na+ and K+ (Zachary 1976). On the contrary, our results showed that, Na+ ions caused a high significant decrease in the activity of phage vB_KsaO-C2.

133 Discussion

Divalent salts such as magnesium chloride and calcium chloride showed different effects on both isolated phages. Mg++ ions showed no significant effect on the activity of phage vB_KsaM-C1, however, it showed a high significant increase in the activity of phage vB_KsaO-C2. Our results are in agreement with Tucker (1961) who explained the role of Mg++ ions in the enhancement of phage growth, replication and activity. Ca++ ions showed a high significant increase in the activity of both phages at concentration greater than 1mM. This is attributed to the role of Ca++ ions in enhancement of phage adsorption to host cell, transfer of nucleic acid through the host cell and working as co-factor for phage replication (Bonhivers and Letellier 1995 and Paunikar et al, 2012). The trivalent salts such as aluminum chloride and ferric chloride showed negative effect on the phage activity. Al3+ and Fe3+ ions showed a significant decrease in the activity of both phages. Our results are in agreement with Matsui et al., (2003) who explained the inhibitory effect of Al3+ ions due to its coagulating effect on viral particles, and Sagripanti et al., (1993) who explained the significant decrease in the activity of bacteriophages, in presence of F3+ ions due to its inactivating effect.

In our study, the effect of some heavy metals salts (cadmium chloride, cobalt chloride, copper chloride, mercuric chloride, silver nitrate and zinc sulphate) was studied on the activity of both isolated bacteriophages. Co++ ions showed a high significant increase in the activity of phage vB_KsaM-C1, this is due to the role of Co++ions in enhancement of viral transformation (Casto et al., 1979). On the other hand, Co++ ions showed a significant decrease in the activity of phage vB_KsaO-C2. Cd++ ions showed a significant decrease in the activity of both isolated bacteriophages. Contrary to our results, Casto et al., (1979) studied the effect of Cd++ ions on viral transformation and showed

134 Discussion positive effect of Cd++ ions on the viral particles due to its enhancement to the virus transformation. Cu++ ions showed a significant decrease in the activity of both isolated bacteriophages. This is attributed to deactivating effect of Cu++ ions to the phage particles (Paunikar et al., 2012). Contrary to our results, Shams El Din (2013) studied the effect of Cu++ ions on the isolated phages and found no significant effect of Cu++ions on the isolated phages. Zn++ ions showed a significant decrease in the activity of both isolated bacteriophages. This is due to weak effect of Zn++ ions on viral transformation in addition to be a mutagenic agent in DNA synthesis (Casto et al., 1979). Hg++ ions showed no significant effect on the activity of phage vB_KsaM- C1, however Hg++ ions showed a high significant decrease in the activity of phage vB_KsaO-C2, this is attributed to the toxic effect of Hg++ ions on proteins causing its deformation and precipitation (Patra et al., 2004). Ag+ ions showed no significant effect on the activity of phage vB_KsaM-C1. In agreement to our results, Shams El Din (2013) studied the effect of Ag+ ions on the isolated phages and recorded no significant effect of Ag+ ions on the isolated phages. On the other hand, Ag+ ions showed a significant decrease in the activity of phage vB_KsaO-C2. This is due to the interaction of Ag+ ions with proteins thiol groups and its destructive effect on DNA replication (Paunikar et al., 2012).

In our study, the effect of monosaccharides (fructose and glucose) and disaccharides (lactose and sucrose) was tested on the activity of both isolated phages. All the tested sugars (fructose, glucose, lactose and sucrose) showed high significant increase in the activity of both isolated phages, in the form of an

135 Discussion increase in the number of plaques. Our results are in agreement with Cromier and Janes (2014) who proved the activating effect of glucose on bacteriophages. They explained that the bacterial cells utilize sugars, not only as a carbon source but also as a building block for the synthesis of amino acids, vitamins, nucleotides and cell wall. Therefore, the addition of sugars to the media modified the growth conditions of bacteria, resulting in an increased number of host bacterial cells. This leads to an increased number of the propagating bacteriophage which is expressed in an increase in the number of plaques.

In our study, the effect of some natural plant extracts, either in the form of aqueous extracts or essential oils was tested at a concentration of 100% on the isolated bacteriophages. The effect of some natural aqueous plant extracts (Camellia sinensis L., Cinnamomum cassia L., Hibiscus rosa- sinensis L., Organum majorana L., Matricaria camomilla L., Mentha piperita L., Pimpinella anisum L. and Zingiber officinale Roscoe) showed different effects on the both isolated bacteriophages. The extracts of Cinnamomum cassia L., Mentha piperita L., Pimpinella anisum L., and Zingiber officinale Roscoe showed a high significant increase in the activity of phage vB_KsaM-C1. On the other hand, these extracts showed a significant decrease in the activity of phage vB_KsaO-C2. The extract of Camellia sinensis L. showed no significant effect on phage vB_KsaM-C1, on the other hand, it showed a significant decrease on the activity of phage vB_KsaO-C2. The extracts of Hibiscus rosa- sinensis L., Matricaria camomilla L. and Organum majorana L. showed a high significant decrease in the activity of both isolated phages.

136 Discussion

In addition, the effect of some natural essential oils (Acacia sp., Allium sativum L., Lactuca sativa L., Lavandula angustifolia Mill., Pimpinella anisum L. and Zingiber officinale Roscoe) on the activity of the isolated phages was studied. Oil extracts of Acacia sp., Pimpinella anisum L. and Zingiber officinale Roscoe showed a high significant increase in the activity of phage vB_KsaM-C1. On the other hand, these oil extracts showed a high significant decrease on phage vB_KsaO-C2. Oil extracts of Allium sativum L., Lactuca sativa L. and Lavandula angustifolia Mill. showed a high significant decrease in the activity of both isolated phages. In agreement to our results, Zahran (2017) studied the effect of some natural plant extracts of Camellia sinensis L., Cinnamomum cassia L., Ellicium verum Hook F., Organum majorana L., Matricaria camomilla L., Mentha piperita L. and Zingiber officinale Roscoe on the isolated phages. The polyphenols with other chemical constituents of these plant extracts have a role in increasing or decreasing the phage activity (AlKhazindar et al., 2016).

In our study, we succeeded to isolate two bacteriophages, vB_KsaM-C1 and vB_KsaO-C2 belonging to Myoviridae and Microviridae respectively, to control potato soft rot disease caused by Enterobacteriacea members.

Application of phage therapy on small scale, supported their role in controlling soft rot disease, by protecting potato slices from rotting symptoms caused by the bacterial inoculum in non treated potato slices. Further studies concerning application of phage therapy on large scale to control soft rot disease in field, will be taken in consideration. However, the application of phage therapy in soil, will fate the disadvantage of phage inactivation by temperature and UV radiation of sunlight, which propose its

137 Discussion use directly on potato tubers before planting or at storage (Czajkowski et al., 2013).

138

Summary

In our study, three Enterobacter isolates (Lelliottia amnigena, Kosakonia sacchari, Enterobacter cloaca) were identified and proved to cause potato soft rot disease to two different types of potato (Solanum tuberosum L. cv. Spunta and Solanum tuberosum L. cv. Lady rosetta).

The bacterial isolates were recorded in the gene bank and were given accession numbers KY427021, KY235364 and KY235363, respectively.

Two bacteriophages were isolated from potato tubers rhizosphere and named vB_KsaM-C1 and vB_KsaO-C2.

Examination of bacteriophages negatively stained by 3% uranyl acetate by Transmission Electron Microscope showed that, the bacteriophage vB_KsaM- C1 possessed icosahedral head (58.04nm), neck (13.13nm(L) X 8.06nm(W)) and tail (96.42nm(L) X 14.34nm(W)). The bacteriophage vB_KsaO-C2 showed polyhedral head (23.45nm). The genome of both isolated bacteriophages was proved to be DNA. Accordingly, bacteriophage vB_KsaM-C1 belongs to Myoviridae, and vB_KsaO-C2 belongs to Microviridae.

The two isolated bacteriophages showed lytic effect on some bacterial strains known to cause potato soft rot disease as Pectobacterium carotovorum subsp. carotovorum, Pseudomonas flourecens and Enterobacter cloaca isolate. Phage vB_KsaM-C1 showed its lytic effect on Bacillus pumilus, while Phage vB_KsaO-C2 showed its lytic effect on Lelliottia amnigena.

139 Summary

The thermal inactivation point was at 65oC for both isolated bacteriophages. The optimum temperature for the bacteriophage vB_KsaM-C1 was at 15oC, while that for the bacteriophage vB_KsaO-C2 was at 5oC.

Studying the effect of pH showed that both isolated bacteriophages sustained their optimum lytic activity at neutral pH values. The bacteriophage vB_KsaM-C1 showed optimum lytic activity at pH (7-8) while bacteriophage vB_KsaO-C2 showed its optimum lytic activity at pH 7.

Studying the longevity in vitro showed that both isolated bacteriophages sustained their lytic activity more than 20 months but with remarkable decrease in phage titre.

In our study, we investigated the effect of different chloride salts (sodium chloride, potassium chloride, calcium chloride, magnesium chloride, aluminum chloride and ferric chloride) on the bacteriophages activity. The effect of monovalent salts of Na+ and K+ ions on the activity of both isolated bacteriophages was studied. K+ ions showed a high significant increase in the activity of both isolated bacteriophages. Na+ ions showed a a high significant increase in the activity of bacteriophage vB_KsaM-C1, however, it showed a high significant decrease in the activity of bacteriophage vB_KsaO-C2. The effect of divalent salts of Ca++ and Mg++ ions on the activity of both isolated bacteriophages was studied. Ca++ ions caused a significant increase in the activity of both isolated bacteriophages. Mg++ ions caused a significant increase in the activity of bacteriophage vB_KsaO-C2 and it has no significant effect on bacteriophage vB_KsaM-C1. The trivalent salts of Al3+ and Fe3+ ions caused a significant decrease in the activity of both isolated bacteriophages.

Studying the effect of some heavy metals salts such as (cadmium chloride, cobalt chloride, copper chloride, mercuric chloride, silver nitrate and zinc

140 Summary sulphate) on the activity of both isolated bacteriophages showed different results. Cd++, Cu++and Zn++ ions showed a significant decrease in the activity of both isolated bacteriophages. However, Co++ ions showed a significant increase in the activity of the bacteriophage vB_KsaM-C1 and significant decrease in the activity of bacteriophage vB_KsaO-C2. Hg++ and Ag+ ions showed no significant effect on the activity of bacteriophage vB_KsaM-C1, while, both showed a significant decrease in the activity of bacteriophage vB_KsaO-C2.

Studying the effect of monosaccharides (fructose and glucose) and disaccharides (lactose and sucrose) on the activity of both isolated bacteriophages, showed high significant increase in activity of both bacteriophages.

The effect of some natural plant extracts (Camellia sinensis L., Cinnamomum cassia L., Hibiscus rosa-sinensis L., Matricaria camomilla L., Mentha piperita L. Organum marjorana L., Pimpinella anisum L. and Zingiberofficinale Roscoe), at a concentration of 100%, on the activity of the isolated bacteriophages were studied. The extracts of Hibiscus rosa-sinensis L. and Organum marjorana L. showed a significant decrease in the activity of both isolated bacteriophages. The extracts of Cinnamomum cassia L., Mentha piperita L., Pimpinella anisum L. and Zingiber officinale Roscoe showed a significant increase in the activity of bacteriophage vB_KsaM-C1, on the other hand, these extracts showed a significant decrease in the activity of bacteriophage vB_KsaO-C2. The extracts of Camellia sinensis L. and Matricaria camomilla L. showed no significant effect on bacteriophages vB_KsaM-C1, however, these extracts showed a significant decrease in the activity of bacteriophage vB_KsaO-C2.

141 Summary

The effect of some natural essential oils extracts (Acacia sp., Allium sativum L., Lactuca sativa L., Lavandula angustifolia Mill., Pimpinella anisum L. and Zingiber officinale Roscoe) on the activity of the isolated bacteriophages was studied. Oil extracts of Allium sativum L., Lactuca sativa L. and Lavandula angustifolia Mill. showed a significant decrease in the activity of both isolated bacteriophages. Oil extracts of Acacia sp., Pimpinella anisum L. and Zingiber officinale Roscoe showed a high significant increase in the activity of bacteriophage vB_KsaM-C1, and showed a significant decrease in the activity of bacteriophage vB_KsaO-C2.

In our study, a small scale application of phage therapy on potato slices, successfully supported the role of the two isolated bacteriophages in controlling potato soft rot disease.

142

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الملخص العربى

فقد اظهرت زيادة كبيرة في نشاط الفاجvB_KsaM-C1، من ناحية أخرى، أظهرت هذه المستخلصات انخفاضا كبيرا في نشاط الفاج vB_KsaO-C2 . اما مستخلصات الشاى و الكاموميل فلم يكن لها اى تأثير على نشاط الفاج vB_KsaM-C1، بينما، أظهرت هذه المستخلصات انخفاضا كبيرا في نشاط الفاجvB_KsaO-C2.

وقد تم ايضا دراسة تأثير بعض الزيوت النباتية العطرية مثل :الصمغ العربى،و الثوم ، والينسون، والخس، والالفندر والزنجبيل. وقد أظهرت بعض المستخلصات الزيتية العطريه من الثوم، و الخس ، و الالفندر انخفاضا كبيرا في نشاط كل من البكتريوفاجات المعزولة. اما المستخلصات الزيتية من الينسون،والصمغ العربى ، و الزنجبيل فقد اظهرت زيادة كبيرة في نشاط الفاجvB_KsaM-C1، و انخفاضا كبيرا في نشاط الفاجvB_KsaO-C2.

و قد تم تطبيق المكافحة البيولوجية لمرض العفن الطرى فى البطاطس على نطاق صغير فى المختبر. و ذلك باستخدام البكتريوفاجات المعزولة على شرائح درنات البطاطس وقد بينت النتائج انه تم تثبيط المرض بنجاح وان شرائح درنات البطاطس لم تصاب بمرض العفن البكتيرى الطرى.

3 الملخص العربى

العدوى قد سجل عند الرقم الهيدروجيني 8-7 وان اقصى نشاط للفاج vB_EsaO-C2عند العدوى قد سجل عند الرقم الهيدروجيني 7 .

وأظهرت دراسة طول العمر في المختبر أن كال من البكتريوفاجات المعزولة استمرت نشاطها التحليلي أكثر من 20 شهرا.

تم دراسة تأثير عدد من امالح الكلورايد على البكتريوفاجات المعزولة مثل :كلوريد الصوديوم و كلوريد البوتاسيوم و كلوريد الكالسيوم و كلوريد الماغنيسيوم و كلوريد االلومنيوم وكلوريد الحديد. وبدراسة تأثير االمالح احادية التكافؤ اليونات +Na و+K اظهرت زيادة كبيرة فى نشاط كال البكتريوفاجات . وتم دراسة تأثير االمالح ثنائية التكافؤ اليونات ++Ca و++Mg و قد وجد ان ايونات ++Ca تسببت فى زيادة كبيره فى نشاط كال البكتريوفاجات . و تسببت ايونات ++Mg فى زيادة نشاط فاج vB_EsaO-C2، ومن ناحية اخرى فان ايونات ++Mg ليس لها اى تأثير على نشاط فاج vB_EsaM-C1. وبدراسة تأثير االمالح ثالثية التكافؤ اليونات Al3+ وFe3+ ، وجد انها قد ادت الى انخفاض شديد فى نشاط كال البكتريوفاجات .

وبدراسة تأثير بعض المعادن الثقيلة مثل: كلوريد الكادميوم و كلوريد الكوبالت و كلوريد النحاس و كلوريد الزئبق ونترات الفضة و كبريتات الزنك على نشاط كل من البكتيروفاجات المعزولة ، تبين ان ايونات++Cd و++Cu و ++Zn تسبب انخفاض شديد فى نشاط كال البكتيروفاجات . و قد تسبب ايون ال ++Co فى حدوث زيادة فى نشاط فاج vB_KsaM-C1، بينما تسبب فى حدوث انخفاض فى نشاط vB_EsaO-C2 . وقد تسببت ايونات ++Hg و+Ag فى انخفاض نشاط فاج -vB_KsaO C2، ومن ناحية اخرى فلم يكن لها تأثير على نشاط فاج vB_EsaM-C1 .

وبدراسة تأثير السكريات االحادية )الفركتوز والجلوكوز( والسكريات الثنائية )الالكتوز والسكروز( على نشاط كال البكتريوفاجات المعزولة، أظهرت زيادة كبيرة جدا في نشاط كال من البكتريوفاجات.

و بدراسة تأثير بعض المستخلصات النباتية الطبيعية بتركيز 100٪ على نشاط البكتيريوفاجات المعزولة أظهرت نتائج مختلفة. وانخفض نشاط كال من البكتريوفاجات المعزولة بشكل كبير في وجود مستخلصات الكركديه و البردقوش. أما مستخلصات القرفة، والينسون، والنعناع والزنجبيل

2 الملخص العربى

الملخص العربى

تم عزل و تعريف ثالث عزالت من عائلة إنتيروباكتر ) Lelliottia amnigena, Kosakonia sacchari and Enterobacter cloaca) من نوعين من البطاطس المصابة ) Solanum tuberosum L. cv. Spunta and Solanum tuberosum L. cv. Lady rosetta) وثبت أن هذه العزالت تسبب مرض العفن الطرى فى البطاطس و تم تسجيل العزالت البكتيرية في بنك الجينات وأعطيت أرقام التسجيل التالية: KY235364 ،KY427021 و KY235363 على التوالي. تم عزل اثنين من البكتريوفاجات vB_KsaM-C1 وvB_KsaO-C2 من التربة المحيطة لدرنات البطاطس .وقد تم فحص البكتريوفاجات بعد صبغها بصبغة 3٪ استات اليورانيل السالبة تحت المجهر االليكترونى ، و قد تبين ان فاجvB_EsaM-C1 يمتلك رأس متعددة الوجوه بقطر 58.04 نانومتر ورقبة )13.13 نانومتر طول x 8.06 نانومتر عرض( و ذيل )96.42 نانومتر طول x 14.34 نانومتر عرض(. اما فاج vB_EsaO-C2 فيمتلك رأس مستديرة بقطر 23.45 نانومتر. وقد ثبت أن الجينوم لكل من البكتيريا المعزولة هو الحمض النووي(DNA(. ووفقا لذلك، ينتمى فاج vB_EsaM-C1 لعائلة Myoviridae ، بينما ينتمى فاج vB_EsaO-C2 لعائلة Microviridae

وقد اظهر كال من البكتريوفاجات المعزولة تأثير تحليلي على بعض سالالت البكتريا المسببة لمرض العفن الطرى فى البطاطس مثل: .Pectobacterium carotovorum subsp carotovorum, Pseudomonas flourecens and Enterobacter cloaca isolate.

وقد سجل ان كال من البكتريوفاجات المعزولة فقدت قدرتها على العدوى عند درجة حرارة 65oC . اما درجة الحرارة المثلى القصى نشاط تحليلى لفاج vB_EsaM-C1 فقد كانت 15oC .بينما درجة الحرارة المثلى القصى نشاط تحليلى لفاج vB_EsaO-C2 فقد كانت 5oC

و بدراسة تأثير الرقم الهيدروجيني تبين أن كال من البكتريوفاجات المعزولة استمر نشاطها التحليلي األمثل في قيم الرقم الهيدروجيني المتعادل. فقد وجد ان اقصى نشاط للفاج vB_EsaM-C1 عند

الملخص العربى المستخلص العربى

نهاية التخفيف عند 7-10، وقد وأظهرت دراسة طول العمر في المختبر أن البكتريوفاج المعزول استمر نشاطه التحليلي أكثر من 20 شهرا ، و فقد قدرته على العدوى عند درجة حرارة 65oC ، و ان اقصى نشاط للفاج عند العدوى قد سجل عند الرقم الهيدروجيني 8-7. اما الفاج vB_EsaO-C2 فيمتلك رأس مستديرة بقطر 23.45 نانومتر. و قد تم تحديد نقطة نهاية التخفيف عند 9-10 ، وقد أظهرت دراسة طول العمر في المختبر أن البكتريوفاج المعزول استمر نشاطه التحليلي أكثر من 20 شهرا ، و فقد قدرته على العدوى عند درجة حرارة 65oC ، و ان اقصى نشاط للفاج عند العدوى قد سجل عند الرقم الهيدروجيني 7. وقد تم اختبار تأثير عدد من االمالح األحادية والثنائية و الثالثة التكافؤ، والمعادن الثقيلة وأنواع مختلفة من السكاريات االحادية والسكاريات الثنائية وأنواع مختلفة من المستخلصات النباتية والزيوت العطرية على البكتريوفاجين المعزولين. وقد تم تطبيق المكافحة البيولوجية لمرض العفن الطرى فى البطاطس باستخدام البكتريوفاجات المعزوله على نطاق صغيرفى المختبر باستخدام شرائح درنات البطاطس وتم تثبيط المرض بنجاح وحماية شرائح درنات البطاطس من العفن البكتيرى الطرى.

الساده المشرفون: التوقيع

ا.د. السيد طارق عبد السالم .………………………… استاذ علم الفيروسات- قسم النبات و الميكروبيولوجى- كلية العلوم- جامعة القاهره

د. مها محمد الخاذندار ...... استاذ مساعد علم الفيروسات- قسم النبات والميكروبيولوجى- كلية العلوم- جامعة القاهرة

ا.د. نيفين صالح جويلى رئيس قسم النبات و الميكروبيولوجى

المستخلص العربى

اسم الطالب: ايمان عبد الحفيظ الديب عبد الحميد

عنوان الرسالة: عزل و تعريف البكتريوفاجات التى تصيب البكتريا المسببه لمرض العفن الطرى فى البطاطس

الدرجة : الماجيستير فى العلوم )النبات و الميكروبيولوجى(

محصول البطاطس من المحاصيل األكثر أهمية من الناحية االقتصادية، وتعتبر المحصول الغذائي الرئيسي الرابع في العالم . وتتعرض البطاطس للعديد من االمراض والحشرات التي تسبب خسارة كبيرة في محصول البطاطس . و من أهم األمراض الخطيرة التى تصيب البطاطس هو مرض العفن الطرى البكتيرى . في دراستنا تم عزل البكتيريا الرئيسية المسببة لمرض العفن الطرى من نوعين من البطاطس المصابة: Solanum tuberosum L. cv. Spunta and Solanum tuberosum L. cv. Lady .rosetta تم التعرف على العينات البكتيرية المعزولة عن طريق االختبارات الكيميائية الحيوية وأكدت من قبل التسلسل الجزيئي لتكون سالالت من Lelliottia amnigena, Kosakonia sacchari and Enterobacter cloaca تم تسجيل العزالت البكتيرية في بنك الجينات وأعطيت أرقام التسجيل التالية: KY427021، KY235364 و KY235363 على التوالي. في دراستنا، تم عزل و تعريف اثنين من البكتيريوفاجات لمكافحة البكتيريا المسببه لمرض العفن الطرى فى البطاطس . تم جمع عينات التربة من منطقة زراعة البطاطس من حقل مزروع بالبطاطس في الجيزة، مصر. تم عزل اثنين من البكتريوفاجات وتنقيتها وتعريفها باستخدام المجهر اإللكتروني، ونقطة نهاية التخفيف ، وطول العمر في المختبر، تأثير الحرارة ، وتأثير الرقم الهيدروجيني والمدى العائلي. وقد تم تسمية كال من الفاجات المعزولة vB_EsaM-C1 وvB_EsaO-C2 . ووجد ان الفاج vB_EsaM-C1 يمتلك رأس متعددة الوجوه بقطر 58.04 نانومتر ورقبة )13.13 نانومتر طول x 8.06 نانومتر عرض( و ذيل )96.42 نانومتر طول x 14.34 نانومتر عرض(. و قد تم تحديد نقطة

كلية العلوم

قسم النبات و الميكروبيولوجى

عزل و تعريف البكتريوفاجات التى تصيب البكتريا المسببه لمرض العفن الطرى فى البطاطس

رسالة مقدمة الى كلية العلوم جامعة القاهرة كمتطلب جزئى للحصول على درجة الماجيستير فى العلوم

)الميكروبيولوجى(

إعداد ايمان عبد الحفيظ الديب عبد الحميد

بكالوريوس علوم ( الكيمياء والميكروبيولوجى ( ۲۰۱۲

قسم النبات و الميكروبيولوجى

كلية العلوم-جامعة القاهرة

الساده المشرفون:

ا.د. السيد طارق عبد السالم استاذ علم الفيروسات- قسم النبات و الميكروبيولوجى- كلية العلوم- جامعة القاهره التوقيع ………………………… د. مها محمد الخازندار استاذ مساعد علم الفيروسات- قسم النبات والميكروبيولوجى- كلية العلوم- جامعة القاهرة التوقيع………………………… ا.د. نيفين صالح جويلى رئيس قسم النبات و الميكروبيولوجى التوقيع………………………

كلية العلوم

قسم النبات و الميكروبيولوجى

عزل و تعريف البكتريوفاجات التى تصيب البكتريا المسببه لمرض العفن الطرى فى البطاطس

رسالة مقدمة الى كلية العلوم جامعة القاهرة كمتطلب جزئى للحصول على درجة الماجيستير فى العلوم )الميكروبيولوجى(

إعداد

ايمان عبد الحفيظ الديب عبد الحميد بكالوريوس علوم ( الكيمياء والميكروبيولوجى ( ۲۰۱۲

قسم النبات و الميكروبيولوجى

كلية العلوم-جامعة القاهرة

۲۰۱۷