Control of Root Rot of Chickpea Caused by Sclerotium Rolfsii by Different Agents and Gamma Radiation

Tanta University Faculty of Science Botany Department

Control of root rot of chickpea caused by Sclerotium rolfsii by different agents and gamma radiation.

A thesis submitted to Faculty of Science – Tanta University In partial fulfillment of the requirements for the degree of master in Microbiology (Mycology) Submitted by Rasha Mohammed Fathy El- Said B.Sc. Microbiology- 2004- Al- Azhar University 2012 Supervisors Prof. Dr. Abd El Wahab Anter Ismail. Head of Integrated control department, Giza Research Institute.

Prof. Dr. Ahmed Ibrahim El- Batal. Professor of Applied Microbiology and Biotechnology, National Center for Radiation Research & Technology (NCRRT).

Prof. Dr. Hanan Mahmoud Mubarak. Associate. Prof. of Mycology, Botany Department, Faculty of Science, Tanta University. Supervisors

Prof. Dr. Abd El Wahab Anter Ismail Head of Integrated control department, Giza Research Institute.

Prof. Dr. Ahmed Ibrahim El- Batal Professor of Applied Microbiology and Biotechnology, National Center for Radiation Research & Technology (NCRRT).

Prof. Dr. Hanan Mahmoud Mubarak, Associate. Prof. of Mycology, Botany Department, Faculty of Science, Tanta University.

Head of Botany Department

Prof. Dr. Hassan Fared El-Kady.

This thesis has not been previously submitted for any degree at this or any other University.

Rasha Mohammed Fathy

TO WHOM IT MAY CONCERN

This is to certify that Ms. Rasha Mohamed Fathy has attended and passed successfully the following post-graduate courses (theoretical and practical) as a partial fulfillment of the requirement for the degree of Master of Science (Microbiology) Botany Department, Faculty of Science, Tanta University during the academic year 2004/2005.

The courses cover the following topics:

1- General and applied bacteriology, the use of microorganisms in preparation of leathers, methods and instruments used in microbiology.

2- Biochemistry, fermentation chemistry and immunological reactions.

3- Plant pathology, virology and special fungi.

4- Phycology, physiology of algae and physiology of fungi.

5- Biostatistics.

6- German language.

7- Computer science. This certificate is issued at here own request.

Head of Botany Department

Prof. Dr. Hassan Fared El-Kady

CURRICULUM VITAE

Name: Rasha Mohamed Fathy El-Said

Locality: Tanta - Gharbia Governorate.

Nationality: Egyptian.

Occupation: Dministrator at National Center for Radiation Research & Technology (NCRRT) - Atomic Energy Authority – Egypt.

Qualification: B.Sc. Degree in Microbiology (2004), Excellent, Faculty of Science, Al-Azhar University.

Attended and passed successfully the post graduate courses in partial fulfillment of M. Sc. (2005)

Head of Botany Department

Prof. Dr. Hassan Fared El-Kady

ACKNOWLEDGMENT

Firstly my unlimited thanks to Allah, the most merciful, the most greatful, real helper and real support for all.

I am also deeply thankful to Prof. Dr. Abd El Wahab Anter Ismail Head of Integrated control department, Giza Research Institute for suggestion the topic, supervising the work and revising the manuscript. And also for his great effort throughout the work.

I am also indebted to Prof. Dr. Ahmed Ibrahim El- Batal Professor of Applied Microbiology and Biotechnology, National Center for Radiation Research & Technology (NCRRT) for supervising this work and for his kind and valuable helping in radiation experiments.

I want to express about my keen thanks and my great gratitude to Prof. Dr. Hanan Mahmoud Mubarak, Associate. Prof. of Mycology, Botany Department, Faculty of Science, Tanta University for supervising this work, encouragement during practical work and for her keen interest and unfailing help.

I wish to express my appreciation to Prof. Dr. Yehia A.G. Mahmoud Professor of Mycology, Botany department, Faculty of Science, Tanta University, for his cooperation and help during the progress of this work, I would like to truly thank him for his stimulating, care, support and encouragement through out the course of this thesis. I want also to express about my deepest appreciation and my special thanks to whom working at Mycology lab. at Botany Department, Faculty of Science, Tanta University and National Center for Radiation Research & Technology (NCRRT) lab. for their sincere help.

Finally, I must offer my deepest appreciation and very special thanks to all members of my family for their excellent help, and continues encouragement to make this thesis to arise.

Rasha

Abstract

Sclerotium rolfsii causes root rot disease in several crops including chickpea that result in low yield. Artificial infection of chickpea seedlings by S. rolfsii in vitro demonstrated that different tissues of the plant completely disintegrated by fungal infection. In vitro and green house pot experiments demonstrated that inducers in combination with fungicides, oils and bioagents resulted in about 80 % suppression of root rot disease. Treatments have no phytotoxic effect on chickpea seedlings at low doses. Gliocladium virens and Gliocladium deliquescens were effective as biocontrol agents against Sclerotium rolfsii.

The percent of survival plants, fresh weight, dry weight and plant height of chickpea plants increased with different treatments with inducers compared with the control. Chlorophyll a, b, and total chlorophyll amounts increased to the maximum values. The activity of two plant enzymes, peroxidase and polyphenol oxidase increased.

In this study, gamma irradiation of chickpea seeds at doses 5, 10, 15, 20, 25 and 30 Gy have negative effect on survival, plant height, fresh weight and dry weight of chickpea. The effect of gamma irradiation at doses 0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 kGy on the antagonistic effect of Gliocladium virens and Gliocladium deliquescens against S. rolfsii were investigated. The results revealed that gamma irradiation increase the antagonistic effect of Gliocladium virens and Gliocladium deliquescens against S. rolfsii .

Abstract

Effect of gamma irradiation at doses of 0, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 5 kGy on the mycelial growth and pathogenicity of S. rolfsii were investigated. The results revealed that gamma irradiation at doses 0.25 up to 3.0 kGy increase the pathogenicity of S. rolfsii but gamma irradiation at dose 5.0 kGy completely inhibited the growth of S. rolfsii.

Extracellular polygalacturonase was characterized and purified by precipitation with 70 % ammonium sulfate, dialysis and gel filtration through Sephadex 75 for irradiated and unirradiated S. rolfsii isolates. The purified enzyme exhibited its maximum activity at gamma irradiation dose 3.0 kGy compared with the control. The molecular weight was determined by SDS- PAGE for irradiated and un-irradiated isolates to be a single band at 72 kDa.

Key words: Sclerotium rolfsii, chickpea, root rot, biocontrol, gamma irradiation, polygalacturonase.

Contents

Page

Introduction…………………………………………………………… 1 Preface………………………………………………………... 1 Aim of the work…………………………………………………… 5 Review of literature…………………………………………... 6 Chickpea (Cicer arietinum)…………………………………. 6 Sclerotium rolfsii………………………………………… 8 Effect of the fungicides, oils, inducers and bioagents on the growth of the pathogenic fungus S. rolfsii……………………………… 11 1. Effect of fungicides……………………………………… 11 2. Effect of oils……………………………………………….. 13 3. Effect of inducers…………………………………………... 17 4. Effect of bioagents………………………………………… 20 Phytotoxicity of the tested fungicides, oils and inducers on the length of shoot and root system………………………………… 22 Effect of some combinations of fungicides, biocontrol agents, and oils on fungi……………………………………………………. 25 Effect of fungicides, oils, biocontrol agents on chlorophyll content and plant chemical constituents……………………………………. 26 Polygalacturonase content in Sclerotium rolfsii……………………. 28 Effect of gamma radiation…………………………………………... 30 Types of radiation…………………………………………… 30 1- Ionizing radiation……………………………………… 31 2- Non- ionizing radiation………………………………… 31 Effect of ionizing radiation on microorganisms...... 31 Factors affecting the sensitivity of microorganisms to radiation…… 32 Effect of gamma radiation on microorganisms………………………… 33 1- The direct action…………………………………………….. 33 2- The indirect action…………………………………………… 33 Materials and Methods…………………………………………….. 36 Tested fungicides…………………………………………………… 36 1-Vitavax-200……………………………………………….. 36 2-Monceren – T……………………………………………… 37 The tested oils……………………………………………………….. 37 Potato dextrose agar medium……………………………………… 38 Tested inducers………………………………………………………. 38 Tested fungi…………………………………………………………… 38 1- Mode of artificial infection of Cicer arietinum as a host tissue in vitro……………………………………………………………. 39 a) Preparation of inoculum…………………………………… 39 b) Plant materials………………………………………………. 39 c) Examination of the infected tissues ………………………. 40 d) Light microscopy…………………………………………… 40 2- Biological tests ( In vitro studies )………………………………….. 40 2.a- Antagonism between pathogenic fungus and antagonistic fungi (biocontrol agents)………………………………………… 40 2. b- Fungicidal activity of fungicides, oils and inducers……...... 41 2.c- Phytotoxicity test………..………………………… 42 2.d- Joint toxic effect vitavax fungicide combined with inducers and oils against S. rolfsii…………………………………………. .. 43 3- Greenhouse experiments ( In vivo )…………………………………. 44 3.a- Pathogenecity test…………………………………………….. 44

3.b- Preparation of inoculum and soil infestation…………………. 44 3.c- Effect of infection by S. rolfsii on growth parameters

of Cicer arietinum…………………………………………. 45

4- Effect of fungicides, oils, inducers, bioagents and their combinations

on the infected Cicer arietinum plants under green house conditions……………………………………………… 46

5- Effect of the different treatments on chlorophyll content in leaves of Cicer arietinum plants………………………………… 47

6- Determination of peroxidase and polyphenol oxidase activity in the treated Cicer arietinum plants in vitro……………….………… 48

6.a- Preparation of crude enzyme……………………………… 48

6.b- Enzymes assay…………………………………………… 48

6.c- Peroxidase assay……………………………………..…… 48

6.d- Polyphenol oxidase assay………………………...... 49

7- Effect of gamma irradiation……………………………………. 49

7. a- Effect of gamma irradiation on the antagonism between

Gliocladium virens and Gliocladium deliquescence against

S. rolfsii in vitro on the control of root rot disease………………. 50

7. b- Effect of gamma irradiation of chickpea seed germination on control of root rot disease caused by S. rolfsii……………………… 50

7. c- Effect of different doses of gamma irradiation on the antagonistic action of Gliocladium virens and Gliocladium deliquescens against S. rolfsii in vivo……………….. 51

7. d- Effect of gamma irradiation on morphological characteristics, internal anatomical structure and pathogenecity of S. rolfsii………. 51

7.e- Electrophoretic analysis of unirradiated and gamma irradiated Sclerotium rolfsii total potein by SDS – PAGE……………………… 52

i. Extraction of S. rolfsii protein…………………………………. 52

ii. Electrophoresis stock solutions…………………………………... 52

A) Acrylamide stock solution (kept in dark at 4° C)…………….. 53

B) Sodium dodecyl sulphate (10 % W/ V SDS)………………… 53

C) Ammonium persulphate solution (1.5 % W/ V APS )……… 53

D) Buffers………………………………………………….. 53

iii. Separating and stacking gels preparations………………….. 54

• Separating gel……………………………………. 54 • Stacking gel………………………………………. 55

iv. Samples preparation ………………………………………… 55

v. Gel running…………………………………………………… 55

vi. Staining the gel………………………………………………. 56

7.f- Protein assay…………………………………………………….. 56

7. g- Extraction and purification of polygalacturonase………………… 57

a) Production of crude extract containing the

extracellular polygalacturonase…………………………………… 57

b) Assay of exo- polygalacturonase extracted from unirradiated and gamma irradiated Sclerotium rolfsii isolates…………………... 58 c) Purification of polygalacturonase produced by unirradiated

and gamma irradiated Sclerotium rolfsii isolates…………………… 58

8- Statistical analysis…………………………………………………. 59

Results …………………………………………………………… 60

I- In vitro experiments:………………………………………… 60

1- Mode of infection of Cicer arietinum in vitro (artificial infection to

Cicer arietinum as a host tissue)……………………………………. 60 Stages of sclerotia formation………………………………………… 60 Mode of infection of Sclerotium rolfsii to Cicer arietinum:………… 62 1.a- Infection of seed (seed rot)…………………………………… 62 1.b- Infection of stem ……………………………………………. 64 1.c- Infection of the root ………………………………………… 67 2- Effect of tested treatments on the growth of pathogenic and antagonistic fungi………………………………………………. 69 2-a Effect of the tested fungicides on the growth of Sclerotium rolfsii…………………………………………………. 69 2. b- Effect of fungicides on the growth of the antagonistic fungi:…... 72 2. c- Effect of oils (clove and mint oils) on the growth of Sclerotium rolfsii…………………………………….………… 75 2. d- Effect of oils on the growth of Gliocladium deliquescens and Gliocladium virens in vitro…………………………………. 77 2. e- Effect of inducers (copprus KZ and starner) on the growth of S. rolfsii, G. deliquescens and G. virens in vitro……………… 77 2. f- Inhibitory percentage of Sclerotium rolfsii under the effect of antagonistic fungi (T. hamatum, G. deliquescens and G. virens) on PDA medium in vitro (Three days old ) …………………….. 78 2. g- Effect of culture filtrate of Gliocladium virens and Gliocladium deliquescens on the growth of Sclerotium rolfsii……………… 80 3. Phytotoxicity of the tested compounds on Cicer ariertinum seedlings in vitro…………………………………………………. . 82 3.a- Fungicides…………………………………………………. 83 3.b- Oils………………………………………………………… 83 3.c- Inducers…………………………………………………… 83 4- Joint toxic effect of the tested compounds against pathogenic fungus…………………………………………………… 87 4.a- Joint toxic effect against Sclerotium rolfsii………………… 88 4.b- Joint toxic effect of the tested compounds with bioagents against pathogenic fungus…………………………… 89 II- In vivo experiments:…………………………………………. 93 1 .a- Effect of different antifungal treatments on survival plants (%)… 92 1 .b- Effect of different antifungal treatments with bioagents on survival plants (%) …………………………………………………. 95 2.a- Effect of different antifungal treatments on growth parameters…. 98 A) Plant height (cm) ……………………………………… 98 B) Fresh weight (gm) ………………………………………. 98 C) Dry weight (gm) …………………………………………. 98 2. b- Effect of different antifungal treatments with bioagents on growth parameters……………………………………………. 103 A) Plant height (cm)…………………………………………… 103 B) Fresh weight (gm) ………………………………………… 103 C) Dry weight (gm) ………………………………………… 103 3. a- Effect of different antifungal treatments on chlorophyll content in Cicer arietinum leaves…………………………………………… 108 A) Chlorophyll (a) ………………………………………. 108 B) Chlorophyll (b) …………………………………………… 108 C) Total chlorophyll …………………………………………. 108 3. b- Effect of different antifungal treatments with bioagents on chlorophyll content in Cicer arietinum leaves…………………… 113 A) Chlorophyll (a) ………………………………………… 113 B) Chlorophyll (b) ………………………………………… 113 C) Total chlorophyll ………………………………… 113 4. a- Effect of different antifungal treatments and their combinationson enzymes activity…………………………………. 118 A) Peroxidase enzyme ………………………………………… 118 B) Polyphenol oxidase …………………………………………. 118 4. b- Effect of different antifungal treatments with bioagents on enzymes activity ……………………………………………… 122 A) Peroxidase enzyme ……………………………………… 122 B) Polyphenol oxidase ……………………………………….. 122 5- Effect of different treatments (Fungicides, oils and inducers) on the efficiency of S. rolfsii germination and pathogenecity in 7 days ………………………………………………………………. 126 III - Effect of gamma irradiation:………… ……………………………… 127 1. a- Effect of different doses of gamma irradiation on the antagonistic effect of Glioccladium virens against S. rolfsii in vitro (3 days incubation)… 127 1. b- Effect of different doses of gamma irradiation on the antagonistic effect of Glioccladium deliquescens against S. rolfsii in vitro(3 days incubation). 129 2. a- Effect of gamma irradiation doses on Cicer arietinum seed germination…. 131 2. b- Effect of gamma irradiation doses on growth parameters of Cicer arietinum seeds……………………………………………….. 132 3. a- Effect of gamma irradiated Gliocladium virens on chickpea plants inoculated in soil infested with S. rolfsii…………………………………… 134 3. b- Effect of gamma irradiated Gliocladium deliquescens on chickpea plants inoculated in soil infested with S. rolfsii……………… 136 4. a- Effect of gamma irradiation on external morphology of S. rolfsii………………………………………………………. 137 4. b- Effect of different gamma irradiation doses on pathogenecity and infection process of S. rolfsii after 7 days in vitro………………... 140 4. c- Effect of gamma irradiation on pathogenecity of S. rolfsii to Cicer arietinum in vivo…………………………………………. 141 4. d- SDS- PAGE for total protein profile of unirradiated and irradiated isolates of S. rolfsii…………………………………………… 142 4. e- Purification of polygalacturonase produced by gamma irradiated and unirradiated isolates of S. rolfsii………………… 145 Discussion ……………………………………………………….. 155 1- Mode of infection of Sclerotium rolfsii to Cicer arietinum……… 155 1. a- Infection of seed ……………………………………… 155 1. b- Infection of stem ……………………………………… 156 1. c- Infection of the root ………………………………… 157 2- Effect of tested treatments on the growth of the pathogenic fungus S. rolfsii…………………………………………………. 157 2.a- Effect of fungicides on the growth of Sclerotium rolfsii in vitro. 157 2.b- Effect of oils on the growth of Sclerotium rolfsii in vitro…… 162 2.c- Effect of inducers on the growth of Sclerotium rolfsi in vitro. 164 2.d- Effect of antagonistic fungi (Gliocladium deliquescens, Gliocladium virens and Trichderma hamatum) on the growth of Sclerotium rolfsii…………………………………. 166 2. e- Effect of culture filtrate of Gliocladium virens and Gliocladium deliquescens on the growth of Sclerotium rolfsii………… 168 3-Effect of Sclerotium rolfsii on the pathogenecity of Cicer arietinum …………………………………………… 169 4- Phytotoxicity of the tested compounds on Cicer arietinum seedlings in vitro……………………………………………… 172 4.a – Fungicides ………………………………………… 172 4. b – Oils ……………………………………………… 175 4. c - Inducers …………………………………………. 177 5- Joint toxic effect of the tested compounds against pathogenic fungus Sclerotium rolfsii………………………………… 179 II- Green house experiments (In vivo) …………………… 181 1- Effect of different antifungal treatments and their combinations on survival plants ………………………………………… 181 2- Effect of different antifungal treatments and their combinations on different growth parameters of Cicer arietinum………………………………………. ……… 183 3- Effect of different antifungal treatments and their combinations on chlorophyll (a), chlorophyll (b) and total chlorophyll Cicer arietinum …………………………………………… 185 4- Effect of different antifungal treatments and their combinations on enzyme activity (peroxidase and polyphenol oxidase)……. 186 III- Effect of different gamma irradiation ………… 188 1- Effect of gamma irradiation on the antagonism between Gliocladium virens and Gliocladium deliquescence against S. rolfsii…………… 188 2- Effect of gamma irradiation on Cicer arietinum seed germination…. 189 3- Effect of different gamma irradiation doses on pathogenecity and infection process of S. rolfsii………………………… 191 4- Purification of polygalacturonase produced by gamma irradiated and unirradiated isolates of S. rolfsii. …………………… 193 Summary …………………………………………………… 196 Conclusion …………………………………………………………. 200 References ………………………………………………………… 201 Arabic summary

List of Tables

Table Page Table (1): Inducers compound used. 38

Table (2): Effect of fungicides (vitavax and monceren- T) on the 70 linear growth of the pathogenic fungus Sclerotium rolfsii on PDA medium in vitro after three days.

Table (3): Effect of Vitavax and Monceren -T on the growth of 73 antagonistic fungus Gliocladium deliquescens on PDA medium in vitro (3 days old).

Table (4): Effect of Vitavax and Monceren -T on the linear growth of 74 antagonistic fungus Gliocladium virens on PDA medium in vitro.

Table (5): Effect of oils on the linear growth of Sclerotium rolfsii on 75 PDA medium in vitro after three days.

Table (6): Inhibitory percentage of Sclerotium rolfsii under the effect 79 of antagonistic fungi in vitro.

Table (7): Effect of G. virens and G. deliquescens culture filtrates on 80 the percentage of inhibition of S. rolfsii after different incubation periods.

Table (8): Phytotoxicity of the tested compounds on shoot system of 84 Cicer arietinum 14 days old seedlings (Inhibition percentage of shoot length).

Table (9): Phytotoxicity of the tested compounds on root system of 86 Cicer arietinum 14 days old seedlings (Inhibition percentage of root length).

Table (10): Percentage of inhibition (I%) and Co-Toxicity Factor of 88 treatments against pathogenic fungus Sclerotium rolfsii.

Table (11): Percentage of inhibition (I %) and Co-Toxicity Factor of 90 treatments with bioagents against pathogenic fungus Sclerotium rolfsii. Table (12): Effect of different antifungal treatments and their 93 combinations on survival plants of Cicer arietinum in vivo under green house conditions. Table (13): Effect of different antifungal treatments with bioagents 96 on survival plants of Cicer arietinum in vivo under green house conditions. Table (14): Effect of different antifungal treatments and their 99 combinations on different growth parameters of Cicer arietinum in vivo. Table (15): Effect of different antifungal treatments with bioagents 104 on different growth parameters of Cicer arietinum in vivo .

Table (16): Effect of different antifungal treatments and their 109 combinations on chlorophyll (a), chlorophyll (b) and total chlorophyll of Cicer arietinum in vivo.

Table (17): Effect of different antifungal treatments with bioagents 114 chlorophyll a, chlorophyll b and total chlorophyll content of Cicer arietinum in vivo.

Table (18): Effect of different antifungal treatments and their 119 combinations on enzyme activity (peroxidase and polyphenol oxidase) in vivo.

Table (19): Effect of different antifungal treatments with bioagents 123 on enzyme activity (peroxidase and polyphenol oxidase) in vivo.

Table (20): Effect of different doses of gamma irradiation on the 128 antagonistic effect of Glioccladium virens against S. rolfsii in vitro. Table (21): Effect of gamma irradiation doses on the antagonistic 130 effect of Glioccladium deliquescens against S. rolfsii in vitro.

Table (22): Effect of gamma irradiation doses on seed germination. 131 Table (23): Effect of gamma irradiations doses on growth parameters 133 of Cicer arietinum in vivo.

Table (24): Effect of gamma irradiated GV on chickpea plants 135 inoculated in soil infested with S. rolfsii.

Table (25): Effect of gamma irradiated GD on chickpea plants 136 inoculated in soil infested with S. rolfsii.

Table (26): Effect of gamma irradiation on pathogenecity of S. rolfsii 141 to Cicer arietinum in vivo.

Table (27): SDS- PAGE band survey for the total proteins extracted 144 from irradiated and unirradiated isolates of S. rolfsii.

Table (28): Purification profile of polygalacturonase from 146 unirradiated S. rolfsii (control).

Table (29): Purification profile of polygalacturonase from S. rolfsii 148 (irradiated isolate at dose 0.25 kGy).

Table (30): Purification profile of polygalacturonase from S. rolfsii 149 (irradiated isolate at dose 0.5 kGy). Table (31): Purification profile of polygalacturonase from S. rolfsii 150 (irradiated isolate at dose 1.0 kGy).

Table (32): Purification profile of polygalacturonase from S. rolfsii 151 (irradiated isolate at dose 1.5 kGy).

Table (33): Purification profile of polygalacturonase from S. rolfsii 152 (irradiated isolate at dose 2.0 kGy).

Table (34): Purification profile of polygalacturonase from S. rolfsii 153 (irradiated isolate at dose 2.5 kGy).

Table (35): Purification profile of polygalacturonase from S. rolfsii 154 (irradiated isolate at dose 3.0 kGy).

List of Figures

Figure Page Fig. (1): Effect of vitavax and monceren- T on the linear growth of 71 Sclerotium rolfsii in vitro. Fig (2): Effect of fungicides on Sclerotium rolfsii represented as 71 regression lines. Fig. (3): Effect of vitavax and monceren –T on the linear growth of 73 Gliocladium deliquescens.

Fig. (4): Effect of vitavax and monceren –T on the linear growth of 74 Gliocladium virens.

Fig. (5): Effect of clove and mint oils on the linear growth of 76 Sclerotium rolfsii in vitro.

Fig. (6): Effect of oils on Sclerotium rolfsii represented as regression 76 lines.

Fig. (7): Inhibitory percentage of Sclerotium rolfsii under the effect 79 of antagonistic fungi in vitro.

Fig. (8): Effect of culture filtrates of Gliocladium virens and G. 81 deliquescens on inhibition percentage of Sclerotium rolfsii after different incubation periods.

Fig. (9): Effect of tested compounds (ICR50R) on shoot system of 85 chickpea seedlings.

Fig. (10): Effect of tested compounds (ICR50R) on root system of 87 chickpea seedlings. Fig. (11): Effect of joint toxic effect on the linear growth of 89 Sclerotium rolfsii. Fig. (12): Effect of joint toxic effect with bioagents on the linear 91 growth of Sclerotium rolfsii. Fig. (13): Effect of different antifungal treatments on survival plants 94 of Cicer arietinum in vivo.

Fig. (14): Effect of different antifungal treatments with bioagents on 97 survival plants of Cicer arietinum in vivo.

Fig.(15): Effect of different antifungal treatments and their 100 combinations on plant height of Cicer arietinum in vivo.

Fig.(16): Effect of different antifungal treatments and their 101 combinations on fresh weight of Cicer arietinum in vivo.

Fig (17): Effect of different antifungal treatments and their 102 combinations on dry weight of Cicer arietinum in vivo. Fig. (18): Effect of different antifungal treatments with bioagents on 105 plant height of Cicer arietinum in vivo.

Fig. (19): Effect of different antifungal treatments with bioagents on 106 fresh weight of Cicer arietinum in vivo.

Fig. (20): Effect of different antifungal treatments with bioagents on 107 dry weight of Cicer arietinum in vivo.

Fig. (21): Effect of different antifungal treatments and their 110 combinations on chlorophyll (a) of Cicer arietinum in vivo.

Fig. (22): Effect of different antifungal treatments and their 111 combinations on chlorophyll (b) of Cicer arietinum in vivo.

Fig. (23): Effect of different antifungal treatments and their 112 combinations on total chlorophyll of Cicer arietinum in vivo.

Fig. (24): Effect of different antifungal treatments with bioagents on 115 chlorophyll (a) of Cicer arietinum in vivo.

Fig. (25): Effect of different antifungal treatments with bioagents on 116 chlorophyll (b) of Cicer arietinum in vivo.

Fig. (26): Effect of different antifungal treatments with bioagents on 117 total chlorophyll of Cicer arietinum in vivo.

Fig.(27): Effect of different antifungal treatments and their 120 combinations on peroxidase activity of Cicer arietinum in vivo. Fig.(28): Effect of different antifungal treatments and their 121 combinations on polyphenol oxidase activity of Cicer arietinum in vivo.

Fig.(29): Effect of different antifungal treatments with bioagents on 124 peroxidase activity of Cicer arietinum in vivo.

Fig.(30): Effect of different antifungal treatments with bioagents on 125 polyphenol oxidase activity of Cicer arietinum in vivo.

Fig. (31): Effect of gamma irradiation on the antagonistic effect of 128 Glioccladium virens against S. rolfsii.

Fig (32): Effect of gamma irradiation on the antagonistic effect of 130 Glioccladium deliquescens against S. rolfsii .

Fig. (33): Effect of gamma irradiation on survival plants (%). 132

Fig. (34): Effect of gamma irradiation on number of germinated 132 seeds. Fig. (35): Effect of gamma irradiation doses on plant height. 133

Fig. (36): Effect of gamma irradiation doses on fresh weight. 134

Fig. (37): Effect of gamma irradiation doses on dry weight. 134

Fig. (38): Effect of gamma irradiated GV on chickpea plants 135 inoculated in soil infested with S. rolfsii.

Fig. (39): Effect of gamma irradiated Gliocladium deliquescens on 137 chickpea plants inoculated in soil infested with S. rolfsii.

Fig. (40): Percentage of live sclerotia and live hyphae at control. 139

Fig. (41): Percentage of live sclerotia and dead hyphae at (0.25 up to 139 3 kGy) doses.

Fig. (42): Percentage of dead sclerotia and dead hyphae at dose 5 139 kGy.

Fig. (43): Effect of gamma irradiation doses on the pathogenecity of 142 Sclerotium rolfsii to Cicer arietinum.

Fig. (44): Progress of polgalacturonase specific activity through 147 different stages of purification (unirradiated control).

Fig. (45): Progress of polgalacturonase specific activity through 148 different stages of purification (irradiated isolate at dose 0.25 kGy).

Fig. (46): Progress of polgalacturonase specific activity through 149 different stages of purification (irradiated isolate at dose 0.5 kGy).

Fig. (47): Progress of polgalacturonase specific activity through 150 different stages of purification (irradiated isolate at dose 1.0 kGy).

Fig. (48): Progress of polgalacturonase specific activity through 151 different stages of purification (irradiated isolate at dose 1.5 kGy).

Fig. (49): Progress of polgalacturonase specific activity through 152 different stages of purification (irradiated isolate at dose 2.0 kGy).

Fig. (50): Progress of polgalacturonase specific activity through 153 different stages of purification (irradiated isolate at dose 2.5 kGy).

Fig. (51): Progress of polgalacturonase specific activity through 154 different stages of purification (irradiated isolate at dose 3.0 kGy).

List of Photos

Photo Page Photo (1): shows Sclerotium rolfsii on PDA medium. 60 Photo (2): Microscopic examination of Sclerotium rolfsii after one 60 day. Photo (3): Microscopic examination of Sclerotium rolfsii after 60 two days Photo (4): Transfer section in sclerotia of Sclerotium rolfsii. 60

Photo (5): Seeds of Cicer arietinum (non infected control). 62

Photo (6): Transfer section of seeds of Cicer arietinum shows 62 cotyledons of Cicer arieinum rich in starch grains.

Photo (7): Seeds of Cicer arietinum infected with Sclerotium 62 rolfsii, after one month.

Photo (8): Transfer section of one month age infected seed of 62 Cicer arietinum which shows black infection cushion.

Photo (9): Transfer section of infected seed of Cicer arietinum 62 after two months. Photo (10): Infected seed of Cicer arietinum with white rot after 62 three months. Photo (11): T.S. of seed of Cicer arietinum infected with white 62 rot after three months. Photo (12): 10 days old seedling of Cicer arietinum. 65

Photo (13): 10 days old seedling of Cicer arietinum infected with 65 S. rolfsii Photo (14): T.S. of transition zone of infected Cicer arietinum. 65 Infected region is occupied by microsclerotiole.

Photo (15): T.S. of infected transation zone showing infection of 65 cortex by microsclerotia Photo (16): T.S. of infected transition zone showing infected 65 xylem vessels. Photo (17): T.S. of the infected transition zone of Cicer arietinum 65 seedlings showing that all the cells are completely digested by the fungus Photo (18): White rot on the root of Cicer arietinum caused by S. 67 rolfsii after one week from infection.

Photo (19): Longitudinal section of the infected root of Cicer 67 arietinum colonized by infected hyphae of Sclerotium rolfsii.

Photo (20): L.S of infected root of Cicer arietinum showing rod 67 shaped sclerotia at cortex region.

Photo (21): L.S of infected root of Cicer arietinum showing 67 xylem vessels that are occupied by the fungal elements.

Photo (22): L.S of infected root of Cicer arietinum showing all 67 the xylem vessels of the host are colonized by the fungus.

Photo (23): Vertical section of rotted root of Cicer arietinum after 67 7 days from infection.

Photo (24): Antagonistic effect of Gliocladium deliquescens 78 against S. rolfsii. Photo (25): Antagonistic effect of Gliocladium virens on S. rolfsii 78 in vitro Photo (26): Antagonistic effect of Trichoderma hamatum against 78 S. rolfsii Photo (27): Effect of vitavax on Cicer arietinum seedlings. 82

Photo (28): Effect of mint oil on Ciecr arietinum seedlings. 82

Photo (29): Effect of clove oil on Cicer arietinum seedlings. 82

Photo (30): Effect of starner on Cicer arietinum seedlings. 82

Photo (31): Effect of copprus KZ on Cicer arietinum seedlings. 82

Photo (32): SDS- PAGE band survey for the total cellular 143 proteins extracted from unirradiated and irradiated isolates of S. rolfsii.

Photo (33): Sodium dodecyl sulphate polyacrylamide gel 145 electrophoresis of polygalacturonase from S. rolfsii

Introduction

INTRODUCTION Preface

A wide variety of plants and trees are affected by root rot disease. This insidious family of infections, most of which eventually prove fatal, consists of several different pathogens, all of which are fungal and thrive in damp environments. Both indoor and outdoor plants are susceptible to root rot, although the former are more likely to be infected because they tend to be overwatered and are often in pots or other containers that provide inadequate drainage where the excess water makes it very difficult for the roots to get the air that they need, causing them to decay.

A plant with root rot will not normally survive, but can often be propagated so it will not be lost completely. Plants with root rot should be removed and destroyed. Some root rot pathogens target tree stumps, from which they spread to living trees.

Chickpea was one of the first legume crops domesticated. Today it is a key component of cropping systems in many parts of Asia and Africa, providing families of resource-poor farmers with a valuable source of dietary protein. Chickpea is also becoming established in some agriculturally advanced nations in response to a growing world demand. Knights (2004) describes the chickpea species, its uses, and role in farming systems around the world.

- 1 - Introduction

Chickpea (Cicer arietinum) is the most important commercial crop playing a key role in economic and social affairs in Egypt, it is an important food crop in many regions in the world. It is affected by various diseases. Damping- off and root- rot diseases are the most important diseases that affect plant stand causing great losses in chickpea crop, total nitrogen and protein contents in chickpea seeds.

Sclerotium rolfsii represents both unity and diversty in fungi. It may be a plant pathogen, a parasite, a symmbiont, or a saprophyte. Its pathogenesis is complex; it has a heterogeneity of strains and a diversity in host rang. It occurs throughout the world and can damage any part or all of a plant. Neither semiarid nor aquatic plants escape its destruction, given the appropriate strain. It is known to cause seed decay, damping- off, seedling blight, root rot, crown rot, as well as such stem infections as soreshing and wirestem, hypocotyl cankers, bud rot, foliage blight and storage rots (Baker,1970).This fungus may also form mycorrhizae on vanilla roots, which may be beneficial, but it also can cause severe root decay of vanilla (Alconero and Santiago, 1969). Of all these diseases, root rot is without doubt the principal disease caused by S. rolfsii.

The role of enzymes in penetration is unknown. Sclerotium rolfsii produces cutinolytic enzymes (Linskines and Haage, 1963) which could degrade the cuticle. Pectinases and cellulases may also function in penetration, presumably after the cuticle is penetrated or destroyed. However, in roots, where cuticle is lacking or different from that on hypocotyls, these enzymes or others may play a role (Dodman and Flentje,

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1970). Both Kerr (1956) and Wyllie (1962) demonstrated that S. rolfsii, when separated from host tissues by a cellophane membrane that perimitted fungal metabolites to pass through, produced injury to underlying tissues.

Bateman (1970) postulated that in the initial phases of pathogenic attack, the pathogen may be an aggressive member of a complex that can initially elaborate "attacking mechanisms" to foster invasion of the host. If the host fails to respond or responds too slowly on defense, the host dies. Because of the diversity within species in pathogenicity, host range, and plant parts infected, it is somewhat difficult to generalize however, the fact that pre- and post-emergence seedling blights are common, that resistance increases with age of seedlings, and that breeding for resistance to this fungus has generally been unsuccessful, especially for root infections, this species, for all its diversity, fits the category of a pathogen- dominant fungus.

Plant and their constituents have become of great value as potent, harmless and easily available fungi toxicant in contrast to synthetic chemical fungicides which often impose various undesirable effects. With the increase a awareness of the toxic hazards effects of chemicals to crops due to their phytotoxic residual and pollutive effects consumers and environmental have emphasized the importance of indigenous products in plant disease control (Ahmed and Grainge, 1982, Narayansamy et al., 1983 and Sivaprakasam, 1994).

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Essential oils have been widely used for bactericidal, virucidal, fungicidal, antiparasitical, insecticidal, medicinal and cosmetic applications, especially nowadays in pharmaceutical, sanitary, cosmetic, agricultural and food industries. Because of the mode of extraction, mostly by distillation from aromatic plants, they contain a variety of volatile molecules such as terpenes and terpenoids, phenol-derived aromatic components and aliphatic components.

Biological control of plant pathogens is becoming an important for plant diseases management and several successful attempts have been made to control the pathogens by using Trichderma spp and /or Gliocladium spp., which attack the mycelium that causes plant diseases. Different antagonistic fungi varied in their action. This variation may be due to difference in the ability of each antagonist to grow, and to produce toxic substances. It is also correlated with the antagonistic ability of parasitizing the hyphae of the pathogens and the antibiosis potential.

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Aim of the work

This study is aiming to investigate the following : • Studying the pathogenecity of Sclerotium rolfsii • Control of root rot of chickpea by different agents: . Fungicides . Oils . Inducers . Bioagents

• Studying joint toxic effect between different treatments. • Estimation phytotoxicity of all treatments on Cicer arietinum plants.

• Studying the effect of treatments In vivo.

• Evaluation the effect of gamma irradiation on pathogenecity of Gliocladium virens and Gliocladium deliquescens against Sclerotium rolfsii In vitro and In vivo.

• Evaluation the effect of gamma irradiation on viability of sclerotia and the mycelium, polygalacturanase enzyme activity, pathogenecity of Sclerotium rolfsii In vitro and In vivo.

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Review of literature

Vegetable crops are grown worldwide as a source of nutrients and fiber in the human diet. Fungal plant pathogens can cause devastation in these crops under appropriate environmental conditions. Vegetable producers confronted with the challenges of managing fungal pathogens have the opportunity to use fungi and yeasts as biological control agents. Several commercially available products have shown significant disease reduction through various mechanisms to reduce pathogen development and disease. Production of hydrolytic enzymes and antibiotics, competition for plant nutrients and niche colonization, induction of plant host defense mechanisms, and interference with pathogenicity factors in the pathogen are the most important mechanisms. Biotechnological techniques are becoming increasingly valuable to elucidate the mechanisms of action of fungi and yeasts and provide genetic characterization and molecular markers to monitor the spread of these agents (Zamir, et al., 2003)

Chickpea (Cicer arietinum):

Chickpea (Cicer arietinum) is an ancient self-pollinated legume crop believed to have originated in south-eastern Turkey and the adjoining part of Syria. The major goals of chickpea breeding are to increase production either by upgrading the genetic potential of cultivars or by eliminating the effect of diseases, insects, drought and cold. Selection techniques for pest resistance and agronomic characters have been developed. Over 50 cultivars

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Review of literature have been developed for winter sowing in the Mediterranean basin and for the rice fallow in South Asia. Cultivars with heavy shoot biomass, long reproductive phase, multiple-stress resistance, or early maturity are required to meet farmers' needs. Chickpea is a good source of zinc, and protein. It is also very rich in dietary fibers and hence is a healthy food source especially as a source of carbohydrates for persons with insulin sensitivity or diabetes. It is low in fat, and most of the fat content is mono unsaturated.

Raw whole seeds contain per 100 g: 357 calories, 4.5 – 15.6 % moisture, 14.9 – 24 % protein, 0.8 – 6.4 % fat, 2.1 – 11.7 grams fiber, 2.4 – 8 % ash, 140 – 440 g calcium, 190 – 382 mg phosphorus, 9 mg iron, 0.225 mg β– carotene equivalent, 0.21 – 1.1 mg thiamin, 0.12 – 33 mg riboflavin and 1.3 – 2.9 mg niacin.

One hundred grams of mature boiled chickpea contains: 164 calories, 2.6 grams of fat (of which only 0.27gram is saturated), 7.6 grams of dietary fibers, and 8.9 grams of proteins. Also, contain 38.59 % carbohydrates, 3 % fibers, 4.8 -5.5 % oil, 3 % ash, 0.2 % calcium, and 3 % phosphorus.

Chickpea is affected by various plant diseases. Damping- off, wilting and root- rot diseases are considered among the most important diseases that affect plant stand causing great losses in chickpea yield.

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Review of literature

The primary causes of damping- off and root-rot diseases are Sclerotium rolfsii, Pythim spp, Fusarium solani and Sclerotium sclertineum

Sclerotium rolfsii

S. rolfsii is a soil-borne plant pathogen of world wide occurrence that infects more than 500 plant species (Aycock, 1966 and Punja, 1985). Mostly S. rolfsii diseases have been reported on dicotyledonous hosts, but several monocotyledonous species have also been infected (Aycock, 1966 and Mordue, 1974). Humid weather is conductive to sclerotial germination and mycelial growth. Consequently the diseases caused by the fungus are more serious in tropical and subtropical regions than in hot regions. The large number of sclerotia produced by S. rolfsii and their ability to persist in the soil for several years, as well as the profuse growth rate of the fungus make it well suited facultative parasite and a pathogen of major importance throughout the world (Punja et al., 1982).

Hypocotyls apparently are more susceptible than root to infection by S. rolfsii and lima bean (Warren, 1973). However, Flentje (1957) distinguished root attacking and stem-attacking strains and found that root attacking strains attacked all hosts tested but stem strains showed marked selectivity.

Meristematic tissues of seedlings are susceptible to S. rolfsii, and the hyphyae growing either from infected seed or from soil invade all immature seedling tissues causing death prior to or just after emergence. As tissues are

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Review of literature mature, they become increasingly resistant. In bean this has been attributed to the conversion of pectin to calcium pectate which is unaffected by the polygalactouronase produced by the fungus (Bateman and Lumsden, 1965) but in cotton due to the presence in older seedlings of catechin in an oxidized form which may inhibit the same enzyme (Hunter, 1974). In snap bean, susceptibility may be due to polygalactouronase trans-eliminase which may partially degrade pectate (Ayers et al., 1966).

Dodman and Flentje (1970), Bateman (1970) and Parmeter (1970) indicated that plant pathogenic fungi might penetrate plants in various ways: (1) Through the intact plant surface by means of complex infection structures (infection cushions) which is characteristic of different isolates; (2) Through natural openings and through wound. Isolates usually pentrate the host in only one way, but it is not uncommon for some isolates to penetrate the same host in several ways. Dodman and Flentje (1970) reported dome-shaped infection caushion to be formed on hypocotyls of radish and that infection pegs penetrate the host tissues beneath the epidermis. Sometimes flattened tips of hyphae function as appressoria prior to penetration. The infection pegs apparently penetrate cells mechanically as described by Christou (1962), Dodman and Flentje (1970) and Van Etten et al. (1968).

Sclerotium rolfsii may penetrate the host mechanically or by means of enzymes, depending upon interpretation of events.

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Review of literature

The lack of conclusive evidence for mechanical or chemical penetration, or a combination of both, may further indicate that the mode of penetration varies for each type of penetration and conditions under which it is initiated. This is may be an especially important factor in the consideration of hypocotyls (or other aerial portions of the plant) or root infection. Following intial penetration, the pathogenic mechanisms in S. rolfsii are varied and inadequately explored. The degree of inter and intracellular mamification varies with the strain of S. rolfsii and the host, but generally intercellular often precedes intracellular invasion. Tissue maceration seemed to be a characteristic symptom in the early phases of pathogenicity in chickpea. Apparently, S. rolfsii is capable to produce cell wall degrading enzymes and tissue macerating enzymes as well as producing phytotoxic metabolites. Browning of the host cell walls appear to be true whether injury precedes or accompanies hyphal penetration (Bateman, 1970).

Boosalis (1975) reported browning of cells in advance of penetration by hyphae in soybean roots and stems, plants with only cortical infections generally recover but those in which vascular invasion occurred were often destroyed outright (Van Etten et al., 1968).

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Review of literature

Effect of the fungicides, oils, inducers and bioagents on the growth of the pathogenic fungus S. rolfsii.

1. Effect of fungicides:

Fungicides are agents of natural or synthetic origin which act to protect plants against invasion by fungi and/or to eradicate established fungal infection. Herbicides, insecticides and plant growth regulators, form the battery of agrochemicals or pesticides that is available to protect crops and maintain their yield potential, measured as quality or quantity of produce. Some fungicides (ronilan 50 NP, vinclozolin 0.1%), metiltopsin 70 NP (thiophanate-methyl, 0.1 and 0.2 %) fundasole 50 Np (benomil 0.1%), sumilex 50 NP (procimidon, 0.1 and 0.2 %) affected on the charcoal rot fungus Sclerotium bataticola or Macrophomina phaseolina. The fungicides metal- topsin 70 NP suppressed the growth of mycelia, but the preparation ronilan 50 NP did not. (Alexander, 2000).

Using fungicide Rovral 50 WP reduced infection of groundnut seedling with Sclerotium rolfsii and Rhizoctonia solani. Rovral 50WP gave the best control of seedling mortality and enhanced the growth of groundnut seedling (Raihan et al., 2003).

Six fungicides, benomyl, sancozeb, thiovit, dithane, carbandazim and topsin-M were tested against Sclerotium rolfsii by food poison method. At low concentration, no fungicide inhibited the growth of S. rolfsii,

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Review of literature however, at high concentration dithane and sencozeb significantly reduced the growth (Fouzia and Saleem, 2006).

Tamire et al., (2007) tested the effect of five fungicides: tebuconazole, benomyl, mancozeb, captan and thiram on white rot, Sclerotium cepivorum that infect local garlic cultivars at the Debre Zeit and Bakelo areas of North Shewa, Ethiopia. Treatment of garlic tebuconazole gave a significant reduction in initial and final white rot incidence, progress rate, and severity on bulbs. Captan and benomyl gave 16 % and 24 % in disease reduction, respectively, over the control at Bakelo area. At Debre Zeit area these fungicides decreased final disease incidence by 40- 44%. The highest increase in marketable yield was obtained from garlic treated with tebuconazole (43–73%), captan (48–77%) and benomyl (42–64%) more than the control. Thiram reduced the disease initially, but subsequently did not prove effective.

All fungicides led to a significant reduction in deoxynivalenol, although there was substantial between-study variability. For deoxynivaleno, metconazole was the most effective treatment, All fungicides, with the exception of propiconazole, were significantly more effective than tebuconazole for control of deoxynivalenol (Paul et al., 2008).

Also, the management of early leaf spot Cercospora arachidicola, late leaf spot Cercosporidium personatum and stem rot Sclerotium rolfsii of peanut Arachis hypogaea in the southeastern USA is heavily dependent upon sterol biosynthesis inhibitor (SBI) and quinone outside inhibitor (QoI)

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Review of literature fungicides. Effective new fungicides with different modes of action could improve overall disease control and extend the utility of the current fungicides. (Culbreath et al., 2009).

David et al., (2009) tested the efficacy of fourteen selected fungicides against Phaeomoniella chlamydospora and Phaeoacremonium aleophilum in vitro by mycelial growth and conidial germination assays. Azoxystrobin, carbendazim and tebuconazole were the most effective fungicides against P. chlamydospora, while carbendazim and didecyldimethylammonium chloride were the most effective against P. aleophilum.

Muneeb et al., (2011). Found that Fungicide carbendazim at 100, 200, 500 ppm caused maximum percent inhibition of Fusarium oxysporum, F. solani and Rhizoctonia solani that isolated from the wilted chickpea (Cicer arietinum) plants under in vitro conditions. Carbendazim was applied as seed treatment reduced disease incidence significantly. Seed treatment with carbendazim increased seed germination by (71.24%)

2. Effect of oils:

Constant use of synthetic pesticides for disease control has resulted in several environmental problems such as long persistence period (Beye, 1978) pollutive effects (Dubey and Mall, 1972), phytotoxity, teratogenecity (Javoraska, 1978) and carcinogencity (Epstein et al., 1967). These factors emphatic sized for new methods to control disease (Wilson et al., 1987)

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Review of literature

Essential oils have been largely employed for their properties already observed in nature, i.e. for their antibacterial, antifungal and insecticidal activities. At present, approximately 3000 essential oils are known, 300 of which are commercially important especially for the pharmaceutical, agronomic, food, sanitary, cosmetic and perfume industries. Essential oils or some of their components are used in perfumes and make- up products, in sanitary products, in dentistry, in agriculture, as food preservers and additives, and as natural remedies. For example, d-limonene, geranyl acetate or d-carvone are employed in perfumes, creams, soaps, as flavour additives for food, as fragrances for household cleaning products and as industrial solvents. Moreover, essential oils are used in massages as mixtures with vegetal oil or in baths but most frequently in aromatherapy. Some essential oils appear to exhibit particular medicinal properties that have been claimed to cure one or another organ dysfunction or systemic disorder (Silva et al., 2003; Hajhashemi et al., 2003; Perry et al., 2003).

The great number of constituents, essential oils cause to have no specific cellular targets (Carson et al., 2002). As typical lipophiles, they pass through the cell wall and cytoplasmic membrane, disrupt the structure of their different layers of polysaccharides, fatty acids and phospholipids and permeabilize them. Essential oils are volatile, natural, complex compounds characterized by a strong odor and are formed from aromatic plants as secondary metabolites. They are usually obtained by steam or hydro-distillation. Volatile oils are known for their antiseptic effect, i.e. bactericidal, virucidal and fungicidal, and medicinal properties and their

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Review of literature fragrance; they are used in embalment, preservation of foods and as antimicrobial, analgesic, sedative, anti-inflammatory, spasmolytic and locally anesthesic remedies. In nature, essential oils play an important role in the protection of the plants as antibacterials, antivirals, antifungals, insecticides and also against herbivores by reducing their appetite for such plants (Ipek et al., 2005). Studies have been carried out by the plant pathologists in different countries of the world to identify plants with antifungal properties.

Ismail and Ahmed (2000) showed that extracts of cumin fruits, anise fruits, black pepper fruit, terms seeds, fenugreek seeds, soybean seeds, apricot stones, granhollyhock leaves and water hyacinth leaves were effective against Rhizoctonia solani, Sclerotium rolfsii, Fusarium moniliforme and Macrophomina phaseolina that causing damping off and cotton seedlings. Green house experiments showed that cumin fruits, black pepper fruits extract significantly increased survival plants, while the extracts of anise fruits and water hyacinth leaves had a moderate effect increasing survival plants.

Nguefack et al., (2004) investigated five essential oils (EO) extracted from Cymbopogon citratus, Monodora myristica, Ocimum gratissimum, Thymus vulgaris and Zingiber officinale for their inhibitory effect against three food spoilage and mycotoxin producing fungi, Fusarium moniliforme, Aspergillus flavus and Aspergillus fumigatus. Five strains of each fungus were tested. The EO from O. gratissimum, T. vulgaris and C. citratus were the most effective and prevented conidial germination and the

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Review of literature growth of all three fungi on corn meal agar at 800, 1000 and 1200 ppm, respectively. Moderate activity was observed for the EO from Z. officinale between 800 and 2500 ppm, while the EO from M. myristica was less inhibitory.

The antifungal effects of essential oils of oregano (Origanum syriacum var. bevanii) and fennel (Foeniculum vulgare) were evaluated against Sclerotinia sclerotium (Soylu et al., 2007). In eukaryotic cells, essential oils can act as prooxidants affecting inner cell membranes and organelles such as mitochondria. Depending on type and concentration, they exhibit cytotoxic effects on living cells but are usually nongenotoxic. In some cases, changes in intracellular redox potential and mitochondrial dysfunction induced by essential oils can be associated with their capacity to exert antigenotoxic effects (Bakkali et al., 2008). Mustard essential oil (EO) affected the cell membrane of Escherichia coli O157:H7 and Salmonella typhi. Mustard EO affects the concentration of intracellular component, such as ATP in both bacteria and affects the pH suggesting that cytoplasmic membrane is involved in the antimicrobial action of mustard EO. Mustard EO can be used as an effective antimicrobial agent (Mélanie et al., 2009). Global oil resources and the future global oil supply/demand balance were examined. Global oil resources have been investigated on three levels; country, company and field levels (Jan and Filip 2009).

Antifungal activity of six essential oils Clove, Cedar wood, Lemon grass, Peppermint, Citronella and Nutmeg oils was tested in vitro on

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Review of literature

Phomopsis azadirachtae the causative agent of destructive die-back disease of neem. All the used six oils showed significant antifungal activity against the tested pathogen. The results indicated that the citronella and lemongrass showed 100% inhibition of mycelial growth at 2,500 ppm. Hence, the results of the present investigations indicate that plant essential oils possess antifungal activity and can be exploited as an ideal treatment for future plant disease management to eliminate fungal spread (Nagendra et al., 2010).

3. Effect of inducers:

Plant resistance provides an environmentally and economically appropriate means for disease control that can be easily included within an integrated disease management strategy. In fact, the use of natural resistance for the management of fungal diseases in chickpea may be enhanced by various inducers. Protection of chickpea against root rot disease caused by Sclerotium rolfsii was shown to be associated with the induction of the synthesis of the phytoalexins medicarpin and maackiain and the related isoflavones formononetin and biochanin A. Fathi et al., (2000) showed that michael adducts of ascorbic acid with α,β-unsaturated carbonyl compounds have potent inhibitors of protein phosphatase 1 (PP1) without affecting cell viability at the respective concentrations. Higher concentrations can partially inhibit PP2A activity and concomitantly induce apoptotic cell death. A nitrostyrene adduct of ascorbic acid proved to be a more potent and effective inhibitor of PP2A as well as a stronger inducer of apoptosis

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Review of literature

Sarma et al., (2002) showed successful reduction of collar rot of chickpea (Cicer arietinum) caused by a soil-borne pathogen, Sclerotium rolfsii, through enhanced synthesis of phenolic compounds in the host. Demonstration the sensitivity of S. rolfsii towards higher concentrations of ferulic acid occured. Abiotic inducers include chemicals which act at various points in the signaling pathways involved in disease resistance, as well as water stress, heat shock, and pH stress. Resistance induced by these agents (resistance elicitors) is broad spectrum and long lasting, but rarely provides complete control of infection, with many resistance elicitors providing between 20 and 85% disease control (Dale et al., 2005).

Mahmoud et al., (2006b). Studied the effectiveness of three chemical

inducers resistance, hydrogen peroxide (HR2ROR2R) at 0.25, 0.5 and 1%, Bion (benzo 1,2,3) thiodiazole-7-carbothioic– methlye ester) and salicylic acid at 2, 4 and 8 mM in greenhouse under artificial inoculations. All tested inducers significantly reduced damping-off, wilt and root rot incidence, Salicylic acid at 4 mM and Bion at 8 Mm followed by Hydrogen peroxide at 0.25 % gave the highest effect on all parameters of disease incidence.

Four chemicals salicylic acid, sodium salt of salicylic acid, isonicotinic acid, and DL-β-amino-n-butyric acid that they are inducers and the yeast antagonist Cryptococcus flavescens separately or together can reduce Fusarium head blight (FHB) of wheat in the greenhouse severity compared to the non-treated disease control. (Shouan et al., 2007).

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Review of literature

Efficacies of some plant resistance elicitors, chitosan, salicylic acid and hydroquinone as seed treatments against soil borne pathogenic fungi Rhizoctonia solani and Fusarium solani, Fusarium oxysporum and Sclerotium rolfsii that attack lupine plants showed significant reduction in the fungal growth in vitro. The greenhouse experiments showed that S. rolfsii and R. solani followed by F. solani and F. oxysporum were the most aggressive root rot fungi. Chitosan at 8 g/ L increasing the percentage of healthy plants to record 72.5, 80.9, 62.7and 64.3%, when seeds were grown in soil infested with of F. solani, F. oxysporum, R. solani and S. rolfsii, respectively (Ali et al., 2009).

Maurya et al., (2009) investigated the total protein content of seven isolates of Sclerotium rolfsii and the effect of the total protein of different isolates on growth promotion and management of collar rot disease of chickpea. Among different concentrations of fungal protein, 0.21 and 0.28 mg/ml showed maximum efficacy in plant growth promotion. Also, plant mortality was reduced significantly compared to control following treatment of fungal protein. Phenolic acid content of plant leaves after spray of fungal protein increased compared to control. The fungal protein has an important role in inducing resistance in plants.

Biological control and represents an interesting strategy to stimulate the defense system of the plant especially when applied together. He used Trichoderma harzianum, and hormonal inducers salicylic acid as a new strategy to enhance tomato defense response against wilt disease caused by

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Review of literature

Fusarium oxysporum f. sp. Lycopersici under greenhouse conditions (Amel et al., 2010).

4. Effect of bioagents:

In recent years, concerns have been raised over the effects of the overuse of agricultural pesticides on the environment and human health. Biocontrol agents can serve as an alternative to chemicals in integrated pest management systems. Although the adoption of biocontrol agents is strongly affected by the socio-economic environment in, which they are to be applied and by farmers’ attitudes, these factors have been poorly investigated in development programs. (Riccarda et al., 2008).

Antagonisms may operate by using the pathogen as a food source. If the pathogen is a fungus then the antagonists is a mycoparasite and usually possesses chitinase to break down the walls of the fungus. If the pathogen is an oomycete (e.g. Pythium or Phytophthora) then cellulases (s) are needed.

The best known mycoparasite is the fungus Gliocladium spp. which has been suggested as a biocontrol agent against many soil pathogens (Parker et al., 1985) and is one of the few agents at present commercially available. The hyphae of Trichoderma may penetrate resting structures such as sclerotia or may parasitize growing hyphae. In the later case the hyphae grow alongside the pathogen and send out side branches that coil around the hyphae.

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Review of literature

Penetration of the wall has been shown in some cases and assumed in others. Other soil fungi can coil round hyphae of pathogens and produce death of the later, some times with out obvious evidence of holes in the attacked hyphae. Three species of Trichoderma (Trichoderma viride, Trichoderma harzianum, and Trichoderma virens) were tested against four isolates of fusarium wilt (Fusarium oxysporum) of chickpea, Cicer arietinum representing four different races commonly prevalent in India. T. viride isolated from Ranchi followed by T. harzianum (Ranchi) and T. viride isolated from Delhi inhibited maximum mycelial growth of the pathogen. They also enhanced seed germination, root and shoot length, and decreased wilt incidence under green house condition (Sunil et al., 2007).

Collar rot of chickpea (Cicer arietinum) is caused by the soil-borne pathogen Sclerotium rolfsii. An integrated approach was adopted by using vermicompost and an antagonistic strain of Pseudomonas syringae possessing plant growth-promoting characteristics. The vegetation diversity is fundamental to biological control when it serves as a source of habitat and nutritional resources (Sahni et al., 2008).

Isolated actinomycetes can reduce in vivo the incidence of root rot induced by Sclerotium rolfsii on sugar beet. These isolates were subsequently tested for their ability to inhibit sclerotial germination and hyphal growth of S. roflsii. The most important inhibitions were obtained by the culture filtrate from the isolates J-2 and B-11, including 100% inhibition of sclerotial germination and 80% inhibition of hyphal growth. Isolate J-2 was most effective and allowed a high fresh weight of sugar beet roots to be

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Review of literature obtained. Streptomyces isolates J-2 and B-11 showed a potential for controlling root rot on sugar beet and could be useful in integrated control against diverse soil borne plant pathogens (Errakhi et al., 2009).

Trichoderma atroviride is an experimental biocontrol agent that is active against the fungus Armillaria mellea. T. atroviride had an effect on the soil microflora during the first two weeks following inoculation. However, later, environmental conditions had a higher influence on the surveyed communities. Soil depth had a strong influence on the composition and biodiversity of fungal communities (Federica et al., 2009).

Gaur and Sharma (2010) recorded that Trichoderma viride and T. harzianum were effective inhibiting by 76.53 and 72.78 % of growth of dry root rot pathogen, Rhizoctonia bataticola of cotton respectively. These two bioagents also proved their superiorityover rest of the bioagents in controlling root rot disease in pots under cage house conditions. Trichoderma viride and T. harzianum reduce root rot incidence of 35.78 and 38.98 % in pots when seeds and soil were treated with T. viride and T. harzianum respectively.

Phytotoxicity of the tested fungicides, oils and inducers on the length of shoot and root systems.

Phytotoxicity is a term used to describe the toxic effect of a compound on plant growth. Such damage may be caused by a wide variety of compounds, including trace metals, pesticides, salinity or allelopathy,

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Review of literature which is the process used by a plant to release toxic chemicals into the ground to kill neighbouring plants.

Phytotoxicity is common in the application of urea due to the limited ability for soil to fully convert the fertilizer to ammonium in order for mass flow uptake to occur. Organic compost enables more effective uptake of nitrogen due to higher prevalence of aerobic microbial activity.

Sampedro et al., (2004) recorded that the phytotoxicity of dry olive mill residue decreased when it was incubated in the presence of saprobic fungi on the growth of soybean (Glycinemax) and tomato (Lycopersicum esculentum). Some of the saprobic fungi as F. oxysporum and F. lateritum eliminate completely the phytotoxicity of dry olive mill residue.

Chemical sprays were used to help in overcoming dormancy is a wide-spread practice in warm-climate countries that grow temperate fruit crops which require exposure to chilling to overcome the dormant period of the buds. Two main problems confront the use of such chemicals: human and environmental toxicity and phytotoxicity especially in stone-fruit species. As to phytotoxicity, many of the efficient chemicals in use like Dormex, and mineral oils pose a risk to the tree under specific environmental conditions. Experiments initiated with new dormancy breaking agents to achieve strong effects, low phytotoxicity and minimal human hazard. Thidiazuron was reported to have a dormancy breaking effect (Erez et al., 2006).

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Review of literature

Leachates from an operating and a closed landfill showed phytotoxicity by seed germination and root elongation tests using seeds of Brassica chinensis and Lolium perenne. Their EC50s (the effective dose for 50% inhibition) ranged from 3% to 46% v/v, which varied remarkably with the operating status of the landfills. The seed bioassay provided a conservative estimate of the phytotoxicity of landfill leachate (Chenga and Chu, 2007).

Twenty essential oils extracted from various aromatic plants have antifungal activity in vitro against the soil-borne pathogens, Rhizoctonia bataticola and Sclerotium rolfsii by poisoned food technique. The most significant antifungal activity was exhibited by essential oils extracted from Acorus calamus, Curcuma longa, Pimpinella anisum and Vetiveria zizanioides. These oils did not show any phytotoxic effect on the germination of chickpea seedlings (Praveen et al., 2009).

Aranda et al., (2010) found that the physical extractions of dry olive mill residue gave an aqueous and an exhausted fraction with less phytotoxicity for tomato than the original samples. The indigenous arbuscular mycorrhizal and saprobe fungi were able to decrease the phytotoxicity of exhausted fractions inoculated with Trametes versicolor and Pycnoporus cinnabarinus on tomato.

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Review of literature

Effect of some combinations of fungicides, biocontrol agents and oils on fungi:

Combi-product of carbendazim + pankaj iprodione registered maximum inhibition of Fusarium oxysporum collected from various parts of state in vitro followed by companion (carbendazim + mancozeb). (Ashok 2005).

Ansari et al., (2006) found that the white grub, Hoplia philanthus (Coleoptera scarabaeidae) is a major pest of turf and ornamental plants in Belgium. The combination of lethal concentration of the entomopathogenic nematodes Heterorhabditis megidis or Steinernema glaseri with the entomopathogenic fungus Metarhizium anisopliae caused additive or synergistic mortality to H. philanthus in the laboratory and greenhouse.

Both of ZnSOR4 R(10−4 mM) and oxalic acid (4 mM) the chemicals provided significant protection to chickpea against collar rot disease compared to control (100% plant mortality) when used alone as well as in combination with Pseudomonas syringae and vermicompost. However,

ZnSOR4R was more effective than oxalic acid against S. rolfsii. The findings indicate the utility of integration of the above factors in managing collar rot efficiently (Sangita et al., 2008).

Somnath et al., (2009) tested the efficacy of a prepackaged combined formulation, companion (carbendazim 12 % + mancozeb 63 % WP), sole application of carbendazim 50 % WP, manozeb 75% WP and

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Review of literature methyl jasmonate (MeJA), an inducer of systemic acquired resistance on disease severity and their role in post-infectional defense responses in chilli seedlings against Sclerotium rolfsii. At 15 days after sowing maximum defense against fungal infection was exhibited by Companion comparably followed by the sole application of carbendazim and mancozeb.

Five soil fungi Acremonium implicatum, Chrysosporium queenslandicum, Chrysosporium pannicola, Malbranchea pulchella and Verticillium lecanii, when used in combination with Chrysosporium keratinophilum showed feather and keratin azure degradation. (Itisha and Kushwaha, 2011)

Effect of fungicides, oils, biocontrol agents on chlorophyll content and plant chemical constituents:

Harpin protein was applied to the peppers (Capsicum annuum) grown under natural conditions. These plants were subjected to artificial inoculation with Botrytis cinerea, which causes fruit spoilage in peppers. There were changes in vegetative growth, total chlorophyll content in leaves, leaf colour and percentage of rotten fruits after treatments. The number of leaves per plant value was quite low in all cultivars. Leaf chlorophyll values exhibited significant decline in the plants subjected to B. cinerea treatment in all cultivars. However, the chlorophyll content in the plants subjected to harpin protein + B. cinerea treatment was low (Akbudak et al., 2006).

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Review of literature

Chlorophyll content of Begonia Beg saturated at an N supply of 28.6 mM when affected by Botrytis cinerea disease. (Pitchay et al., 2007)

Pyung (2008) recoded that glufosinate ammonium is a herbicide that

diminished developments of rice (Oryza2T sativa)2T blast and brown leaf spot

in bar2T -2T transgenic rice caused slight chlorosis and diminished chlorophyll content.

Chlorophyll fluorescence in high frequency multipoint scanning mode may enable identification of nitrogen(N)-deficiency and pathogen infections such as leaf rust (Puccinia recondita) and powdery mildew (Blumeria graminis) in winter wheat. Fluorescence readings at 690 nm (F690) and 730 nm (F730) were taken in the light under constant environmental conditions at leaf and canopy level. Throughout the experiment, N-deficient wheat plants displayed lower chlorophyll content and increased F690/F730 ratio. Pathogen infected plants showed a significantly enhanced fluorescence ratio, associated with chlorophyll degradation in the infected areas, only after appearance of visual symptoms. The results of cross-validation analysis indicated that with chlorophyll fluorescence measurements, samples with pathogen infections may be misrecognised as N deficiency and vice versa (Kuckenberg et al., 2009).

Deoxynivalenol (DON) has a role in development of Fusarium head blight (FHB), it effects of the toxin on uninfected barley tissues. Content of chlorophylls a and b and of total carotenoid pigment was reduced. DON had a toxic effect, damaging plasmalemmas in treated tissues before chloroplasts

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Review of literature began to lose pigment. Cycloheximide, which likes DON, inhibits protein synthesis, also bleached some tissues and delayed senescence of others. Thus, the effects of DON probably relate to its ability to inhibit protein synthesis (Bushnel et al., 2010).

Polygalacturonase content in Sclerotium rolfsii:

Polygalacturonase is an enzyme that catalyzes the hydrolysis of specific linkages in galacturonides and other polysaccharides.

A novel lysozyme exhibiting antifungal activity and with a molecular mass of 14.4 kDa in SDS–polyacrylamide gel electrophoresis was isolated from mung bean (Phaseolus mungo) seeds using a procedure that involved aqueous extraction, ammonium sulfate precipitation, ion exchange chromatography on CM-Sephadex, and high-performance liquid chromatography. The specific activity of the lysozyme was 355 U/mg at pH 5.5 and 30 °C. The lysozyme exhibited a pH optimum at pH 5.5 and a temperature optimum at 55 °C. For the first time, that a novel plant lysozyme exerted an antifungal action toward Fusarium oxysporum, Fusarium solani, Pythium aphanidermatum, Sclerotium rolfsii, and Botrytis cinerea, in addition to an antibacterial action against Staphylococcus aureus (Shaoyun et al., 2005).

Ratul et al., (2006) studied properties of cell-wall protein (CWP) of Fusarium oxysporum f. sp. ciceri and Macrophomina phaseolina, CWPs were extracted and tested against chickpea. Chickpea seedlings were

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Review of literature exposed to CWPs showed enhanced synthesis of phenol, pathogenesis- related protein and activities of phenylalanine ammonia lyase and peroxidase relative to water-treated controls. Both Fusarium wilt and charcoal rot diseases of chickpea were significantly reduced in seedlings following CWP treatment. However, the same CWPs failed to antagonise the same pathogens in petri dish assays. Sclerotium rolfsii produced extraordinary high amounts of polygalacturonases (PGs). (Schnitzhofer et al., 2007).

Polygalacturonases (PGs), enzymes that hydrolyze the homogalacturonan of the plant cell wall, are virulence factors of several phytopathogenic fungi and bacteria. On the other hand, PGs may activate defense responses by releasing oligogalacturonides (OGs) perceived by the plant cell as host-associated molecular patterns. (Simone Ferrari et al.,

2008)8T 8T

b-Xylosidase production was maximal for the mutant Pichia stipitis grown on xylan as the sole carbon source. b-Xylosidase was purified from

culture supernatant by (NH4)R2RSOR4R precipitation and a hydrophobic interaction chromatography on phenyl sepharose. Optima of pH and temperature were 5.0 and 50ْ C, respectively. The enzyme was inhibited by 3+ 2-mercaptoethanol (100%) and FeP P (80%), and moderately affected by 2+ + 2+ CuP ,P AgP ,P NH R4R and MgP P and SDS. The purified xylosidase hydrolyzed xylobiose and xylo-oligosaccharides and it did not exhibit activity against cellulose, starch, maltose and cellobiose (Pervin et al., 2008).

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Review of literature

Extracted and purified polygalacturonase (PG) from decayed ‘Golden Delicious’ apple fruit inoculated with Penicillium solitum. The enzyme was active with optimal activity between pH 4 and 5. PG was highly active at 20 and 37 gel filtration, and cation exchange chromatography were used to purify the enzyme. Divalent cations affected PG enzyme activity; Mg and Fe increased its activity, whereas Ca and Mn reduced activity in vitro. The enzyme exhibits both exo and endo activity. Purified PG incubated with intact apple fruit tissue in vitro caused a 30% reduction in mass after 48 h, suggesting a role in P. solitum-mediated decay of apple fruit (Wayne et al., 2009).

Rashad et al., (2011) illustrated that the xylanase and polygalacturonase of fruit spoilage fungi Fusarium oxysporum (banana and grape), Aspergillus japonicus (pokhara and apricot), Aspergillus oryzae (orange), Aspergillus awamori (lemon), Aspergillus phoenicis (tomato), Aspergillus tubingensis (peach), Aspergillus niger (apple), Aspergillus flavus (mango), Aspergillus foetidus (kiwi) and Rhizopus stolonifer (date) had the highest level contents as compared to the cellulase and α- amylase

Effect of gamma radiation: Types of radiation:

Radiation in general is the emission of any rays or particles from a source (Uvanov and Isaacs, 1986). It can be divided into two types, ionizing radiation and non- ionizing radiation.

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Review of literature

1- Ionizing radiation Ionizing radiation characterized by its very high-energy content and great penetration power, it can strip off electrons from atoms of the material through which it passes (Silliker et al., 1980). Ionizing radiation includes X- rays, γ- rays, β- rays (high speed electrons), α- rays (positively charged nucleus or heliull 1 atom), protons and neutrons (Coggle, 1983). X- rays and γ- rays consist of very short wave lengths, the latter being produced from 60 radioactive sources such as COP P and the former from a machinery system. β- rays were originally produced from radioactive isotopes but had a little penetration power.

2- Non- ionizing radiation. Non ionizing radiation is an electromagnetic radiation of long wavelengths, including ultraviolet, ultrasonic, microwave, radio frequency and optical radiation. Non- ionizing radiation causes excitation of atoms i.e, an alteration of electrons within their orbits, but does not possesses enough energy to eject electrons to produce ions, and thus it is not an ionizing radiation (Shleien, 1992).

Effect of ionizing radiation on microorganisms.

Ionizing radiation such as gamma rays emitted from the excited 60 nucleus COP P has been studied extensively for its effect on microorganisms (Board, 1983). Ionizing is not a selective process so any atom or molecule in the path of radiation may be ionized. The predominate constituents of the cell stand a better chance of being ionized and therefore affected (Grosch

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Review of literature and Hopwood, 1979). The death of microorganisms is a consequence of the ionizing action of high- energy radiation. Almost all the studies indicated that the lethal damage to microbial DNA resulting in loss of ability to reproduce is the primary cause of lethality. However, damage to other sensitive and critical cell structures, e.g. membranes may also have a similar effect. Dose levels that are lethal to microorganisms do not necessarily cause inactivation to enzymes, proteins or any other large molecules. In general, microorganisms differ greatly in their sensitivity to irradiation (Silliker, et al., 1980).

Factors affecting the sensitivity of microorganisms to radiation.

Different microorganisms have different degrees of radiosensitivity when they are exposed to ionizing radiations. Several factors are also involved in determining the degree of radiosensitivity (EL, Nagar, 1995). These factors are physical factors which include the radiation dose, radiation rate, type of radiation the linear energy transfer and the energy of radiation. Biochemical factors such as the presence of water, increased oxygen concentration and other radio- sensitizers might contribute to higher radiosensitivity. Biological factors mainly include cell division and cell cycle also affects the response of microorganisms to radiation.

One of the major effects of radiation on fungi is the distribution in the timing of cell division; it is the most obvious and immediate consequence of radiation. Other transient morphological and physiological

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Review of literature effects have been described by (James and Nasim, 1987) were cell enlargement, malformation, enzymatic changes, cell wall changes and alteration of permeability and complete inactivation of cells.

Data that inhibition of metabolic processes by radiation is associated with damage of the plasma membrane (James and Nasim, 1987).

Effect of gamma radiation on microorganisms.

Gamma radiation has been shown to exert its lethal effect on microorganisms through two methods of action; direct and indirect.

1- The direct action It is the one in which the ionizing event occurs within the microorganisms in or near the genetic material or any other vital structure exerting the damage directly, that is why subsequent division becomes impossible (Abo Elkhair, 1986).

2- The indirect action It is the one in which the ionizing event occurs outside the microorganisms in the intracellular fluids. The indirect action is dependent on the water content of microorganisms, the presence of dissolved oxygen and protective organic matter in its environment and the physiological state of the microorganism itself (Abo Elkhair, 1986). Ionizing energy can exert its effect on the microorganisms through the reaction products of the radiolysis of water (indirect action) diffusing into the cell; its radiolysis is

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Review of literature also of importance. The radiolysis of water results in hydroxyl, hydrogen and hydroperoxyl radicals as well as some hydrogen peroxide being produced if oxygen is present (Yarmnenko, 1988).

Gamma (γ-) irradiation was used as a method for soil sterilization for laboratory experiments over other sterilization techniques. There was literature dating back over 50 years to investigate the chemical and biological effects on gamma irradiated soils and to determine its practicality for sterilising soils which will subsequently be used for experimental purposes. Typically, γ-irradiation at 10 kGy will eliminate actinomycetes, fungi and invertebrates in most soils. The majority of soil bacteria are eliminated by 20 kGy, however, a dose higher than 70 k Gy may be required to kill certain radio-resistant bacteria.

We recommend prior to experimentation that the radiosensitivity of soils is determined to ensure the desired chemical and biological effects are achieved. Gamma irradiation may not be an appropriate method for all experiments as it can influence soil chemical properties, in particular soil nitrate and ammonium levels. Where chemical stability is required we recommend sterilizing soils air-dry rather than moist (McNamara et al., 2003). Gamma irradiation was effective as a method of decontamination of maize containing Fusarium verticillioides under controlled conditions of relative humidity (RH) (97.5%) and water activity. Maize grains inoculated with a spore suspension of F. verticillioides were irradiated to 2, 5, and 10 kGy. the risk of exposure to fumonisins by irradiating maize to 5 or 10 kGy may be decrease. However, at the dose of 2 kGy, the survived fungi

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Review of literature

(36%) can produce more fumonisins than the fungi in the control unirradiated samples under the same conditions (Ferreira et al., 2007). Different gamma radiation doses affect on the growth of Alternaria alternata in artificially inoculated cereal samples. Seeds and grains were divided into four groups: Control Group (not irradiated), and Groups 1, 2 6 and 3, inoculated with an A. alternata spore suspension (1×10P P spores/ mL) and exposed to 2, 5 and 10 kGy, respectively. Fungal morphology after irradiation was analyzed by scanning electron microscopy (SEM). The results showed that ionizing radiation at a dose of 5 kGy was effective in reducing the growth of A. alternata. However, a dose of 10 kGy was necessary to inhibit fungal growth completely. SEM made it possible to visualize structural alterations induced by the different γ-radiation doses used (Braghini et al., 2009).

Gamma rays influences suppressive effect on root rot fungi such as Macrophomina phaseolina (Tassi) Goid, Rhizoctonia solani Kühn and Fusarium spp., and inducive effect on growth parameters of mung bean (Vigna radiata L.). Seeds of mung bean were treated with gamma rays (60 Cobalt) at time periods of 0 and 4 minutes and stored for 90 days at room temperature to determine its effect on growth parameters and infection of root infecting fungi. All treatments of gamma rays enhanced the growth parameters as compared to untreated plants. Infection of M. phaseolina, R. solani and Fusarium spp., were significantly decreased on mung bean seeds treated with gamma rays. Gamma rays significantly increased the growth parameters and controlled the root rot fungi up to 90 days of storage of seeds (Naheed et al., 2010).

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Materials and methods

MATERIALS AND METHODS

In the present study Cicer arietinum (chickpea seeds cultivar, Giza 3) was selected as a host plant. It was kindly supplied from El- Gemmeza Agricultural Research Station (El – Gharbia- Egypt) and was used as a host plant for the tested fungus causing root- rot disease. The used pathogenic soil borne fungus was obtained from Plant Pathology Research Institute, Agricultural Research Center, Giza, Egypt. The experiments were conducted under both laboratory and greenhouse conditions.

Tested fungicides: 1- Vitavax-200 (75% W.P.) Consists of (37.5% Carboxin + 37.5% thiram): a)Carboxin (37.5% W.P.)

5, 6 dihydro-2 methyl – 1, 4 oxathi – ine – carboxanilide . Trade name: Vitavax Common name: Carboxin

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Materials and methods

b) Thiram ( 37.5% W.P. )

Bis ( Dimethyl thio- carbamoyl ) disulphide Common name: TMTD

2- Monceren – T (47% W.P.) Consists of (15 % pencycuron + 32% Thiram) . a) Pencycuron (15 % W.P.) 1 – (4-chloro – benzyl) – 1-cyclopentyl -3- phenylurea . b) Thiram ( 32 % W.P. )

The tested oils Both of clove (Syzygium aromaticum) and mint (Mentha spp.) oils were used to examine their antifungal effect on pathogenic and antagonistic fungi throughout these experiments. They were supplied from Phytochemistry Department- faculty of Pharmacy- Tanta University. After several preliminary experiments concentrations: (0.01, 0.1, 1.0, 5.0, 10.0, 25.0 and 50.0 ppm) were used.

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Materials and methods

Potato dextrose agar medium : Two hundred grams of sliced (washed and peeled) potatoes was boiled in water for 30 minutes and then decanting or straining the broth through cheesecloth. Distilled water is added such that the total volume of the suspension is 1 litre. 20 grams dextrose and 20 grams agar powder is then added .The medium was sterilized by autoclaving at for 15 minutes (Jong & Edwards, 1991).

Tested inducers: Two inducers were used throughout the present study obtained from Plant Pathology Research Institute, Agricultural Research Center, Giza, Egypt (illustrated in table1).

Table (1): Inducers compound used:

Rate of application Commercial name Chemical composition per one liter dist water

Oxalinic acid (20 %) W.P 2 g / L Starner

Copprus KZ Copprus oxide(50 %)W.P 3 g / L

Tested fungi : One pathogenic and Three antagonistic fungi were used in these experiments namely : i) The pathogenic fungus : Sclerotium rolfsii.

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Materials and methods

ii) The antagonistic fungi : Gliocladium viriens, Gliocladium deliquesens and Trichoderma hamatum.

1- Artificial infection of Cicer arietinum as a host tissue in vitro. a) Preparation of inoculum:

Sterilized water with 0.1 % Tween- 80 was added on 3 days old culture Sclerotium rolfsii as a pathogenic fungus fragmented mycelia on PDA. The fragmented mycelium and fungal suspension was used in infection processes. b) Plant materials Cicer arietinum seeds were cultivated in sand for 10 days. Segments, 10 cm long were excised from Cicer arietinum (10 days old). The cut ends were sealed with warm paraffin wax, and the segments were arranged on a glass racks within a plastic boxes lined with a moist tissue paper (O'Conell et al. 1985). For each experiment, 4 segments were inoculated with 2 droplets of fungal suspension in three different places. In the meantime Cicer arietinum, the whole roots were washed in running tap water. The roots were washed with sterilized distilled water several times and dried carefully. The cut ends were sealed with a warm paraffin wax, and the segments were placed over a glass plate within a plastic box under humid conditions. For each experiment, 4 segments were

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Materials and methods

inoculated each with 2 droplets of fungal suspension in three different places. The bioassay boxes were incubated at 28°C for 7 days. Control plant materials (roots and hypocotyls) were inoculated with droplets of sterilized water instead of fungal suspension. c) Examination of the infected tissues

After the establishment of the infection in hypocotyls and root of the tissues in the inoculated area. Infection was examined by cutting thin longitudinal and transverse sections of hypocotyls and root using a single edge razor blade, and removed carefully using fine forceps. The cut tissues were placed on a slide in a distilled water or mounted in glycerol and covered with a cover slide. d) Light microscopy

The infected tissues were examined by using a normal microscope (bright field illumination) connected with a video camera attached to a computer unit.

2- Biological tests ( In vitro studies ).

2. a- Antagonism between pathogenic fungus and antagonistic fungi (biocontrol agents).

Petri dishes each containing 15 ml of PDA medium were used. Each Petri dish was divided into two equal halves, the first half was inoculated

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Materials and methods

with a disk (0.5 cm in diameter) of the tested antagonistic fungus and the second half was inoculated with a similar disk of the pathogenic fungus. Plates inoculated only with the pathogenic fungi acted as control. Each treatment was replicated four times. All Petri dishes were incubated at 28º C and observed daily. After 3 days incubation, when the pathogenic fungi almost covered the surface of the medium in control treatment, the percentage of inhibition (I %) was calculated.

2. b- Fungicidal activity of fungicides, oils and inducers.

Inhibitory effect of fungicides, oils and inducers were tested in vitro. A laboratory study was performed to examine the sensitivity of both pathogenic and antagonistic fungi to the tested fungicides, oils and inducers. After several preliminary experiments to choose suitable concentrations seven concentrations of each fungicide, oil and inducer were used as follows: 0.01, 0.1, 1.0, 5.0, 10, 25 and 50 ppm, respectively. The required concentrations were obtained by adding the appropriate amount of stock solution used, to 100 ml portions of autoclaved potato dextrose agar medium (PDA) which was cooled to about 45ºC (not solidified), four Petri dishes were used as replicates for each concentration and four were left without any treatment as control. After solidification of the medium, each dish was inoculated centrally with a mycelial disk (0.5 cm in diameter) taken from the cultures of each fungus (3 days – old). Plates were incubated at 28ºC and colony diameters were measured till untreated control had just covered the plate. Linear growth was measured daily in cm. The average of two perpendicular diameters were recorded. The percentage of inhibition (I %) was calculated according to Topps and Wain equation (1957) as follows:

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Materials and methods

I % = (A – B) / A x 100

Where:

I % = Inhibition percentage.

A = Mean diameter of growth in the control.

B = Mean diameter of growth in treatment.

Regression lines of the results were drawn probit – log paper and the median inhibition concentration (IC50, the concentration of each treatment that is required to cause fungitoxic inhibition of the tested fungus by 50 % in comparison with the control) was detected.

2.c- Phytotoxicity test.

This test was carried out by El- Nawawy et al., (1972). It was carried out by germinating seeds of chickpea in water moisted material for 3-4 days. Selected germinating seeds were inserted in test tubes each containing plain agar (15 gm/ L) with their rootlets immersed slightly on the surface of agar containing the required concentrations from the tested compounds (fungicides, oils, and inducers respectively). Three replicates were conducted for each treatment. Control tubes were prepared by the same way without adding any of the tested compounds. Test tubes were incubated at ambient temperature. Shoot and root length were measured every 24 hours for 7 days. The phytotoxic effects on shoot and root system was determined as percentage of inhibition in the root and shoot length (I %) when the

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Materials and methods

control sample reach to the end of the test tube, using, the following equation :

I % = ((A – D)/ A) X100

Where:

I % = Inhibition percentage.

A = Length of untreated shoot or root system.

D = Length of treated shoot or root system.

2.d- Joint toxic effect of vitavax combined with inducers and oils against S. rolfsii.

IC25 of carboxin/ thiram (vitavax) which is a traditional fungicide (has the highest effect on the pathogenic fungus and the lowest effect on antagonistic fungi) was mixed with IC25 from each of the used compounds alone (inducers and oils). Each mixture was used to control each of the tested pathogenic and antagonistic fungi. In case of using vitavax as a traditional fungicide in enhancing, the efficacy of the biocontrol agents IC25 from the fungicide was added to PDA medium before solidification.

(IC25, the concentration of each treatment that is required to cause fungitoxic inhibition of the tested fungus by 25 % in comparison with the control).

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Materials and methods

The joint toxic effect of the different combinations was evaluated by the following equation Mansour et al., (1966):

Co- Toxicity factor = (Observed I % - Expected I %) / Expected I % X 100

This factor (C.T.F.) was used to categorize the results into 3 categories:

- A positive factor (C.T.F.) of 20 or more means synergistic effect.

-A negative factor (C.T.F.) of -20 or more means antagonistic effect.

-An intermediate value of (-20 to +20) was considered as an additive effect.

3- Greenhouse experiments ( In vivo ):

3.a- Pathogenecity test:

Isolate of Sclerotium rolfsii was used separately in initial pathogenecity experiment. The inoculum was mixed with sterilized soil at the rate of 3% soil (30 gm fungal inoculum / 1 kg soil), 2 % (20 gm fungal inoculum / 1 kg soil), 1 % (10 gm fungal inoculum / 1 kg soil), ½ % (5 gm fungal inoculum / 1 kg soil), 1/4 % (2.5 gm fungal inoculum / 1 kg soil), 1/8 % (1.25 gm fungal inoculum / 1 kg soil).

3.b- Preparation of inoculum and soil infestation:

Isolate of Sclerotium rolsii, from the previous stock culture was grown in barley grain medium a mixture of 1: 3 (wheat bran / sand), which is a suitable substrate for mycelial growth and multiplication. Glass bottles

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Materials and methods

(500 ml) each containing 200 gm of a mixture of 1: 3 (wheat bran / sand) was moistened with water and autoclaved for 30 minutes. The prepared bottles were inoculated separately with each of pathogenic and antagonistic fungi separately which had been grown on PDA for 3 days. Sterilized sandy clay soil was compacted into 25 cm diameter plastic pots, each pot contained 1 kg soil. Soil infestation was carried out one week before sowing with the used pathogenic fungus (at the rate of 1/4 %). Soil was kept moist to allow sclerotial germination.

3.c- Effect of infection by S. rolfsii on growth parameters of Cicer arietinum.

Ten Cicer arietinum seeds were sowed in each plastic pot and replicated 3 times for each particular treatment. Pre- and- post emergence root rot were recorded 15 and 45 days after sowing for each treatment respectively, as follows :

(Number of infected plants / Total plant number) X 100

Percentage of survival plants (% survival) was recorded 45 days after sowing for each treatment as follows:

(Number of uninfected plants / Total plant number) X 100

Plant height, fresh and dry weight were recorded 45 days after sowing.

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Materials and methods

4- Effect of fungicides, oils, inducers, bioagents and their combinations on the infected Cicer arietinum plants under green house conditions.

This study was carried out in vivo (under green house conditions) to show the best treatments obtained from In vitro experiments and their mixtures on root rot disease of Cicer arietinum plants.

To design this experiment 24 pots replicated 3 times were used. Each pot contains 1 kg of clay and sand soil (90: 10 %) sterilized by autoclaving on three successive days. One week early before sowing, each pot (from 24 pots) was inoculated with 2.5 gm of pathogenic fungus per 1 kg sterilized soil and irrigated. 12 pots from total 24 pots were inoculated with 2.5 gm/ kg soil wheat bran preparation from GV and GD, respectively, one week early before sowing. Each pot from1 to 24 was cultivated with 10 seeds from the host plant Cicer arietinum and was treated by the following treatments Pot1: vitavax 2: Clove oil 3: mint oil 4: copprus KZ 5: starner 6: GV 7: GD 8: vitavax + clove oil 9: vitavax + mint oil 10: vitavax + copprusKZ 11: vitavax + starner 12: vitavax + GV 13: vitavax + GD 14: clove oil + mint oil 15: copprus KZ + starner 16: GV + GD 17: clove oil + GV 18: clove oil + GD 19: mint oil + GV 20: mint oil + GD 21: copprus KZ + GV 22: copprus KZ + GD 23: starner + GV 24: starner + GD

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Materials and methods

5- Effect of the different treatments on chlorophyll content in leaves of Cicer arietinum plants

Chlorophyll content in Cicer arietinum plants leaves after 45 days from sowing was determined according to Moran and Porath (1980). A known area (two disks, each 1 cm²) from the fourth leaf from the top was taken and extracted by 5 ml of N,N- dimethylformamide, in dark bottles and then the intensity of the colour was measured at λ = 647 and 664 nm wave length using spectrophotometer (Corning Scientific Instruments, Model 410).

Chlorophyll content was calculated using the following equations (Moran and Porath, 1980).

Chl. a = 12.46 A664 – 2.49 A647 Mg/ ml.

Chl. b = -5.6 A664 + 23.26 A647 Mg/ ml.

Total chl. = 7.04 A664 + 20.27 A647 Mg/ ml.

A664 is the absorption at λ = 664 nm.

A647 is the absorption at λ = 647 nm.

Then chlorophyll was calculated as assigned to the area of the leaf.

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Materials and methods

6- Determination of peroxidase and polyphenol oxidase activity in the treated Cicer arietinum plants in vitro.

6.a- Preparation of crude enzyme

Cicer arietinum leaves for each treatment were collected and prepared for the measurement of peroxidase and polyphenol oxidase enzyme activity. The collected leaves were separately homogenizes in a mortar with 0.1 M sodium phosphate buffer at pH 7.1 at the rate of 2 ml/ gm fresh weight leaves for 1 minute. Then these triturated tissues were filtered through four layers of cheesecloth and the filtrate was centrifuged at 3000 rpm for 20 minutes. The clear supernatant was collected and considered as a crude extract for enzymes assay.

6.b- Enzymes assay

Peroxidase and polyphenol oxidase enzymes were expressed as units in mg protein, where 1 unit is defined as the amount of enzymes converting one mole of substrate to product during 1 minute.

6.c - Peroxidase assay

According to Zapata et al., (1992) peroxidase enzyme activity was spectrophotometrically determined by measuring the oxidation of galic acid in the presence of H2O2 at λ = 470 nm. The sample cuvette contained 0.5 ml. of 0.1 M sodium phosphate buffer (pH 5.8), 0.3 ml of 7.2 mM galic acid,

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Materials and methods

and 0.1 ml. of 11.8 mM H2O2 and 0.3 ml crude enzyme and distilled water to a final volume of 3 ml. The changes of observation were recorded at λ = 470 nm. The enzyme activity was calculated according to the following equation:

Enzyme activity = K x ( Δ A/ min.).

Where: K (extension coefficient) is 26.6 mM / cm at λ = 470 nm for galic acid.

Δ A / min is the change in absorbency per one minute.

6.d - Polyphenol oxidase assay

To determine polyphenol oxidase enzyme activity the reaction .M. catechol and 1 ml ³־mixture contained 2 ml. enzyme extract, 1 ml. of 10 of 0.2 M. sodium phosphate buffer (at pH = 7) then the reaction mixture was brought to a final volume of 6 ml. with distilled water (Ismail et al., 1995). The activity of polyphenol oxidase was expressed according to the following equation:

Enzyme activity = K x (Δ A/ min.).

Where K (extension coefficient) is 0.272 m M/ cm at λ = 490 nm for catechol.

72T - Effect of gamma irradiation:

- 49 -

Materials and methods

All irradiation processes were carried out at the National Center for Radiation Research and Technology (NCRRT).

The used irradiation facility was Co- 60 Gamma Chamber 4000- A India. The source gave average dose rate 3.696 kGy/ hr at the time of experiments.

7. a- Effect of gamma irradiation on the antagonism between Gliocladium virens and Gliocladium deliquescence against S. rolfsii in vitro on the control of root rot disease.

Petri dish contain 15 ml PDA medium its half was inoculated with 0.5 cm of the pathogenic fungus and the second half was inoculated with G. virens and/ or G. deliquescence irradiated by gamma irradiation at doses 0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 kGy, respectively. Petri dishes were incubated at 28º C. The percentage of inhibition (I %) was calculated.

7. b- Effect of gamma irradiation of chickpea seed germination on control of root rot disease caused by S. rolfsii.

This method was carried out according to Kumar and Singh (1996). Plastic pots each containing 1 kg of soil cultivated with 10 of irradiated chickpea seeds that subjected separately to gamma irradiation at doses 5, 10, 15, 20, 25 and 30 Gy. The control was prepared by the same way with unirradiated seeds. Three replicates were used for each dose to study the effect of seed gamma irradiation on control of root rot disease.

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Materials and methods

7. c- Effect of different doses of gamma irradiation on the antagonistic action of Gliocladium virens and Gliocladium deliquescens against S. rolfsii in vivo.

In the experiments each treatment were replicated 3 times. Each pot contains 1 kg of soil. Each pot was inoculated with 2.5 gm/ kg soil wheat bran preparation from pathogenic and gamma irradiated GV and/ or GD antagonistic fungi at doses 0.25, 0.5, 1.0, 1.5 and 2.0 kGy one week early before sowing and irrigated. Each pot cultivated with 10 seeds from the host plant Cicer arietinum. The experiments were conducted together with control.

7. d- Effect of gamma irradiation on morphological characteristics, internal anatomical structure and pathogenecity of S. rolfsii.

The fungal strain S. rolfsii was grown on PDA medium for three days and subjected to gamma irradiation at doses (0.25, .05, 1.0. 1.5, 2.0, 2.5, 3.0 and 5.0 kGy). The effect of different doses of gamma irradiation on the internal anatomical structure of sclerotia was studied compared by unirradiated control. Artificial infection was made for Cicer arietinum in vitro to test the ability of gamma irradiation to inhibit or stimulate the pathogenecity of the fungus to cause root rot disease compared by unirradiated (control). Also testing the ability of irradiated isolates on causing pathogenecity to Cicer arietinum in vivo (in green house).

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Materials and methods

7. e- Electrophoretic analysis of unirradiated and gamma irradiated Sclerotium rolfsii total potein by SDS – PAGE.

Polyacrylamide gel electrophoresis (PAGE) was used to determine quantitative and qualitative changes that occur in the soluble proteins of gamma irradiated and unirradiated Sclerotium rolfsii isolates where, S. rolfsii was grown on PDA medium for three days and subjected to gamma irradiation at doses (0.25, .05, 1.0. 1.5, 2.0, 2.5 and 3.0 kGy). i. Extraction of S. rolfsii protein:

Protein was extracted from S. rolfsii (Laemmli, 1970) which grown on liquid PDA media. After that Sclerotium mycelial growth was harvested from the culture media, suspended in 100 ml distilled water. 10 ml 0.2 M NaOH was incubated for 5 min at room temperature, pelleted, resuspended in 50 ml SDS sample buffer ( 0.06 M Tris HCl pt ) = 6.8, 5 % glycerol, 2 % SDS, 4 % Bromoptoethanol, 0.0025 % bromophenol blue). Then centrifuged at 10.000 rpm for 20 min and supernatants containing water soluble protein fraction were transferred to clean tubes and stored at 20° C. Protein content of supernatant was estimated according to the method of ( Bradford, 1976). ii. Electrophoresis stock solutions:

The stock solutions used for protein electrophoresis were as follow:

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Materials and methods

A) Acrylamide stock solution (kept in dark at 4° C):

Acrylamide 29.2 g

Bis acrylamide 0.8 g

Deionized distilled water up to 100 ml

Any insoluble materials were removed by filtration through whatman filter paper No .1

B) Sodium dodecyl sulphate (10 % W/ V SDS).

Sodium dodecyl sulphate (5g) was dissolved in 25 ml deionized distilled water.

C) Ammonium persulphate solution (1.5 % W/ V APS ).

Ammonium persulphate (0.15 g) was dissolved in 10 ml deionized distilled water and kept at 4° C. The solution is unstable and must be made just before use.

D) Buffers: a) Separating gel buffer (1.5 ml Tris HCl, pH 8.8 kept in dark at 4° C). 1.17 g of Tris base was dissolved in 50 ml deionized distilled water and adjusted to pH 8.8 using concentrated HCl. The final volume was made up to 100 ml.

b) Stacking gel buffer (0.5 Tris HCl, pH 6.8 kept in dark at 48 hr). 6.05 g of Tris base was dissolved in 50 ml deionized distilled water and adjusted to pH 6.8 using concentrated HCl. The final volume was made up to 100 ml.

- 53 -

Materials and methods

c) Electrophoresis buffer (pH = 8.3- 8.5).

The tank buffer consists of 3 g Tris base, 14.4 g glycine and 1 g sodium dodecyl sulphate dissolved in 1000 ml deionized distilled water. iii. Separating and stacking gels preparations :

Vertical slab (18 x 16) gel electrophoresis apparatus Hoefer SE 600 series was used. All glass plates were washed with deionized distilled water then surface sterilized with ethanol spacers of 1.5 mm were used. Preparation of gel was made as described by (Laemmli, 1970). 11 % SDS – PAGE is prepared as follow:

. Separating gel

Acrylamide stock solution 30 % 11 ml

1.5 ml Tris HCl, pH 8.8 7.5 ml

10 % (W/ V) SDS 0.3 ml

1.5 % (W/ V) APS 1.5 ml

Deionized distilled water 9.7 ml

TEMED 15 Ml

Separations gel solutions was instantly swirled, then poured simultaneousiy to a height of 1.5 cm below the bottom of the comb and left to polymerize for at least 30 min. Separating gels were overlaid with lam of water which removed before the stacking gels solution was poured.

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Materials and methods

. Stacking gel

Acrylamide stock solution 30 % 3.5 ml

1.5 ml Tris HCl, pH 8.8 7.5 ml

10 % (W/ V) SDS 0.3 ml

1.5 % (W/ V) APS 1.5 ml

Deionized distilled water 17.8 ml

TEMED 30 Ml

Stacking gels solutions was quickly poured over the separating gels and combs were used. Gels were used to polymerize for 30 min before running. iv. Samples preparation

Sodium dodecyl sulphate (SDS) was added to the sample at a rate 4 mg SDS / 1 mg protein, mixed with 50 Ml 2 mercaptoethanol then boiled at 100° C in water bath for 3- 5 min.5) Loading of the samples 20 Ml of the crude protein solution was applied to the wells of the stacking gel. The sample covered with electrode buffer few drops of bromophenol blue (4 mg / 100 ml deionized distilled water) were added to the electrode buffer (tracking dye) Molecular weight protein markers (SIGMA) were used as a stander. v. Gel running

Electrophoresis was performed in avertical slab mold (Hoefer Scientific Instruments, Sanfrancisco, CA USA, Model LKB 2001,

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Materials and methods

measuring 16 x 18 x 0.15 am 4 L of the running buffer was poured in to the running tank and pre cooled by flooding cold water (4° C) through cooling tubes. Hr run buffer (800 ml) was added to the upper tank just before running.

Electrophoresis was carried out at 30 millampere (mA) at 10° C or 3 hours till the samples reach 0.5 cm from the bottom of gel. Gels were removed from the apparatus and placed in plastic tank (contain 50 % ethanol and 10 % acetic acid freshly prepared) then stained. vi. Staining the gel

Staining solution used in this study was prepared by dissolving 0.2 g Coomassie Brilliant blue (CBB) R- 250 in 30 ml methanol and 10 ml acetic acid, then completed to 100 ml with distilled water, then destaining solution was prepared by mixing 10 ml acetic acid, 20 ml methanol then completed to 100 ml with distilled water after stainig processes, gels were photographed.

7.f- Protein assay:

The protein concentrations in different enzyme preparations were measured according to Bradford (1976) as follows:

Commassie Brilliant blue G.250 dye reagent 100 mg were dissolved in 50 ml of 95 % ethanol, 100 ml of 85 % phosphoric acid, the whole mixture was diluted in one liter with distilled water and filtered. From each supernatant, 0.1 ml added to 5 ml of the dye reagent, shacked for 5 minutes,

- 56 -

Materials and methods

absorbance of mixtures was measured at λ = 595 nm. Records of absorbance indicated total protein concentration in the tested filtrate, after comparing its absorbance with standard protein.

Plotting a standard curve by using absorbance of known concentrations of pure bovine serum albumin solution as a standard protein and stained with the same dye reagent.

7. g- Extraction and purification of polygalacturonase:

The steps that carried out to characterize and purify polygalacturonase from unirradiated and gamma irradiated isolates of Sclerotium rolfsii were as follows: a) Production of crude extract containing the extracellular polygalacturonase:

Unirradiated and gamma irradiated Sclerotium rolfsii isolates were grown in the modified medium (Bateman et al., 1969) containing the -3 following ingredients (g 100 ml ) sodium polypectate, 1; NaNO3,1;

MgSO4. 7H2O, 0.181; K2HPO4, 0.697; KCl, 0.149; Thiamine- HCl, 0.001; -2 and 2 ml of stock solution containing (mg ml ): ZnSO4. 7H2O, 2.85;

MnSO4. 5H2O, 3.1; FeCl3. 2H2O, 8.65. The initial PH of the medium was adjusted to 6.8 and autoclaved. The sterilized flasks were inoculated 6 mm mycelium discs cut from the margins of 7 days old colonies and incubated at 28° C statically.

- 57 -

Materials and methods

b) Assay of exo- polygalacturonase extracted from unirradiated and gamma irradiated Sclerotium rolfsii isolates:

Polygalacturonase of unirradiated and gamma irradiated Sclerotium rolfsii isolates was assayed by following the release of reducing groups from the substrate according to Beg et al., (2000).

Reaction mixture contained 0.5 ml of 1% (w/ v) sodium polypectate as substrate in 1ml 0.1 M acetate buffer (pH 4.8) and 1 ml of enzyme preparation. The reaction was carried out at 30º C for 30 min. Control values was carried out by stopping the reaction mixture by boiling the enzyme for 10 min. 2.5 ml DNS (Di nitro salicylic acid) reagent was added and was boiled for 5 min. After that, the solutions were mixed with 10 ml distilled water. Finally, the OD 540 nm of the mixed solutions was measured and the corresponding sodium polypectate content was determined from the standard.

The activity of enzyme was defined as: The amount of enzyme required to release one Mmol min-1 as the standard glucose under the given assay conditions. c) Purification of polygalacturonase produced by unirradiated and gamma irradiated Sclerotium rolfsii isolates.

Cell- free culture liquid from unirradiated and gamma irradiated Sclerotium rolfsii isolates were dialyzed overnight at 4° C against 0.05 M sodium acetate buffer (pH 4.5). Enzyme protein was precipitated by ethanol after standing overnight at 4° C contained most of the enzyme activity. This

- 58 -

Materials and methods

precipitate was collected by centrifugation and re suspended in 0.05 M acetate buffer (pH 4.5). The salts remaining in the enzyme protein solution were removed by dialysis against the Tris HCl (50 mM, pH = 8) to be partially purified to the next step.

The dialyzed ammonium sulphate solution was applied to gel filtration through Sephedex G- 75 as described by Foldin (1961). Eluates (5 ml fraction) were collected and assayed for its protein content using Spectrophotometer 240. The purity of the enzyme was confirmed by sodium dodecyl sulphate, (SDS- PAGE) according to Laemmli (1970).

8- Statistical analysis:

All the previous results were statistically analyzed according to Finney, (1952). Analysis of variance and LSD (Least significant difference) was used if differences seemed to be found (Snedecor, 1965). Correlation for the control mortality was made by using Abbott`s formula.

- 59 - Results

Results

I- In vitro experiments: 1- Mode of infection of Cicer arietinum in vitro (artificial infection to Cicer arietinum as a host tissue). In the present investigation Cicer arietinum was used as a host plant to be infected with Sclerotium rolfsii in vitro. The growing stage of Sclerotium rolfsii and the steps in the formation of sclerotia was observed as shown in photos (1, 2, 3 and 4).

Stages of sclerotia formation:

Photo (1) Photo (2)

Photo (3) Photo (4)

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Photo (1): shows Sclerotium rolfsii on PDA medium (Arrows indicate hyphae of S. rolfsii, white undeveloped sclerotia and brown mature sclerotia). (X 40)

Photo (2): Microscopic examination of Sclerotium rolfsii after one day from incubation on PDA medium in vitro.

Photo (3): Microscopic examination of Sclerotium rolfsii after two days from incubation on PDA medium in vitro.

Photo (4): Transfer section in sclerotia of Sclerotium rolfsii (Arrow indicate microsclerotiole surround with pseudo paranchymatic cells).

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Mode of infection of Sclerotium rolfsii to Cicer arietinum:

1.a- Infection of seed (seed rot)

Photo (5) Photo (6)

Photo (7) Photo (8)

Photo (9)

Photo (10) Photo (11)

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Photo (5): Seeds of Cicer arietinum (non infected control). (Arrow indicate non infected seeds) (X 40).

Photo (6): Transfer section of seeds of Cicer arietinum shows cotyledons of Cicer arieinum rich in starch grains 86 % of its constituents.

Photo (7): Seeds of Cicer arietinum infected with Sclerotium rolfsii, after one month from infection. All seeds show external white rot (arrow indicate infected seeds).

Photo (8): Transfer section of one month age infected seed of Cicer arietinum which shows black infection cushion (dome like structure) emerging from sclerotia of Sclerotium rolfsii and it is found between starch grains of Cicer arietinum (arrow indicate black infection cushion of S. rolfsii that appear after one month).

Photo (9): Transfer section of infected seed of Cicer arietinum after two months age from infection with S. rolfsii. shows many large black dome like infection cushion, the cells of the cotyledons and starch grain are wholly disintegrated (arrow indicate large black dome like infection cushion appear after two months) (X 40).

Photo (10): Infected seed of Cicer arietinum with white rot after three months of infection with S. rolfsii. It shows that the seed is white, wrinkled and its internal content is converted into brown liquid. (arrow indicate infected seed after three months)

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Photo (11): T.S. of seed of Cicer arietinum infected with white rot after three months from infection. The photo shows that the fungus digest all the seed content (the cells and starch grains) and the fungus convert the digested content into black dome like sclerotia or cushion to remain in the soil. After that all the internal component has changed into black sclerotia (arrow indicate black dome like sclerotia after three months).

1. b- Infection of stem

Sclerotium rolfsii infect seedlings at the transition zone (the nearest zone to the soil). S. rolfsii is a soil born pathogenic fungus. It disintegrates the transition zone of Cicer arietinum and causes pre and post emergence damping off. It also causes white rot on the transition zone region as evidenced from photo (13).

- 64 - Results

Photo (12) Photo (13)

Photo (14) Photo (15)

Photo (16) Photo (17) (X-40)

- 65 - Results

Photo (12): 10 days old seedling of Cicer arietinum (arrow indicate healthy plant).

Photo (13): 10 days old seedling of Cicer arietinum infected with S. rolfsii (arrow indicate infected transition zone).

Photo (14): T.S. of transition zone of infected Cicer arietinum. Infected region is occupied by microsclerotiole (that get out from sclerotia) (arrows indicate infected transition zone occupied by microsclerotiole) (X- 40).

Photo (15): T.S. of infected transation zone showing infection of cortex by microsclerotia. Small sclerotia or microsclerotiole are found between cortical cells (arrow indicate microsclerotiole between cortical cells).

Photo (16): T.S. of infected transition zone showing infected xylem vessels. Xylem vessels are blocked by the fungus. Microsclerotiole occupied and colonized xylem vessels (arrows indicate microsclerotiole in xylem vessles).

Photo (17): T.S. of the infected transition zone of Cicer arietinum seedlings showing that all the cells are completely digested by the fungus. Microsclerotioles now change to large black microsclerotia that remain in the soil for next infection (arrow indicate microsclerotioles of the fungus in transition zone).

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1. c- Infection of the root

Sclerotium rolfsii invades root of Cicer arietinum from the upper region. It causes white rot and maceration to the root as evidenced from photo (20). The lower region of the root has discolourization or browning and at last undergo rotting.

Photo (18) Photo (19) (X- 40)

Photo (20) Photo (21) (X- 40)

Photo (22) Photo (23) (X- 40)

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Photo (18): White rot on the root of Cicer arietinum caused by S. rolfsii after one week from infection (arrow indicate infected root) (X- 40).

Photo (19): Longitudinal section of the infected root of Cicer arietinum colonized by infected hyphae of Sclerotium rolfsii. Microsclerotiole also appears on the piliferous layer of the root. The cells of the root show discolouization or browning (arrow indicate microsclerotiole in the root). Photo (20): L.S of infected root of Cicer arietinum showing rod shaped sclerotia at cortex region. Rod shaped sclerotia extend along the root and secret enzymes that digest the cells (arrow indicate rod shape sclerotia).

Photo (21): L.S of infected root of Cicer arietinum showing xylem vessels that are occupied by the fungal elements. The photo also shows the disintegrated cells of the root (rotted zone of the root) (arrow indicates fungal elements in xylem vessels) (X 40).

Photo (22): L.S of infected root of Cicer arietinum showing all the xylem vessels of the host are colonized by the fungus. (Infection after 7 days) (Arrow indicates blocking xylem vessels).

Photo (23): Vertical section of rotted root of Cicer arietinum after 7 days from infection. The photo shows that all the cells are digested, lyzed, disintegrated, and destroyed by the fungus. At the end of infection the fungus form numerous large black dome shaped sclerotia and survive in the soil for a long time till the next infection (arrow indicate large black dome shaped sclerotia).

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2- Effect of tested treatments on the growth of pathogenic and antagonistic fungi. The antifungal activity of the tested fungicides (vitavax and monceren- T), oils (clove and mint oils) and inducers (starner and copprus KZ) was evaluated against pathogenic fungus (Sclerotium rolfsii) and the antagonistic fungi (Gliocladium deliquescens and Gliocladium virens) in vitro.

2. a- Effect of the tested fungicides on the growth of Sclerotium rolfsii.

Data in table (1) and fig. (1) showed that the linear growth of S. rolfsii was completely inhibited at 10 ppm of vitavax, while at 0.01 ppm the lowest effect against the same fungus was obtained ( I % = 36.33 ). Vitavax was

effective against the tested fungus Sclerotium rolfsii. IC R50R (concentration giving 50 % inhibition) for Sclerotium rolfsii was 0.9 ppm. Monceren- T had

a toxic effect on the growth of pathogenic fungus (Its IC R50R was 1.2 ppm).

- 69 - Results

Table (1): Effect of fungicides (vitavax and monceren- T) on the linear growth of the pathogenic fungus Sclerotium rolfsii on PDA medium in vitro after three days.

Fungicides Inhibition percentage (I %) Conc.

(ppm) The pathogenic Vitavax Monceren -T fungus 0.01 36.33 30.8 0.1 42.44 38.32 1 49.00 55.55 Sclerotium rolfsii 5 63.33 59.11 10 100 100 25 100 100 50 100 100

ICR50 0.9 1.2 LSD 0.40 0.42

*** = Highly significant at (p ≤ 0.01)

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vitavax

monceren- T

100

20 Inhibition percentage( I%) 0 0.01 0.1 1 5 10 25 50

Conc ( ppm )

Fig. (1): Effect of vitavax and monceren- T on the linear growth of Sclerotium rolfsii in vitro.

100

vitavax

moncerenT

50 Percentage of Inhibition (I%) (I%) Inhibition of Percentage 0 0 2 4 6 8 10 12 ppm

Fig (2): Effect of fungicides on Sclerotium rolfsii represented as regression lines

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2. b- Effect of fungicides on the growth of the antagonistic fungi:

In vitro sensitivity tests of the tested fungicides on the growth of Gliocladium deliquescens and Gliocladium virens are illustrated in tables (2, 3) and figs. (3, 4).

Vitavax gave moderate toxic effect, Its ICR50R for Gliocladium

deliquescens was 30.3 ppm and ICR50R for Gliocladium virens was 25.7 ppm

Monceren- T was the most effective fungicide against all the

antagonistic fungi (ICR50R for Gliocldium deliquescens was 8.23 ppm and ICR50R for Gliocladium virens was 5.2 ppm)

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Table (2): Effect of Vitavax and Monceren -T on the growth of antagonistic fungus Gliocladium deliquescens on PDA medium in vitro (3 days old).

Fungicides Inhibition percentage (I %) Conc Antagonistic (pmm) fungus Vitavax Monceren -T 0.01 0.00 0.00 0.1 0.00 0.00 1 0.00 0.00 Gliocladium 5 6.32 38.89 deliquescens 10 11.13 50.00 25 28.19 75.35 50 44.32 100

ICR50 30.3 8.23 LSD 2.20 1.79 ** = Significant at (p ≤ 0.05) *** = Highly significant at (p ≤ 0.01)

vitavax monceren T

100 80 60 Inhibition 40 percentage (I%) 20 0 0.01 0.1 1 5 10 25 50

Conc. (ppm)

Fig. (3): Effect of vitavax and monceren –T on the linear growth of Gliocladium deliquescens.

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Table (3): Effect of Vitavax and Monceren -T on the linear growth of antagonistic fungus Gliocladium virens on PDA medium in vitro.

Fungicides Inhibition percentage (I %) Conc Antagonistic (pmm) fungus Vitavax Monceren -T 0.01 0.00 0.00 0.1 6.68 10.12 1 11.25 16.67 Gliocladium 5 22.23 44.45 virens 10 30.27 57.78 25 49.17 80.98 50 73.45 100 IC50 25.7 5.2 LSD 1.7 4.21 ** = Significant at (p ≤ 0.05)

vitavax monceren T

100 80

(I%) 60 40

Inhibition percentage Inhibition percentage 20 0 0.01 0.1 1 5 10 25 50

Conc. (ppm)

Fig. (4): Effect of vitavax and monceren –T on the linear growth of Gliocladium virens.

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2. c- Effect of oils (clove and mint oils) on the linear growth of Sclerotium rolfsii.

Data in table (4) and fig. (4) show that the tested oils have a toxic effect on the growth of pathogenic fungus in vitro. Toxicity of the oils to the pathogenic fungus increased by increasing the concentration of the oil.

Clove oil was more toxic to the pathogenic fungus (IC R50R = 5.4 ppm)

than mint oil (ICR50R = 6.1 ppm).

Table (4): Effect of oils on the linear growth of Sclerotium rolfsii on PDA medium in vitro after three days.

Oils Inhibition percentage (I %) Conc. The pathogenic (ppm) Clove oil Mint oil fungus 0.01 16.66 21.15 0.1 33.63 29.68 1 39.89 34.52 Sclerotium rolfsii 5 51.36 53.25 10 65.71 59.42 25 73.83 81.60 50 100 100 IC50 5.4 6.1 LSD 10.7 9.06

*** = Highly significant at (p ≤ 0.01)

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Clove oil Mint oil

100

20 Inhibition percentage(I %) Inhibition percentage(I 0 0.01 0.1 1 5 10 25 50

Conc (ppm)

Fig. (5): Effect of clove and mint oils on the linear growth of Sclerotium rolfsii in vitro.

100

Clo

50 M Inhibition percentage ( % I ) 0 0 2 4 6 8 10 12 ppm Clove oil (clo) Mint oil ( M )

Fig. (6): Effect of oils on Sclerotium rolfsii represented as regression lines.

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2. d- Effect of oils on the growth of Gliocladium deliquescens and Gliocladium virens in vitro.

The experiments showed that both of clove and mint oils have no antifungal effect on the growth of G. deliquescens and G. virens.

2. e- Effect of inducers (copprus KZ and starner) on the growth of S. rolfsii, G. deliquescens and G. virens in vitro.

The experiments showed that both of copprus KZ and starner have no antifungal effect on the growth of S. rolfsii, G. deliquescens and G. virens.

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2. f- Inhibitory percentage of Sclerotium rolfsii under the effect of antagonistic fungi (T. hamatum, G. deliquescens and G. virens) on PDA medium in vitro (Three days old).

Photo (24) Photo (25)

Photo (26)

Photo (24): Antagonistic effect of Gliocladium deliquescens against S. rolfsii in vitro after 3 days.

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Photo (25): Antagonistic effect of Gliocladium virens on S. rolfsii in vitro after 3 days.

Photo (26): Antagonistic effect of Trichoderma hamatum against S. rolfsii in vitro after 3 days.

Table (5): Inhibitory percentage of Sclerotium rolfsii under the effect of antagonistic fungi in vitro.

Pathogenic Inhibition percentage fungus (I %)

Antagonistic Sclerotium rolfsii fungi T. hamatum 18.89 G. deliquescens 61.11 G. virens 67.78 LSD 3.27 ** = Significant at (p ≤ 0.05)

40 (I%)

20 Inhibition percentage percentage Inhibition 0 T. hamatum G. viren G.deliquescens against against against S.rolfsii S. rolfsii S. rolfsii

Fig. (7): Inhibitory percentage of Sclerotium rolfsii under the effect of antagonistic fungi in vitro.

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2. g- Effect of culture filtrate of Gliocladium virens and Gliocladium deliquescens on the growth of Sclerotium rolfsii.

Table (6) and fig. (8) shows that culture filtrates of G. virens and G. deliquescens vary in their effect on the linear growth of S. rolfsii. The culture filtrate of G. virens is more inhibitory than the filtrate of G. deliquescens.

The inhibitory effect of the filtrate differ during the incubation periods it records 7.8 % after one week, 61.8 % after two weeks and 89.8%, after three weeks for G. virens and 21.5 % after one week , 48.7 % after two weeks and 62 % after three weeks for G. deliquesces.

Table (6): Effect of G. virens and G. deliquescens culture filtrates on the percentage of inhibition of S. rolfsii after different incubation periods.

Antagonistic fungi Time (week) Gliocladium Gliocladium deliquescens virens 1 7.8 21.5 2 61.8 48.7 3 89.8 62 LSD 1.09 1.37

** = significant at (p ≤ 0.05)

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100

Inhibition percentage (I%) 0 3 weeks 2 weeks Control 1 week

G. virens G. deliquescens

1 week 2 weeks Treatments 3 weeks

Fig. (8): Effect of culture filtrates of Gliocladium virens and G. deliquescens on inhibition percentage of Sclerotium rolfsii after different incubation periods.

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3- Phytotoxicity of the tested compounds on Cicer ariertinum seedlings in vitro.

Cont. 50 25 10 5 1 0.1 (ppm) Cont. 50 25 10 5 1 0.1(ppm) Photo (27) Photo (28)

Cont. 50 25 10 5 1 0.1(ppm) Contr. 50 25 10 5 1 0.1(ppm)

Photo (29) Photo (30)

Cont. 50 25 10 5 1 0.1 (ppm)

Photo (31)

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Photo (27): Effect of vitavax on Cicer arietinum seedlings.

Photo (28): Effect of mint oil on Ciecr arietinum seedlings.

Photo (29): Effect of clove oil on Cicer arietinum seedlings.

Photo (30): Effect of starner on Cicer arietinum seedlings.

Photo (31): Effect of copprus KZ on Cicer arietinum seedlings.

3. a- Fungicide

Results of tables (7, 8) reveal that the growth of Cicer arietinum seedlings is reduced by vitavax (IC50 of shoot is 40 ppm). On the other hand, root system is sensitive to vitavax and IC50 of root is 17.5 ppm. . 3. b- Oils

Tables (7, 8) illustrate that mint oil gave the highest toxic effect (ICR50R of shoot is 3 ppm while IC50 of root system is 5.5 ppm) followed by clove oil

(IC R50 Rof shoot is 16 ppm and IC50 of root is 13 ppm).

Root system is very sensitive to clove oil than mint oil.

3. c- Inducers Both of starner and copprus KZ have stimulatory effect on shoot system of Cicer arietinum tables (7, 8). Starner was the most active inducer followed by copprus KZ.

Starner is more active on root system of Cicer arietinum than copprus KZ.

- 83 - Results

Table (7): Phytotoxicity of the tested compounds on shoot system of Cicer arietinum 14 days old seedlings (Inhibition percentage of shoot length).

Treatments Fungicides Oils Inducers

Mint Clove Copprus Vitavax Starner Conc (ppm) oil oil KZ 0.1 0.00 29.41 4.41 -23.53 -29.41 1 8.52 32.35 5.89 -29.41 -52.94 5 11.88 58.82 35.29 -35.29 -58.82 Shoot 10 17.64 100 57.94 -41.18 -64.7 system 25 29.41 100 64.70 -64.70 -70.59 50 35.29 100 82.35 -88.24 -76.47

ICR50 40 3 16 0.00 0.00

LSD 0.12 0.98 0.12 0.45 0.49

** = significant at (p ≤ 0.05) *** = Highly significant at (p ≤ 0.01)

- 84 - Results

40 35 30 25

values 20

50 15 IC 10 5 0 vitavax clove oil mint oil starner copprus kz

Treatments

Fig. (9): Effect of tested compounds (ICR50R) on shoot system of chickpea seedlings.

- 85 - Results

Table (8): Phytotoxicity of the tested compounds on root system of Cicer arietinum 14 days old seedlings (Inhibition percentage of root length).

Treatments Fungicides Oils Inducers

Mint Clove Copprus Conc (ppm) Vitavax Starner oil oil KZ 0.1 0.00 0.00 20 0.00 0.00 1 0.00 46.67 36.67 0.00 2.93 5 46.33 60.32 50 0.00 0.00 Root 10 53.33 100 60 0.00 0.00 system 25 33.33 100 80 0.00 6.67 50 100 100 86.67 0.00 13.33

ICR50 17.5 5.5 13 0.00 0.00

LSD 0.17 0.35 0.90 - 0.17

** = significant at (p ≤ 0.05) *** = Highly significant at (p ≤ 0.001)

- 86 - Results

18 16 14 12 10 values 8 50

IC 6 4 2 0 vitavax clove oil mint oil starner copprus kz

Treatments

Fig. (10): Effect of tested compounds (ICR50R) on root system of chickpea seedlings.

4- Joint toxic effect of the tested compounds against pathogenic fungus.

The fungal toxic effect of the combination between the tested compounds (fungicide, oils and inducers) were tested according to the results obtained from the previously mentioned testes. According to this results,

table (9) and fig. (11) show that ICR25R values from each of the tested compounds was used in the examined mixtures.

- 87 - Results

4. a- Joint toxic effect against Sclerotium rolfsii. Different joint toxic effects of compounds in pairs on the tested fungus were performed table (9). Results show that vitavax + clove oil gave the highest inhibition percentage, 57.47 % with C.T.F. = 14.94 that reveal an additive effect. On the other hand vitavax + starner gave the lowest inhibition percentage, 22.87 % with C.T.F = -54.25 that reveal an antagonistic effect .

Table (9): Percentage of inhibition (I%) and Co-Toxicity Factor of treatments against pathogenic fungus Sclerotium rolfsii.

Co-Toxicity Compounds I % Effect factor Vitavax + mint 54.02 8.04 Additive

Vitavax + clove 57.47 14.94 Additive

Clove + mint 50.57 1.14 Additive

Vitavax +starner 22.87 -54.25 Antagonistic

Vitavax+ copprus 31.49 -37.01 Antagonistic Mint + starner 42.52 -14.94 Additive

Mint + Copprus-kz 51.72 3.44 Additive

LSD 0.98

** = Significant at (p ≤ 0.05)

- 88 - Results

0 Inhibition percentage (I%) percentage Inhibition

clove + mint vitavax+ mint vitavax+ clove mint+ starner mint+ copprus vitavax+ starner vitavax+ copprus

Treatments

Fig. (11): Effect of joint toxic effect on the linear growth of Sclerotium rolfsii.

4. b- Joint toxic effect of the tested compounds with bioagents against pathogenic fungus

Different joint toxic effect of compounds with bioagents in pairs on the tested fungus is shown in table (10) and fig. (12). Results show that copprus KZ + GV gave the highest inhibition percentage, 71.26 % with C.T.F. = 42.52 that reveal synergistic effect. On the other hand vitavax + GV gave the lowest inhibition percentage, 21.8 % with C.T.F = -54.25 that reveal an antagonistic effect. While most combinations gave additive effect.

- 89 - Results

Table (10): Percentage of inhibition (I %) and Co-Toxicity Factor of treatments with bioagents against pathogenic fungus Sclerotium rolfsii.

Co-Toxicity Compounds I % Effect factor

Vitavax+ GV 21.8 -56.4 Antagonistic

Vitavax + GD 34.48 -31.04 Antagonistic

Clove oil+ GD 47.12 -5.76 Additive

Mint oil + GD 36.78 -26.44 Antagonistic

Clove oil + GV 57.47 14.94 Additive

Mint oil + GV 55.17 10.34 Additive

Copprus KZ +GV 71.26 42.52 Synergistic

Copprus KZ + GD 53.12 6.24 Additive

Starner + GV 66.67 33.34 Synergistic

Starner+ GD 51.72 3.44 Additive

LSD 0.30

** = significant at (p ≤ 0.05)

Gliocladium virens: GV Gliocladium deliquescens: GD

- 90 - Results

80 70 60 50 40 30 20

Inhibition percentage (i%) percentage Inhibition 10 0

Vitavax+vitavax+ GV GD Starner+Starner+ GV GD clove oil+ GD mint oilclove + GD oilMent + GV oil + GV Copprus-kz+GVCopprus-kz+GD

Treatments

Fig. (12): Effect of joint toxic effect with bioagents on the linear growth of Sclerotium rolfsii.

- 91 - Results

II. In vivo experiments:

1 .a- Effect of different antifungal treatments on survival plants (%):

Data in table (11) reveal that copprus KZ, starner, vitavax + copprus KZ, vitavax + starner, copprus KZ + starner gave the highest percentage of survival plants (80,70, 80, 100, 90 %) respectively compared by the infected control, on the other hand mint oil, clove oil + mint oil gave the lowest percentage of survival plants (30, 30 %), respectively, compared by the infected plants .

- 92 - Results

Table (11): Effect of different antifungal treatments and their combinations on survival plants of Cicer arietinum in vivo under green house conditions.

Infected plants (%) Survival plants Treatments Pre- Post- (%) emergence emergence Vitavax 10 10 80 Clove oil 30 30 40 Mint oil 70 ─ 30 Copprus KZ 10 10 80 Starner 20 10 70 GV 50 ─ 50 GD 30 10 60 Vitavax + clove oil 30 10 60 Vitavax + mint oil 30 10 60 Vitavax + copprus KZ 20 ─ 80 Vitavax + starner ─ ─ 100 Clove oil + mint oil 60 10 30 Copprus KZ+ starner ─ 10 90 Healthy control ─ ─ 100 Infected control 20 20 60 LDS 0.22 0.18 0.576 * = non significant at (p > 0.05) ** = significant at (p ≤ 0.05) *** = Highly significant at (p ≤ 0.01)

- 93 - Results

100

Survival plants (%) 20

GV GD

Vitavax mint oil starner clove oil copprus

clove + mint vitavax+ mint vitavax+ clove Healthy control infected control vitavax+ starner vitavax+ copprus copprus+ starner Treatments

Fig. (13): Effect of different antifungal treatments on survival plants of Cicer arietinum in vivo.

- 94 - Results

1. b- Effect of different antifungal treatments with bioagents on survival plants (%):

Data in table (12) show that vitavax + GD, copprus KZ + GD, starner + GV, starner + GD gave the highest percentage of survival plants (100, 100, 100 and 100 %) respectively compared by the infected control. on the other hand, mint + GV, mint oil + GD gave the lowest percentage of survival plants ( 20, 10 % ), respectively, compared by the infected plants .

Gliocladium virens: GV Gliocladium deliquescens: GD

- 95 - Results

Table (12): Effect of different antifungal treatments with bioagents on survival plants of Cicer arietinum in vivo under green house conditions.

Infected plants (%) Survival plants Treatments Pre- Post- (%) emergence emergence Vitavax + GV 10 ─ 90 Vitavax + GD ─ ─ 100 GV + GD ─ 10 90 Clove oil + GV 20 10 70 Clove oil + GD ─ 10 90 Mint oil+ GV 50 30 20 Mint oil+ GD 70 20 10 Copprus KZ + GV 20 10 70 Copprus KZ + GD ─ ─ 100 Starner + GV ─ ─ 100 Starner + GD ─ ─ 100 Healthy control ─ ─ 100 Infected control 20 20 60 LSD 0.13 0.45 0.29

** = significant at (p ≤ 0.05) *** = Highly significant at (p ≤ 0.01) Gliocladium virens: GV Gliocladium deliquescens: GD

- 96 - Results

100

20 Survival plants (%) 0

GV+ GD

Mint oil+GD Vitavax+ GVvitavax+ GD Starner + GVStarner+ GD Mint oil + GV Copprus+ GV clove oil + GVclove oil + GD Healthy control Infected control Copprus-kz+GD

Treatments

Fig. (14): Effect of different antifungal treatments with bioagents on survival plants of Cicer arietinum in vivo.

- 97 - Results

2. a- Effect of different antifungal treatments on growth parameters:

A) Plant height (cm)

Copprus KZ, starner, vitavax + copprus KZ, vitavax + starner, copprus KZ + starner gave the highest plant height (57.5, 55.5, 59.5, 58.4, 60.3 cm) respectively compared by the infected control (30 cm), while mint oil, vitavax + mint oil gave the lowest plant height (39, 37 cm), respectively, compared by the infected control (30 cm) table(13).

B) Fresh weight (g)

Data in table (13) show that copprus KZ, starner, vitavax + copprus KZ, vitavax + starner, copprus KZ + starner gave the highest fresh weight (2.62, 2.23, 2.96, 2.01, 2.94 g) respectively compared by the infected control (1.60 g), while mint oil, clove oil + mint oil gave the lowest fresh weight (1.12, 1.14 g), respectively, compared by the infected control (1.60 g) .

C) Dry weight (g)

Data in table (13) show that copprus KZ, starner, vitavax + copprusKZ, vitavax + starner, copprus KZ + starner gave the highest dry weight (0.54, 0.52, 0.62, 0.55, 0.61 g), respectively, compared by the infected control (0.294 g), while mint oil and clove oil + mint oil gave the lowest dry weight (0.19, 0.18 g) compared by the infected control (0.294 g).

- 98 - Results

Table (13): Effect of different antifungal treatments and their combinations on different growth parameters of Cicer arietinum in vivo.

Plant height Fresh weight Dry weight Treatment (cm) ( g ) ( g ) Vitavax 40 1.34 0.22 Clove oil 43 1.22 0.20 Mint oil 39 1.12 0.19 Copprus KZ 57.5 2.62 0.54 Starner 55.5 2.23 0.52 GV 44.3 1.66 0.33 GD 41.7 1.26 0.21 Vitavax + clove 42.5 1.27 0.24 Vitavax + mint 37 1.53 0.27 Vitavax + Copprus 59.5 2.96 0.62 Vitavax + starner 58.4 2.01 0.55 Clove+ mint 54 1.14 0.18 Copprus+ starner 60.3 2.94 0.61 Healthy control 55 2.3 0.52 Infected control 30 1.60 0.294 LSD 0.24 0.27 1.45 * = Non significant at (p > 0.05) ** = Significant at (p ≤ 0.05) Gliocladium virens: GV Gliocladium deliquescens: GD

- 99 - Results

60 50 40 30 20 Plant height (cm) height Plant 10 0

GV GD

Vitavax mint oil starner clove oil copprus

clove+ mint vitavax+ mint vitavax+ clove Healthy control infected control vitavax+ starner vitavax+ copprus copprus+ starner

Treatments

Fig. (15): Effect of different antifungal treatments and their combinations on plant height of Cicer arietinum in vivo.

- 100 - Results

2.5

1.5

Fresh weight (gm) weight Fresh 0.5

GV GD

Vitavax starner clove oil mint oil copprus clove + mint vitavax+ clovevitavax+ mint Healthy controlinfected control vitavax+ starner vitavax+ copprus copprus+ starner

Treatments

Fig. (16): Effect of different antifungal treatments and their combinations on fresh weight of Cicer arietinum in vivo.

- 101 - Results

0.7 0.6 0.5 0.4 0.3 0.2 Dry weight (gm) 0.1 0

GV GD

Vitavax mint oil starner clove oil copprus

clove + mint vitavax+ mint vitavax+ clove Healthy control infected control vitavax+ starner vitavax+ copprus copprus+ starner

Treatments

Fig. (17): Effect of different antifungal treatments and their combinations on fresh weight of Cicer arietinum in vivo.

- 102 - Results

2. b- Effect of different antifungal treatments with bioagents on growth parameters:

A) Plant height (cm)

Starner + GV, copprus KZ + GV, copprus KZ + GD and starner + GD gave the highest plant height (60.3,59.4, 58.8, 57.3 cm) respectively compared by the infected control (30 cm), while vitavax + GD, vitavax + GV and mint oil + GV gave the lowest plant height (38.3, 41, 41 cm), respectively, compared by the infected control (30 cm) table(14).

B) Fresh weight (gm)

Data in table (14) show that copprus KZ + GV, starner + GV, copprusKZ + GD and starner + GD gave the highest fresh weight (2.96, 2.8, 2.72, 2.02 g) respectively compared by the infected control (1.60 g), while vitavax + GD gave the lowest fresh weight (1.80 g) compared by the infected control (1.60 g) .

C) Dry weight (gm)

Data in table (14) show that copprus KZ + GV, copprus KZ + GD, starner + GV gave the highest dry weight (0.61, 0.57, 0.59 g), respectively, compared by the infected control (0.294 g), while vitavax + GD gave the lowest dry weight (0.23 g) compared by the infected control (0.294 g). Gliocladium virens: GV Gliocladium deliquescens: GD

- 103 - Results

Table (14): Effect of different antifungal treatments with bioagents on different growth parameters of Cicer arietinum in vivo .

Plant height Fresh weight Dry weight Treatment (cm) (g) (g)

Vitavax + GV 41 1.80 0.41 Vitavax + GD 38.3 1.37 0.23 GV + GD 53 1.64 0.31 Clove oil + GV 50.3 1.83 0.42 Clove oil + GD 42 1.98 0.49 Mint oil+ GV 41 1.91 0.46 Mint oil+ GD 44 1.79 0.37 Copprus KZ + GV 59.4 2.96 0.61 Copprus KZ + GD 58.8 2.72 0.57 Starner + GV 60.3 2.8 0.59 Starner + GD 57.3 2.02 0.51 Healthy control 55 2.3 0.52 Infected control 30 1.60 0.294 LSD 0.336 0.96 0.451

* = Non significant at (p > 0.05) ** = Significant at (p ≤ 0.05) Gliocladium virens: GV Gliocladium deliquescens: GD

- 104 - Results

Plant hieght (cm) (cm) hieght Plant 10

GV+ GD

Vitavax+ GVvitavax+ GD Mint oil+GD Starner + GVStarner+ GD Mint oil + GV Copprus+ GV clove oil + GVclove oil + GD Healthy control Infected control Copprus-kz+GD

Treatments

Fig. (18): Effect of different antifungal treatments with bioagents on plant height of Cicer arietinum in vivo.

- 105 - Results

2.5

1.5

Fresh weight (gm) weight Fresh 0.5

GV+ GD

Vitavax+ GVvitavax+ GD Mint oil+GD Starner + GVStarner+ GD Mint oil + GV Copprus+ GV clove oil + GVclove oil + GD Healthy control Infected control Copprus-kz+GD

Treatments

Fig. (19): Effect of different antifungal treatments with bioagents on fresh weight of Cicer arietinum in vivo.

- 106 - Results

0.7 0.6 0.5 0.4 0.3 0.2 Dry weight (gm) 0.1 0

GV+ GD

Vitavax+ GV vitavax+ GD Mint oil+GD Starner + GVStarner+ GD Mint oil + GV Copprus+ GV clove oil + GVclove oil + GD Healthy control Infected control Copprus-kz+GD

Treatments

Fig. (20): Effect of different antifungal treatments with bioagents on dry weight of Cicer arietinum in vivo.

- 107 - Results

3. a- Effect of different antifungal treatments on chlorophyll content in Cicer arietinum leaves.

A) Chlorophyll (a)

As shown in table (15) copprus KZ, starner, vitavax + copprus KZ, vitavax + starner and copprus KZ + starner gave the highest chlorophyll (a) content (0.017, 0.017, 0.017, 0.018 and 0.018 mg/ cm2), respectively, compared by the infected control (0.008 mg/ cm2)

B) Chlorophyll (b)

Table (15) illustrated that copprus KZ, starner, vitavax + copprus KZ, vitavax + starner and copprus KZ + starner gave the highest chlorophyll (b) content (0.007, 0.006, 0.009, 0.007 and 0.006 mg/ cm2), respectively, compared by the infected control (0.003 mg/ cm2),

C) Total chlorophyll

Copprus KZ, starner, vitavax + copprus KZ, vitavax + starner and copprus KZ + starner gave the highest total chlorophyll content (0.024, 0.025, 0.027, 0.026 and 0.031 mg/ cm2), respectively, compared by the infected control (0.011 mg/ cm2) (table 15).

- 108 - Results

Table (15): Effect of different antifungal treatments and their combinations on chlorophyll (a), chlorophyll (b) and total chlorophyll of Cicer arietinum in vivo.

Chlorophyll Chlorophyll Total chlorophyll Treatments 2 2 2 (a) (mg/ cmP )P (b)(mg/ cmP )P (mg/ cmP )P

Vitavax 0.010 0.004 0.014

Clove oil 0.013 0.006 0.020

Mint oil 0.009 0.003 0.012

Copprus KZ 0.017 0.007 0.024

Starner 0.017 0.006 0.025

GV 0.010 0.005 0.015

GD 0.008 0.003 0.011

Vitavax + clove oil 0.011 0.005 0.018

Vitavax + mint oil 0.007 0.002 0.010

Vitavax + Copprus KZ 0.017 0.009 0.027

Vitavax + starner 0.018 0.007 0.026 Clove oil + mint oil 0.012 0.007 0.019 Copprus KZ+ starner 0.018 0.006 0.031 Healthy control 0.012 0.007 0.019 Infected control 0.008 0.003 0.011 LSD 0.12 0.12 0.15 ** = significant at (p ≤ 0.05)

- 109 - Results

0.02

0.015 ) 2 cm

/ 0.01 mg ( 0.005 Chlorophyll a content 0

GV GD

Vitavax mint oil starner Clove oil copprus

Clove+ mint vitavax+ mint vitavax+ clove Healthy control infected control vitavax+ starner vitavax+ copprus copprus+ starner

Treatments

Fig. (21): Effect of different antifungal treatments and their combinations on chlorophyll (a) of Cicer arietinum in vivo.

- 110 - Results

0.01

0.008 ) 2 0.006 cm / 0.004 ( mg

0.002 Chlorophyll b content b content Chlorophyll 0

GV GD

Vitavax mint oil starner Clove oil copprus

vitavax+ mint Clove + mint vitavax+ clove Healthy control infected control vitavax+ starner vitavax+ copprus copprus+ starner

Treatments

Fig. (22): Effect of different antifungal treatments and their combinations on chlorophyll (b) of Cicer arietinum in vivo.

- 111 - Results

0.035 0.03 0.025

) 0.02 2

cm 0.015 / /

mg 0.01 ( 0.005 0 TotalChlorophyll content

GV GD

Vitavax mint oil starner Clove oil copprus

Clove+ mint vitavax+ mint vitavax+ clove Healthy control infected control vitavax+ starner vitavax+ copprus copprus+ starner Treatments

Fig. (23): Effect of different antifungal treatments and their combinations on total chlorophyll of Cicer arietinum in vivo.

- 112 - Results

3. b- Effect of different antifungal treatments with bioagents on chlorophyll content in Cicer arietinum leaves.

A) Chlorophyll (a)

As shown in table (16) copprus KZ + GV, copprus KZ + GD, starner + GV, starner + GD gave the highest chlorophyll (a) content (0.016, 0.018, 0.016 and 0.016 mg/ cm2), respectively, while mint oil + GV gave the lowest chlorophyll (a) content (0.007 mg/ cm2) compared by the infected control (0.008 mg/ cm2).

B) Chlorophyll (b)

Copprus KZ + GV, copprus KZ + GD, starner + GV, starner + GD gave the highest chlorophyll (b) content (0.010, 0.007, 0.012 and 0.007 mg/ cm2) on the other hand mint oil + GV gave the lowest chlorophyll (b) content (0.002 mg/ cm2) compared by the infected control (0.003 mg/ cm2) table (16).

C) Total chlorophyll

Data in table (16) show that copprus KZ + GV, copprus KZ + GD, starner + GV, starner + GD gave the highest total chlorophyll content (0.025, 0.028, 0.024 and 0.026 mg/ cm2) while mint oil + GV gave the lowest total chlorophyll content (0.010 mg/ cm2) compared by the infected control (0.011 mg/ cm2).

- 113 - Results

Table (16): Effect of different antifungal treatments with bioagents chlorophyll a, chlorophyll b and total chlorophyll content of Cicer arietinum in vivo.

Treatments Chlorophyll Chlorophyll Total chlorophyll 2 2 2 a (mg/ cmP )P b (mg/ cmP )P (mg/ cmP )P Vitavax + GV 0.010 0.005 0.015 Vitavax + GD 0.011 0.006 0.018 GV + GD 0.013 0.004 0.018 Clove oil + GV 0.010 0.005 0.016 Clove oil + GD 0.010 0.003 0.014 Mint oil + GV 0.007 0.002 0.010 Mint oil + GD 0.013 0.008 0.015 Copprus KZ + GV 0.016 0.010 0.025 Copprus kZ + GD 0.018 0.007 0.028 Starner + GV 0.016 0.012 0.024 Starner + GD 0.016 0.007 0.026 Healthy control 0.012 0.007 0.019 Infected control 0.008 0.003 0.011 LSD 0.18 0.61 0.81

* = non significant at (p > 0.05) ** = significant at (p ≤ 0.05)

- 114 - Results

0.018 0.016 0.014

) 0.012 2 0.01 cm / 0.008

( mg 0.006 0.004

Chlorophyll a content content a Chlorophyll 0.002 0

GV+ GD

Vitavax+ GV vitavax+ GD Mint oil+GD Starner + GV Starner+ GD Mint oil + GV Copprus+ GV Healthy control Clove oil + GVClove oil + GD Infected control Copprus-kz+GD

Treatments

Fig. (24): Effect of different antifungal treatments with bioagents on chlorophyll (a) of Cicer arietinum in vivo.

- 115 - Results

0.012

0.01

) 0.008 2 cm

/ 0.006

( mg 0.004

0.002 Chlorophyll b content b content Chlorophyll 0

GV+ GD

Vitavax+ GV vitavax+ GD Mint oil+GD Starner + GV Starner+ GD Mint oil + GV Copprus+ GV Healthy control Clove oil + GVClove oil + GD Infected control Copprus-kz+GD

Treatments

Fig. (25): Effect of different antifungal treatments with bioagents on chlorophyll (b) of Cicer arietinum in vivo.

- 116 - Results

0.03

0.025 ) 2 0.02 cm / / 0.015 mg ( 0.01

0.005 Total chlorophyll content Totalchlorophyll 0

GV+ GD

Vitavax+ GV vitavax+ GD Mint oil+GD Starner + GV Starner+ GD Mint oil + GV Copprus+ GV Healthy control Clove oil + GVClove oil + GD Infected control Copprus-kz+GD

Treatments

Fig. (26): Effect of different antifungal treatments with bioagents on total chlorophyll of Cicer arietinum in vivo.

- 117 - Results

4. a- Effect of different antifungal treatments and their combinations on enzyme activity.

A) Peroxidase enzyme

Data in table (17) show that vitavax + starner, starner, copprus KZ, vitavax + copprus KZ, gave the highest peroxidase activity (507.7, 491.1, 486.2 and 485.9 U/ ml), respectively, compared by the infected control which gave (326 U/ ml), while vitavax + mint oil gave the lowest peroxidase activity (131.9 U/ ml) compared by the infected control.

B) Polyphenol oxidase

Data in table (17) show that starner, copprus KZ, vitavax + starner, copprus KZ + starner, vitavax + copprus gave the highest polyphenol oxidase activity (770, 735.1, 753, 741 and 635 U/ mg protein), respectively, compared by the infected control (85.5 U/ ml), while vitavax and vitavax + clove oil gave the lowest polyphenol oxidase activity (135.3 and 125 U/ ml) compared by the infected control.

- 118 - Results

Table (17): Effect of different antifungal treatments and their combinations on enzyme activity (peroxidase and polyphenol oxidase) in vivo.

Polyphenol Peroxidase oxidase Treatments enzymes (unit/ ml) enzyme(unit/ ml)

Vitavax 153.2 135.3 Clove oil 148.9 214.8 Mint oil 244.7 210 Copprus KZ 486.2 735.1 Starner 491.1 770 GV 251.1 162.9 GD 266 148.35 Vitavax + clove oil 240.5 125 Vitavax + mint oil 131.90 144.7 Vitavax + Copprus KZ 485.90 635 Vitavax + starner 507.7 753 Clove oil+ mint oil 475.6 178.6 Copprus KZ+ starner 220.2 741 Healthy control 318.1 459.2 Infected control 126 85.5 LSD 17.3 0.12

** = significant at (p ≤ 0.05) *** = Highly significant at (p ≤ 0.001) Gliocladium virens: GV Gliocladium deliquescens: GD

- 119 - Results

600 500 400 300 200 mg protein) mg 100 Peroxidase activity (U/ 0

GV GD

Vitavax mint oil starner Clove oil copprus

Clove + mint vitavax+ mint vitavax+ clove Healthy control infected control vitavax+ starner vitavax+ copprus copprus+ starner

Treatments

Fig. (27): Effect of different antifungal treatments and their combinations on peroxidase activity of Cicer arietinum in vivo.

- 120 - Results

800 700 600 500 400 300 200

polyphenoloxidase 100 activity(U/ mg protein) 0

GV GD

Vitavax mint oil starner Clove oil copprus

Clove + mint vitavax+ mint vitavax+ clove Healthy control infected control vitavax+ starner vitavax+ copprus copprus+ starner Treatments

Fig. (28): Effect of different antifungal treatments and their combinations on polyphenol oxidase activity of Cicer arietinum in vivo.

- 121 - Results

4 .b- Effect of different antifungal treatments with bioagents on enzyme activity.

A) Peroxidase enzyme

As shown in table (18) copprus KZ + GV, copprus KZ + GD, starner + GV, starner + GD gave the highest peroxidase enzyme activity (498.2, 508.5, 492.2, 481.9 U/ ml), respectively, compared by the infected control, while vitavax + GD gave the lowest peroxidase enzyme activity (86.2 U/ ml), respectively, compared by the infected control.

B) Polyphenol oxidase

Data in table (18) show that copprus KZ + GV, copprus KZ + GD, starner + GV, starner + GD gave the highest polyphenol oxidase enzyme activity (714, 654, 665.1, 680 U/ ml), respectively, compared by the infected control, while clove oil + GD, mint oil + GD gave the lowest polyphenol oxidase enzyme activity (179.3, 162.6 U/ ml), respectively, compared by the infected control.

- 122 - Results

Table (18): Effect of different antifungal treatments with bioagents on enzyme activity (peroxidase and polyphenol oxidase) in vivo.

Polyphenol Treatments Peroxidase enzymes oxidase enzyme (unit/ ml) (unit/ ml ) Vitavax + GV 472.4 462.2 Vitavax + GD 86.2 223.1 GV + GD 346.8 352.5 Clove oil + GV 362.8 232.4 Clove oil + GD 395.8 179.3 Mint oil + GV 369.2 211.2 Mint oil + GD 410.7 162.6 Copprus KZ + GV 498.2 714 Copprus kZ + GD 508.5 654 Starner + GV 492.2 665.1 Starner + GD 481.9 680 Healthy control 318.1 459.2 Infected control 126 85.5 LSD 40.65 0.16

** = significant at (p ≤ 0.05) *** = Highly significant at (p ≤ 0.01)

- 123 - Results

600

500

400

300 protein) 200

100 Peroxidase activity (U/ mg 0

GV+ GD

Vitavax+ GV vitavax+ GD Mint oil+GD Starner + GV Starner+ GD Mint oil + GV Copprus+ GV Healthy control Clove oil + GVClove oil + GD Infected control Copprus-kz+GD

Treatment

Fig. (29): Effect of different antifungal treatments with bioagents on peroxidase activity of Cicer arietinum in vivo.

- 124 - Results

800 700 600 500 400 300

(U/mg protein) 200 100 Polyphenoloxidase activity 0

GV+ GD

Vitavax+ GV vitavax+ GD Mint oil+GD Starner + GV Starner+ GD Mint oil + GV Copprus+ GV Healthy control Clove oil + GVClove oil + GD Infected control Copprus-kz+GD

Treatments

Fig. (30): Effect of different antifungal treatments with bioagents on polyphenol oxidase activity of Cicer arietinum invivo.

- 125 - Results

5- Effect of different treatments (Fungicide, oils and inducers) on the efficiency of S. rolfsii germination and pathogenecity in 7 days.

The results obvious that in control case the infection begins on the third day complete disintegration of Cicer arietinum tissues till the seventh day and showed the following symptoms: The fungus is on the epidermal layer only and did not penetrate inside the host tissue. The fungus inters inside the cortex and form sclerotia. While, the fungus was detected in the cortex and the phloem was not macerated. The fungus colonized in the xylem element and spread to the pith. The whole plant tissue are colonized by the fungus and the fungus made sclerotia in the different tissues. The plant rooted, macerated and completely digested by the fungus. Complete rot in all tissue and appearance of sclerotia in all tissue noticed.

On the other hand, the used treatments (vitavax + mint oil, clove oil + mint oil, vitavax + starner, mint oil + starner) prevent the rot during the first five days from the beginning of infection. While (vitavax+ clove oil and mint oil + copprus KZ) prevent the rot during the first four days from the beginning of infection. Vitavax + copprus KZ was the best treatment used in this expeirment.

The used treatments (vitavax+ mint oil, vitavax + clove oil, clove oil + mint oil, vitava + starner, mint oil+ starner, mint oil + copprus KZ, vitavax + copprus KZ) inhibited the germination of sclerotia,

- 126 - Results

fragmented sclerotia and prevented the pathogenecity of culture filtrate during the first weak from the beginning of the patheogenecity, whereas the same treatments decreased germination of sclerotium (9, 11, 13 and 15 days) from the beginning of the pathogenecity. Treatments mint oil + copprus KZ and vitavax + copprus KZ were the most efficient treatments in decreasing germination of sclerotia, whereas vitavax+ mint oil was less effective treatment compared with the control.

III- Effect of gamma irradiation:

1. a- Effect of different doses of gamma irradiation on the antagonistic effect of Glioccladium virens against S. rolfsii in vitro (3 days incubation).

From the results presented in table (20) and fig. (31) it can be noticed that the antagonistic effect of Gliocladium virens against S. rolfsii increased by increasing gamma irradiation dose up to 1.0 kGy that recorded the highest antagonistic effect compared with control. On the other hand, gamma radiation doses higher than 1.0 kGy decrease the antagonistic effect of G. virens

- 127 - Results

Table (20): Effect of different doses of gamma irradiation on the antagonistic effect of Glioccladium virens against S. rolfsii in vitro.

Gamma irradiation Inhibition percentage doses (kGy) (I%) Unirradiated (control) 50 0.25 55.26 0.5 58.8 1.0 67.78 1.5 56.51 2.0 45.56 2.5 31.21 3.0 0.00 LSD 1.29

*** = Highly significant at (p ≤ 0.05)

80 70 60 50 40 30 20 10 0 Inhibitionpercentage (I%) 1 2 3 0.5 1.5 2.5 zero 0.25

Gamma irradiation doses (kGy)

Fig. (31): Effect of gamma irradiation on the antagonistic effect of Glioccladium virens against S. rolfsii.

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1. b- Effect of different doses of gamma irradiation on the antagonistic effect of Glioccladium deliquescens against S. rolfsii in vitro (3 days incubation).

As shown in table (21) and fig. (32) the antagonistic effect of Gliocladium deliquescens against S. rolfsii increased by increasing gamma radiation dose to 2.0 kGy that recorded the highest antagonistic effect compared with control. On the other hand, gamma radiation doses higher than 2.0 kGy decreased the antagonistic effect of G. deliquescens

- 129 - Results

Table (21): Effect of gamma irradiation doses on the antagonistic effect of Glioccladium deliquescens against S. rolfsii in vitro.

Gamma irradiation Inhibition percentage doses (kGy) ( I%) Unirradiated (control) 48.86 0.25 54.44 0.5 57.78 1.0 61.1 1.5 64.4 2.0 72.2 2.5 51.1 3.0 36.67 LSD 1.5

*** = Highly significant at (p ≤ 0.05)

80 70 60 50 40 30 20 10

Inhibition percentage (I%) Inhibition percentage 0

1 2 3 0.5 1.5 2.5 zero 0.25 Gamma irradiation doses (kGy)

Fig (32): Effect of gamma irradiation on the antagonistic effect of Glioccladium deliquescens against S. rolfsii .

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2. a- Effect of gamma irradiation doses on Cicer arietinum seed germination.

The results presented in table (22) and figs. (33 and 34) show that the number of survival plants decrease from 100 % to 20 % by increasing gamma irradiation doses. Irradiated gamma radiation seeds give different symptoms. Number of ungerminated seeds increasing by increasing gamma irradiation doses from 0 to 8 ungerminated seeds due to the disturbance of seed embryo (sudden shock by gamma radiation- radio shock inhibition) as shown in table (33) and fig. (46). In addition, gamma irradiation decreases vegetative growth and number of terminal and lateral buds on shoot.

Table1T (22): Effect of gamma irradiation doses on seed germination.

Gamma Survival plants Number of irradiation doses (%) ungerminated (Gy) seeds Unirradiated 100 0 (control) 5 40 6 10 30 7 15 30 7 20 30 7 25 30 7 30 20 8 LSD 5.6 1.2

* = non significant at (p > 0.05)

- 131 - Results

100

20 Survival plants (%) plants Survival 0 zero 5 10 15 20 25 30 Gamma irradiation doses (Gy)

Fig. (33): Effect of gamma irradiation on survival plants (%).

number of of number 2

ungerminated seeds 0 zero 5 10 15 20 25 30 Gamma irradiation doses (Gy)

Fig. (34): Effect of gamma irradiation on number of germinated seeds.

2. b- Effect of gamma irradiation doses on growth parameters of Cicer arietinum seeds.

Table (23) and figs. (35, 36 and 37) show that different gamma irradiation doses decreased plant height from 50 cm to 35 cm, decreased plant fresh weight from 2 g to 0.9 g and decreased dry weight from 067 g to 0.29 g.

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Table (23): Effect of gamma irradiations doses on growth parameters of Cicer arietinum in vivo.

Gamma irradiation Plant Fresh Dry weight doses (Gy) height (cm) weight (g) (g) Unirradiated (control) 50 2 0.67 5 47 1.9 0.63 10 45 1.7 0.55 15 45 1.5 0.50 20 40 1.2 0.40 25 37 0.9 0.31 30 35 0.9 0.29 LSD 2.8 0.93 0.05

* = non significant at (p ≤ 0.05)

60 50 40 30 20 10 Plantheight (cm) 0

5 10 15 20 25 30 zero Gamma irradiation doses (Gy)

Fig. (35): Effect of gamma irradiation doses on plant height.

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2.5 2 1.5 1 0.5 Fresh weight (g) 0

5 10 15 20 25 30 zero Gamma irradiation doses (Gy)

Fig. (36): Effect of gamma irradiation doses on fresh weight.

0.8 0.7 0.6 0.5 0.4 0.3 0.2 Dry weight (g) 0.1 0

5 10 15 20 25 30 zero Gamma irradiation doses (Gy)

Fig. (37): Effect of gamma irradiation doses on dry weight.

3. a- Effect of gamma irradiated Gliocladium virens on chickpea plants inoculated in soil infested with S. rolfsii.

As shown in table (24) and fig. (38) survival plants percentage of chickpea plants increase by increasing gamma irradiation doses of Gliocladium virens 0.25 up to dose 1.0 kGy. On the other hand, gamma irradiated Gliocladium virens at dose 1.5 up to 2.5 decrease survival plant percentage. At gamma irradiation dose 3.0 kGy the growth of G. virens completely inhibited. Gamma irradiated Gliocladium virens at dose 1.0 kGy

- 134 - Results gave the highest percentage of survival plants (80 %) compared by the unirradiated (control).

Table (24): Effect of gamma irradiated GV on chickpea plants inoculated in soil infested with S. rolfsii.

Gamma irradiation Infected plants (%) Survival plants doses (kGy) Pre-emergence (%) Unirradiated (control) 40 60 0.25 30 70 0.5 30 70 1.0 20 80 1.5 30 70 2.0 50 50 2.5 60 40 3.0 40 60 LSD 4.3 5.7 ** = significant at (p ≤ 0.05)

Infected plants Survival plants

80 60 40 20 0 1 2 3 0.25 0.5 1.5 2.5 control Gamma irradiation doses (kGy)

Fig. (38): Effect of gamma irradiated GV on chickpea plants inoculated in soil infested with S. rolfsii.

- 135 - Results

3. b- Effect of gamma irradiated Gliocladium deliquescens on chickpea plants inoculated in soil infested with S. rolfsii.

Data in table (25) and fig. (39) show that survival plants percentage of chickpea plants increased by using gamma irradiated Gliocladium deliquescens 0.25 up to dose 2.0 kGy in the infested soil with S. rolfsii. On the other hand, survival plants percentage of chickpea plants decreased at gamma irradiation doses 2.5 and 3.0 kGy. Gamma irradiated GD at dose 2.0 kGy gave the highest percentage of survival plants (90 %) compared by the unirradiated (control).

Table (25): Effect of gamma irradiated GD on chickpea plants inoculated in soil infested with S. rolfsii.

Gamma irradiation Infected plants (%) Survival plants doses (kGy) Pre-emergence (%) Unirradiated (control) 50 50 0.25 40 60 0.5 30 70 1.0 30 70 1.5 20 80 2.0 10 90 2.5 30 70 3.0 50 50 LSD 0.02 0.04 ** = significant at (p ≤ 0.05)

- 136 - Results

Infected plants Survival plants

100 80 60 40 20 0 1 2 3 0.25 0.5 1.5 2.5 control

Gamma irradiation doses (kGy)

Fig. (39): Effect of gamma irradiated Gliocladium deliquescens on chickpea plants inoculated in soil infested with S. rolfsii.

4. a- Effect of gamma irradiation on external morphology of S. rolfsii.

Fig. (40) showed that the sclerotia of unirradiated (control) contain approximately 80% from its constituents microsclerotiole and 20% vegetative mycelia. There was heavy mycelial growth, nemours sclerotia distributed all over the Petri dish, small sclerotia are present and few in number (scarce), maturation of all sclerotia. Sclerotia are dark brown in color.

Gamma irradiation at doses (0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 kGy) have slightly external difference in the growth of S. rolfsii than unirradiated (control), where the irradiated isolates at the previous doses can survive, complete the growth and sclerotial formation fig. (41) that show the

- 137 - Results percentage of dead mycelia in the irradiated isolates (70%) while the percentage of live sclerotiole (30%) that resist doses of irradiation.

On the other hand, sclerotia of S. rolfsii exposed to 5 kGy gamma irradiation showed 80 % dead micrsclerotiole and 20 % dead vegetative hyphae. Weak mycelial growth. Inoculums after and before radiation are the same. Radiation at this dose inhibits sclerotial formation. Gamma irradiated isolates can not complete growth that mean it is a lethal dose. Transfer section of irradiated sclerotia fig. (42) show that the dead fungus consists of 70 % dead hyphae and 30 % dead sclerotiole compared by the control where the viability of the fungus is 100 % .

- 138 - Results

20%

80% live Sclerotia live compact mycelia

Fig. (40): Percentage of live sclerotia and live hyphae at control.

30%

70%

Live radiated sclerotia Dead hyphae

Fig. (41): Percentage of live sclerotia and dead hyphae at (0.25 up to 3 kGy) doses.

30%

70%

Dead sclerotia Dead hyphae

Fig. (42): Percentage of dead sclerotia and dead hyphae at dose 5 kGy.

- 139 - Results

4. b- Effect of different gamma irradiation doses on pathogenecity and infection process of S. rolfsii after 7 days in vitro.

In case on unirradiated control showing infection of tissues by microsclerotia. Small sclerotia or microsclerotiole are found between cortex and xylem cells that block vessels.

While, Gamma irradiation at doses 0.25 up to 3.0 sclerotia, mycelia and fragmented sclerotia (infected elements) on epidermis caused the following symptoms on seedlings of Cicer arietinum: Infection in cortex is noticed, browning in xylem vessels and fibers, disintegration of pith and browning and rotting of seedlings. The fungus completely digested all plant tissues. Gamma irradiation dose 5.0 The fungus die, so no infection symptoms appear. No infection takes place due to the death of the fungus

- 140 - Results

4. c- Effect of gamma irradiation on pathogenecity of S. rolfsii to Cicer arietinum in vivo.

Data in table (28) show that dose of gamma irradiation at 5 kGy completely inhibited the pathogenecity of the fungus, where the percentage of survival plants was 100 % and there is no seed rot disease the same result as the healthy control case. On the other hand, 0.25 up to 3 kGy doses of irradiation recorded survival percentage up to 40 % and infected plant percentage ranged from (60 to 100 %) as in fig (43).

Table (28): Effect of gamma irradiation on pathogenecity of S. rolfsii to Cicer arietinum in vivo.

Isolates Infected plants (%) Survival plants (%) Healthy control 0.00 100 Infected control 60 40 0.25 kGy 60 40 0.5 kGy 70 30 1.0 kGy 70 30 1.5 kGy 80 20 2.0 kGy 90 10 2.5 kGy 90 10 3.0 kGy 100 0.00 5.0 kGy 0.00 100 LSD 6.1 5.8 ** = significant at (p ≤ 0.05)

- 141 - Results

Survival plants Infected plants

100 80 60 40 20 0 1 2 3 5 0.25 0.5 1.5 2.5

HealthycontrolInfected control Gamma irradiation doses (kGy)

Fig. (43): Effect of gamma irradiation doses on the pathogenecity of Sclerotium rolfsii to Cicer arietinum.

4. d- SDS- PAGE for total protein profile of unirradiated and irradiated isolates of S. rolfsii.

The cellular proteins of gamma unirradiated and irradiated isolates of S. rolfsii were electrophoretically analyzed on SDS- PAGE. The extracted proteins were fractionated using one dimensional SDS- PAGE. The SDS- PAGE banding patterns and survey for the cellular proteins bands of the unirradiated and irradiated isolates of S. rolfsii are shown in photo (32) and table (28). The isolates share a number of bands. These bands may be species specific bands. The banding patterns showed a total number of 15 bands with molecular weight range from 20 to 112.5 kDa.

- 142 - Results

210- 170- 130- 95- 83- 72- 62- 56- 43- 34- 26- 20-

Photo (32): SDS- PAGE band survey for the total cellular proteins extracted from unirradiated and irradiated isolates of S. rolfsii.

Data showed that the bands was approximately mono morphic with range from 37.5 % in some isolate to 100 % in other isolates. The frequency of each band was little different whereas the bands with molecular weight of (20, 38, 59 kDa) recorded the highest frequency between the irradiated isolates, on the other hand bands at molecular weight (26, 62, 65, 94 kDa) exhibited the lowest frequency as shown in table (29).

- 143 - Results

Table (29): SDS- PAGE band survey for the total proteins extracted from irradiated and unirradiated isolates of S. rolfsii.

Gamma Band no. irradiated Isolates 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 (kGy) Band mol. weight (kDa)

Control 20 26 32 34 38.5 44 54 59 60.5 62 65 72 83 94 112.5

0.25 1 0 1 1 1 1 1 1 0 0 0 1 1 0 1

0.5 1 0 1 1 1 1 1 1 0 0 1 0 1 0 1

1.0 1 0 0 0 1 1 0 1 1 1 0 1 1 0 1

1.5 1 0 0 0 1 1 0 1 1 1 0 1 0 0 0

2.0 1 0 0 0 0 0 0 1 1 1 0 1 0 0 0

2.5 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1

3.0 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1

5.0 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0

Total 8 3 5 5 7 7 4 7 5 3 3 6 5 2 5

- 144 - Results

4. e- Purification of polygalacturonase produced by gamma irradiated and unirradiated isolates of S. rolfsii.

Table (29) showed that polygalacturnase isolated from unirradiated isolate showed major protein band around 78 kDa while exposing S. rolfsii isolates to gamma irradiation at doses (0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3 kGy) showed one major band around 72 kDa. On the other hand, 5 kGy gamma irradiated isolate show one low intense band around 72 kDa indicating weak activity of polgalacturonase and weakness in the pathogenecity processes.

Photo (33): Sodium dodecyl sulphate polyacrylamide gel electrophoresis of polygalacturonase from S. rolfsii

- 145 - Results

Table (30): Purification profile of polygalacturonase from unirradiated S. rolfsii (control).

Purification enzyme Fold) (%) (ml) - stages Yield ( mg/ ml mg/ Protein (U/ ml) (U/ protein) Volume Purification Specific Enzyme activity Total protein (mg) protein Total activity (Units/ mg Total activity (Units) activity Total

Crude enzyme 100 3 7 700 300 2.3 1 100 preparation Amm. Sulphate 20 1.9 32.3 646 38 17 7.39 92.3 precipitation

Gel filtration 5 0.9 74 370 4.5 82.2 35.7 52.8 (Sephadex G- 75)

Where:

Yield % (crude enzyme) = Total activity (crude enzyme) x 100 / Total activity (crude enzyme).

Yield % (Amm. Sulphate, Sephadex) = Total activity (Amm. Sulphate, Sephadex) x 100 / Total activity (crude enzyme).

- 146 - Results

100

20 specific enzyme activity (U/mg) activity enzyme specific 0 ) 75 -

Sephadex G (

Crude enzyme preparation Amm. Sulphate precipitation Gel filtration

Purification stages

Fig. (45): Progress of polgalacturonase specific activity through different stages of purification (unirradiated control).

- 147 - Results

Table (31): Purification profile of polygalacturonase from S. rolfsii (irradiated isolate at dose 0.25 kGy).

Purification Fold) (%) (ml) - stages Yield ( mg/ ml mg/ Protein (U/ ml) (U/ protein) Volume Purification tal protein (mg) protein tal Specific enzyme enzyme Specific Enzyme activity To activity (Units/ mg Total activity (Units) activity Total Crude enzyme 100 3.5 8.2 800 350 2.28 1 100 preparation Amm. Sulphate 20 2 38.5 770 40 19.25 8.4 96.25 precipitation

Gel filtration 5 1.2 81.38 405 6 67.5 29.6 50.6 (Sephadex G- 75)

activity (U/mg) 20 specific enzyme

Gel filtration Crude enzyme Amm. Sulphate Purification stages

Fig. (46): Progress of polgalacturonase specific activity through different stages of purification (irradiated isolate at dose 0.25 kGy).

- 148 - Results

Table (32): Purification profile of polygalacturonase from S. rolfsii (irradiated isolate at dose 0.5 kGy).

) Purification Fold (%) (ml) - stages Yield ( mg/ ml mg/ Protein (U/ ml) (U/ protein) Volume Purification Specific enzyme enzyme Specific Enzyme activity Total protein (mg) protein Total activity (Units/ mg Total activity (Units) activity Total Crude enzyme 100 2 9.1 900 200 4.5 1 100 preparation Amm. Sulphate 20 1.6 50.6 862 32 31.25 6.9 111.1 precipitation

Gel filtration 5 0.8 94.34 471.5 4 117.7 26.15 52.3 (Sephadex G- 75)

120 100 80 60 40 activity (U/mg)

specific enzyme 20 0

Gel filtration Crude enzyme Amm. Sulphate Purification stages

Fig. (47): Progress of polgalacturonase specific activity through different stages of purification (irradiated isolate at dose 0.5 kGy).

- 149 - Results

Table (33): Purification profile of polygalacturonase from S. rolfsii (irradiated isolate at dose 1.0 kGy).

Purification Fold) (%) (ml) - stages Yield (Units) ( mg/ ml mg/ Protein (U/ ml) (U/ protein) Volume Purification Total activity activity Total Specific enzyme enzyme Specific Enzyme activity Total protein (mg) protein Total activity (Units/ mg Crude enzyme 100 2.5 10.8 1080 250 4.32 1 100 preparation Amm. Sulphate 20 1.7 50.6 1012 34 34.11 7.8 107.4 precipitation

Gel filtration 5 1 105.7 525 5 105 24.30 48.6 (Sephadex G- 75)

120 100 80 60 40 activity (U/mg)

specific enzyme 20 0

Gel filtration Crude enzyme Amm. Sulphate

Purification stages

Fig. (48): Progress of polgalacturonase specific activity through different stages of purification (irradiated isolate at dose 1.0 kGy).

- 150 - Results

Table (34): Purification profile of polygalacturonase from S. rolfsii (irradiated isolate at dose 1.5 kGy).

Purification Fold) (%) (ml) - stages Yield (Units) ( mg/ ml mg/ Protein (U/ ml) (U/ protein) Volume Purification activity activity Total Specific enzyme enzyme Specific Enzyme activity Total protein (mg) protein Total activity (Units/ mg Crude enzyme 100 1 12.2 1220 100 12.2 1 100 preparation Amm. Sulphate 20 0.6 64.1 1188 12 106.8 8.7 105 precipitation

Gel filtration 5 0.2 124.4 622 1 622 50.9 50.9 (Sephadex G- 75)

700 600 500 400 300 200 activity (U/mg)

specific enzyme 100 0

Gel filtration Crude enzyme Amm. Sulphate

Purification stages

Fig. (49): Progress of polgalacturonase specific activity through different stages of purification (irradiated isolate at dose 1.5 kGy).

- 151 - Results

Table (35): Purification profile of polygalacturonase from S. rolfsii (irradiated isolate at dose 2.0 kGy).

Purification Fold) (%) (ml) - stages Yield ( mg/ ml mg/ Protein (U/ ml) (U/ protein) Volume Purification Specific enzyme enzyme Specific Enzyme activity Total protein (mg) protein Total activity (Units/ mg Total activity (Units) activity Total Crude enzyme 100 0.8 15.3 1500 80 18.75 1 100 preparation Amm. Sulphate 20 0.4 72.5 1350 8 180 9.6 96 precipitation

Gel filtration 5 0.1 132.8 664 0.5 1328 70.8 44.26 (Sephadex G- 75)

1500 1200 900 600

activity (U/mg) 300 specific enzyme 0

Gel filtration Crude enzyme Amm. Sulphate

Purification stages

Fig. (50): Progress of polgalacturonase specific activity through different stages of purification (irradiated isolate at dose 2.0 kGy).

- 152 - Results

Table (36): Purification profile of polygalacturonase from S. rolfsii (irradiated isolate at dose 2.5 kGy).

Purification Fold) (%) (ml) - stages Yield ( mg/ ml mg/ Protein (U/ ml) (U/ protein) Volume Purification Specific enzyme enzyme Specific Enzyme activity Total protein (mg) protein Total activity (Units/ mg Total activity (Units) activity Total Crude enzyme 100 3 16.4 1600 300 5.3 1 100 preparation Amm. Sulphate 20 2.2 77.2 1544 44 35.9 6.7 98.7 precipitation

Gel filtration 5 1.4 139.6 698 7 99.7 18.8 43.6 (Sephadex G- 75)

20 activity (U/mg) specific enzyme 0

Gel filtration Crude enzyme Amm. Sulphate Purification stages

Fig. (51): Progress of polgalacturonase specific activity through different stages of purification (irradiated isolate at dose 2.5 kGy).

- 153 - Results

Table (37): Purification profile of polygalacturonase from S. rolfsii (irradiated isolate at dose 3.0 kGy).

Purification Fold) (%) (ml) - Yield stages ml) U/ ( (Units) mg/ ml mg/ Protein ( protein) Volume Purification Total activity activity Total Specific enzyme enzyme Specific Enzyme activity Total protein (mg) protein Total activity (Units/ mg

Crude enzyme 100 2 18.2 1800 200 9 1 100 preparation Amm. Sulphate 20 1.3 89.06 1781 26 69.2 7.6 100 precipitation

Gel filtration 5 0.5 150.7 753.5 2.5 301.4 33.48 41.8 (Sephadex G- 75)

20 activity (U/mg) specific enzyme 0

Gel filtration Crude enzyme Amm. Sulphate

Purification stages

Fig. (52): Progress of polgalacturonase specific activity through different stages of purification (irradiated isolate at dose 3.0 kGy).

- 154 -

Discussion

I- In vitro experiments: 1- Mode of infection of Sclerotium rolfsii to Cicer arietinum. 1. a- Infection of seed

S. rolfsii is one of the most important species that related to mycelia sterilia or fungi imperfecti. It consists of sterile hyphae and sclerotia that are found in different developmental stages. The hyphae of S. rolfsii interweave together and compact to form the beginner of the sclerotia. These stepes are repeated many times to form the sclerotia of S. rolfsii. V.S of sclerotia of S. rolfsii under the light microscope show that sclerotia consists of two zones, the external region is composed of protenious cushion and the inner zone of sclerotia composed of different microsclerotiole. The sclerotia contact with the seed of Cicer arietinum, colonized and populated on the external surface, after that the sclerotia secret macerating, lytic, cellulytic, pectinolytic carbohydrases, lipases and amylase enzymes that involve or that share in digesting or rotting the seeds.

After partial digestion (one month) the starch grains of the seeds are digested by the ratio, nearly 60 % and the sclerotia (microsclerotiole) began to reappear inside or between Cicer arietinum cotyledons internal tissue. After two months from infection, the fungus continue secreting the digestive enzymes and the seed is digested by the ratio nearly, 95 %. After three months from infection, the seeds of Cicer arietinum are completely digested and completely rotted. Therefore, all components of the seeds are

- 155 -

Discussion disappeared completely. At this stage S. rolfsii is exited in the form of microsclerotiole and remain in the infected soil. This investigate the contamination of the cultivated Egyptian soil by microorganisms as it is found in sclerotial form and leads to great loss in the field of agriculture, so that we must control the disease or development of this fungus by using integrated controls .

1. b- Infection of stem

Sclerotium rolfsii infect the seedlings of Cicer arietinum by causing seed rot, wilt, stunting, pre- emergence and post emergence root rot. S. rolfsii infects the seedlings near the soil at the transition zone. At first, sclerotia of the fungus absorb water. The black sclerotioles that contain viable hyphae inside the sclerotia get out and infect the seedlings. These sclerotioles and the fungal hyphae secrete cellulases, pectinases, cutinases, polygalacturanse and many other lytic enzymes that lyse the epidermis of the seedlings and allow the sclerotioles to enter inside the transation zone of the seedlings forming large black sclerotia in between cortical cells at cortex region of Cicer arietinum seedlings.

After that, the fungus invades the xylem elements and secretes muceligenous polysaccharides and oxidates that block xylem vessels preventing the water to rise up to the leaves. This leads to seedling wilt, after that the pathogenic fungus occupied the pith region and lyses all the cells. Finally, all the tissues of the plant macerated at the transition zone so, the seedlings fall on the ground.

- 156 -

Discussion

1. c- Infection of the root The pathogenic fungus progress inside the root tissues by the same manner (by microsclerotioles and hyphae) developing rod shape sclerotia inside the digested root cell. At last, all the plants tissues are digested and lysed. The fungus reform sclerotia again to exist in the soil in the form of sclerotia .

2- Effect of tested treatments on the linear growth of the pathogenic fungus S. rolfsii. Root rot disease is considered as the most economically important and wide spread disease attacking chickpea plants causing great losses in yield of chickpea. For controlling root rot disease, it is logic to start first to test the effects of different substances on the causal organism under laboratory conditions. By this method, we can make screening for the large numbers of substances under test to obtain the effective disease control. Fungicides, oils, inducers and bioagents are used in the present investigation.

2. a- Effect of fungicides on the linear growth of Sclerotium rolfsii in vitro: The control of plant diseases is one of the most important topics. Scientists all over the world are trying to solve the complicating problems of plant diseases. The control of plant diseases has been possible by the use of chemicals or fungicides. A fungicide is an agent or a chemical that kills the fungi. A fungistaticis is an agent that causes fungal growth inhibition but

- 157 -

Discussion does not cause death of the fungus. Both actions can be achieved by the same chemicals on different pathogens.

Fungicides are classified into: • Eradicants fungicides that are applied to a plant already infected with the fungus. • Proctectant fungicides that is applied to plant or seeds before infection. Protectant fungicides begin to act when infective part of the pathogen comes with the plant surface (leaf, stem and root). The lethal action of any fungicide depends on: 1. The concentration of the active component. 2. The contact between fungicide and the pathogen. Different fungicides vary in their ability to resist different fungi. Fungicides are characterized by: a) High toxicity on wt / wt basis (Recommended doze). b) Their ability to be accumulated in fungal cells (lethal doze).

c) Each fungicide has LD50 (LC50 median lethal dose that inhibit 50 % of fungal growth). Site of action of fungicide (Intracellular action). The majority of fungicides act within the cell by inhibiting vital processes.1) To achieve this fungicide must be able to penetrate the cell membrane and inhibit the cell metabolism.

- 158 -

Discussion

2) To achieve this fungicide must have a degree in lipid solubility to be able to penetrate into the cell. Fungicides which have lipid solubility have a high antifungal activity.

Vitavax and monceren- T at concentrations (10, 25, 50 ppm) completely inhibit the growth of Sclerotium rolfsii in vitro. Both of vitavax and monceren- T are systemic fungicides as they destroy the fungus that is found on the Cicer arietinum seeds, seedlings, or mature plant internally or externally. These fungicides achieve the following effects: they inhibit sterol biosynthesis in the fungal plasma membrane. Sterol is important in the biosynthesis of fungal ergosterol that share in maintenance of membrane function. Reduction in ergosterol availability results in membrane disruption and electrolyte leakage.

Vitavax and monceren- T also inhibit glycerophospholipids biosynthesis. Glycerophospholipids are essential in cell membrane metabolism. They act as a barrier that moves ions and macromolecules across the plasma membrane. Vitavax and monceren- T disturb nuclear metabolic processes as they inhibit the biosynthesis of pyrine and pyrimidine and their polymerization as they inhibit polymarase and synthetase enzymes inside the nucleus.

Both of fungicides also inhibit protein biosynthesis as they react with ribosomal subunits and block the binding site that is necessary for protein elongation and termination. They inhibit respiration as they inhibit oxidative phosphorylation, electron transport system, polyamine

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Discussion biosynthesis that is necessary for fungal growth. In addition, vitavax and monceren- T affect on cell wall function as they inhibit chitin biosynthesis and inhibit melanin biosynthesis. They also inhibit Ca+ signaling , Ca+ signaling are necessary for several developmental activities such as hyphal tip extension, branching, sporulation and cytoplasmic movement. Ca+ signaling has a necessary role in PH homoeostasis in fungi controlling enzyme activity and membrane transport in fungal hyphae.

These results were in agreement with Abdel- Aziz et al., (1996) who found that tolclofos methyl/ thiram caused strong inhibition against mycelial growth of Sclerotium rolfsii, Rhizoctonia solani, and Macrophomina phaseolina, while carboxin was non-effective against M. phaseolina and R. solani but showing strong activity against S. rofsii. In the meantime, Viji et al., (1997) reported that the benzimidzole group of fungicides (carbendazim and benomyl) was toxic to the pathogen Sclerotium rolfsii and the antagonistic fungi Gliocladium deliquesense and Trichoderma harzianum while organophosphorus fungicides (edifenphos and Lprobenfos) were more toxic to the pathogen S. rolfsii than antagonists.

Virgen et al., (2000) revealed the efficacy of fungicides (pencycuron, tolcoflos- methyl, fluazinam, azoxystrobin) and biocontrol agent Gliocladium virens against S. rolfsii in vitro. Only pencycuron and tolcoflos- methyl inhibited AG- 3 100 % in vitro.

Moreover, Mukherjee and Tripathi (2000) reported that antracol were the most effective against Sclerotium rolfsii, Rhizoctonia solani and

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Discussion non inhibitory to the antagonists Gliocladium virens even at the concentration of 50 Mg / ml. The present results were closely similar to results of Khosla and Kumar (2003) reported that bavistin (0.2%) controlled from 56.47% disease when applied alone to 82.58% disease control by the combi-treatment of Thiram and Trichoderma viridae talc formulation (0.4 + 0.5%). Trichoderma individually (1.0%) gave 72.9% disease control where as Thiram (0.4%) controlled the disease to an extent of 67.25% on seventeen varieties of strawberry against root rot and blight under natural epiphytotic conditions at Regional Horticultural Research Station.

Meena et al., (2004) show that fungicides mancozeb and carbendazim caused 100% reduction in mycelial growth of Alternaria brassicae over control in vitro. Similar to other investigation Ramal (2005) reported that fungicides hexaconazole, difenoconazole and myclobutanil provided 100 % inhibition of spore germination in Alternaria alternata caused leaf-spotting diseases in apple. A spray schedule comprising the sprays of dodine, carbendazim, hexaconazole + mancozeb and tebuconazole at different stages during growing season of apple was highly effective in controlling the disease with 99.21 per cent disease control in the field.

In the mean time Belgers et al., (2009) tested the sensitivity of nine submersed macrophyte species to the fungicides chlorothalonil, pentachlorophenol, fluazinam, and carbendazim. Carbendazim proved not or only moderately toxic to these macrophytes. Pentachlorophenol and chlorothalonil were more toxic than fluazinam.

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Discussion

2. b- Effect of oils on the growth of Sclerotium rolfsii in vitro.

Oil is a substrate that is normally liquid. Oils are extracted from different parts of plant such as roots, leaves, flowers, fruits and seeds. The highest amount of oils is extracted from seeds (oil seeds). The amount of oil in each plant seed is different from one plant seed to another. Approximately, from 80 - 85 % of seed contents are oily components (oils). Oils are classified into vegetable and animal oils. Mint and clove oils are considered as vegetable oils that have many applications in disease controlling.

Crude of clove and mint oils has a yellow or slightly greenish color. The yellow or orange color is due to the presence of carotenoids in the oil, which are derivatives of isoprene. There are number of different carotenoides (the most important is β- carotene).

Both of clove and mint oils at concentration of 50 ppm completely inhibit the growth of S. rolfsii in vitro. This because clove oil (Syzygium aromaticum) includes high amount of the following antimicrobial compounds (eugenol, eugenol acetate, iso-eugenol and caryophyllene). These compounds inhibit all the metabolic processes of the pathogenic fungus. On the other hand mint oil (Mentha spp.) contains (menthol, menthone, 1,8- cineole, methyl acetate, metho furan, isomenthone, limonene, b-pinene, a-pinene, germacrene-d, trans, sabinene hydrate and pulegone) and all these fractions inhibit fungal division, respiration,

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Discussion respiratory enzymes, nucleic acid metabolism, hyphal cell division growth and plasma membrane structure. So clove and mint oils has high antimicrobial activity.

In the meantime, Dimitra et al., (2003) found that the growth of Botrytis cinerea, Fusarium sp. and Clavibacter michiganensis subsp. Michiganensis was completely inhibited by oregano, thyme, dictamnus and marjoram essential oils at relatively low concentrations (85–300 μg/ml).

Pramila and Dubey (2004) used some natural products such as flavour compounds, acetic acid, jasmonates, glucosinolates, propolis, fusapyrone and deoxyfusapyrone, chitosan, essential oils and plant extracts for the management of fungal rotting of fruit and vegetables.

Moreover, Tullio et al., (2007) determined the activity of some essential oils (thyme red, fennel, clove, pine, sage, lemon balm and lavender) against clinical and environmental fungal strains in vitro.

Thyme red and clove were found to be the oils with the widest spectrum of activity against all fungi tested. Essential oil products of the foliage of Eucalyptus (family Myrtaceae) posses a wide spectrum of biological activity including antimicrobial and fungicidal. This oil is used in the filed of the environmental and toxicological researches to reduce the problem of increasing microbes' resistance (Daizy et al., 2008).

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Discussion

Also, Martin et al., (2009) found that essential oils from 25 species of medicinal plants inhibit growth of six important pathogenic and toxinogenic fungal species, Fusarium oxysporum, Fusarium verticillioides, Penicillium expansum, Penicillium brevicompactum, Aspergillus flavus and Aspergillus fumigatus. The superior antifungal activity was in the case of Pimenta dioica.

Nafiseh et al., (2011) reported that Eucalyptus camaldulensis essential oil suppressed the mycelial growth of postharvest pathogenic fungi, Penicillium digitatum, Aspergillus flavus, Colletotrichum gloeosporioides and soilborne pathogenic fungi, Pythium ultimum, Rhizoctonia solani, Bipolaris sorokiniana.

2. c- Effect of inducers on the growth of Sclerotium rolfsi in vitro: Inducers are stimulatory compounds that stimulate the growth of Cicer arietinum seedlings and has no effect on fungi at all (they have no antimicrobial activity) as they did not interfere with the metabolic processes of the pathogenic fungus and the antagonistic fungi. The effect of inducers in green house is concerned with raising the immunity of the plant, enforces the ability of the plant to face the disease and resist the pathogenic fungus.

Gu Keyu (2002). Found that lipopolysaccharide (LPS) is a ubiquitous component of Gram-negative bacteria that can induce defense- related responses in plants.

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Discussion

Another study reported that the amino acid proline elicit resistance in pearl millet Pennisetum glaucum against downy mildew disease caused by Sclerospora graminicola both under greenhouse and field conditions. Proline treatment to seeds enhanced the seed germination of pearl millet in comparison with the control. Proline protected the pearl millet plants from downy mildew by offering 58% protection under greenhouse and 67% protection under field conditions. Proline was effective in enhancing vegetative and reproductive growth of the plants, as evidenced by the increase in height, fresh weight, leaf area, 1000-seed weight and grain yield in comparison with the control plants (Sathyanarayana et al., 2004).

In a similar work Ruz et al., (2007) found that phosphonate derivatives and benzothiadiazole which act as plant defense inducers were effective in fire blight control in pear and apple, under controlled environmental conditions. Plant defense inducers reduced disease levels at 40–60%.

In the mean time Niladevia and Prema (2008) studied the production of extracellular laccases by Streptomyces psammoticus. Pyrogallol and para- anisidine proved to be the best inducers for laccase production by this strain and the enzyme yield was enhanced by 50% with these inducers.

Also, Mahmoud et al., (2009) reported that microelements mixture of copper, iron, zinc, manganese at 200 ppm followed by copper sulphate at 0, 50, 100 and 200 ppm the same concentrate gave the best effect on reducing of peanut pod rot occurrence of aflatoxigenic fungi (Aspergillus

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Discussion flavus and A. parasiticus). Among biochemical changes the increase in phenol contents (free, conjugate and total phenols) and the increase in oxidative enzyme activity (peroxidase, polyphenoloxidase and catalase), increase in sugars content (reducing, non- reducing and total sugars), total free amino acids and crude protein.

Plant defence responses were induced in four different varieties of Arachis hypogaea (J-11, GG-20, TG-26 and TPG41) using the fungal components of Sclerotium rolfsii and the levels of defence-related signal molecule salicylic acid (SA) compared to that of control plants (Nandini et al., 2010).

2. d- Effect of antagonistic fungi (Gliocladium deliquescens, Gliocladium virens and Trichderma hamatum) on the growth of Sclerotium rolfsii.

Gliocladium deliquescens and Gliocladium virens antagonized the growth of S. rolfsii in vitro after 3 days as it prevented the formation of sclerotia and inhibited the different stages in sclerotial formation.

S. rolfsii form a strip zone, abarriage or elevated zone in order to antagonize G. deliquescens (the contact zone between S. rolfsii and G. deliquescens).

After 6 days from incubation G. deliquescens grow above S. rolfsii causing lyses of the hyphae and cell wall of S. rolfsii (mycoparasitism).

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Discussion

Therefore, it inhibits the growth of S. rolfsii. For this reason, G. deliquescens is used as a biocontrol to control root rot disease of Cicer arietinum in the field that is caused by S. rolfsii.

On the other hand, Trichoderam hamatum has no antagonistic effect on the growth of S. rolfsii in vitro after 3 days as recorded in our results.

After 6 days from incubation mature sclerotia of S. rolfsii were formed, that mean T. hamatum has no effect on S. rolfsii.

Meena et al., (2004) found that Trichoderma viride reduced 82% in mycelial growth of Alternaria brassicae over control in vitro.

Our results are similar to (Latunde, 2007) who reported that mycelium powder of Trichoderma koningii significantly controlled symptoms of damping off, blight and wilting caused by Sclerotium rolfsii in tomato. Moreover, sclerotial counts decreased in these soils and those sclerotia found had been parasitized by T. koningii.

Five isolates Bacillus subtilis, Pseudomonase fluorescens (Pf 5), (Sp1), (Sp2) and (Ss2) caused moderate to strong inhibition on mycelium growth to the four tested pathogens (Rhizoctonia solani, Sclerotium rolfsii, Fusarium solani and Macrophomena phaseolina) causing damping-off, wilt and peanut root rot. Pseudomonase fluoressens (Pf 5) followed by Bacillus subtilis (BS1) and Bacillus sp. (Sp2) caused the widest inhibition zone almost to tested pathogens. In greenhouse experiment, the most effective

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Discussion isolates in reducing peanut damping-off, wilt and peanut root rot were P. fluorescens (Pf 5) followed by B. subtills (BS1) and Bacillus sp (Sp2) (Mahmoud et al., 2006a).

In addition, Ibrahim et al., (2008) reported that Bacillus subtilis and Pseudomonase fluorescens caused moderate to strong inhibition to pathogens (Rhizoctonia solani, Sclerotium rolfsii, Fusarium solani and Macrophomena phaseolina) that causing peanut root rots in vitro. In greenhouse trial P. fluorescens reduced peanut pre-emergence damping-off by (87.5 %), while B. subtills gave 100 % reducing peanut post-emergence damping-off.

Saman (2009) showed that a combination of Bacillus subtilis with Pseudomondas strains can lead to greater plant protection against Rhizoctonia solani and Sclerotium rolfsii than the biocontrol exhibited by these strains used separately.

2. e- Effect of culture filtrate of Gliocladium virens and Gliocladium deliquescens on the growth of Sclerotium rolfsii . Both of Gliocladium virens and Gliocladium deliquescens antagonize the growth of Sclerotium rolfsii. Gliocladium virens produce many antibiotics such as gliovirin, gliotoxin and siderophores that are used for iron acquisition.

G. deliquescens never secrete gliovirin so its antagonistic activity is less that of G. virens.

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Discussion

G. virens and G. deliquescens produce extracellular B- (1-3) glucanase and chitinase enzymes which have a key role in lysing of S. rolfsii cell wall.

G. virens and G. deliquescens are active as hyperparasites and they lower the inoculum intensity of the pathogen in vitro and in vivo. G.virens and G. deliquescens antagonize S. rolfsii by competition, mycoparasitism and other forms of direct exploitation.

The mode of action (or the antagonistic effect) of G. virens and G. deliquescens increase by increasing time. The highest effect was recorded after three weeks as the concentration of the antibiotics and the fungal metabolites in the culture media increase. The lowest effect was recorded after one week from incubation with S. rolfsii.

3- Effect of Sclerotium rolfsii on the pathogenecity of Cicer arietinum.

In the present investigation, Cicer arietinum seeds were cultivated and germinated in two successive years 2007 and 2008 in order to test the most optimum climatic conditions that are necessary for Cicer arietinum germination, seedlings, maturation and fruiting through the different months in the year.

November was the most suitable month in the year that is suitable for Cicer arietinum growth and fruiting. From these results we concluded that Cicer arietinum seeds gave the highest growth where the embryos

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Discussion inside the seeds are available and the germinating enzymes record their highest activity in (November) at temperature 24◦C – 27◦C and at relative humidity 42 %.

On the other hand, January and February record the least percent of germination (Cicer arietinum). In January and February, the seeds of Cicer arietinum rot inside the soil due to the low temperature. As low temperature, inactivate the germinating enzymes inside the seeds and this leads to losing the viability of the seeds. Therefore, the seeds became unable to germinate and disintegrated in the soil-by-soil microflora (microorganisms that are normally existed in the soil).

Our results reveal that the best soil suitable for Cicer arietinum seedlings germination was (1/2 clay + 1/2 sand). For soil sterilization, soil sterilized by formaline recorded no germination because formaline has phytotoxic effect on seeds, while, soil sterilized by autoclave recorded the highest growth (100%) as the sterilization lower the percentage of soil microorganisms that lead to disintegration of the seeds.

In the present investigation, we found that Sclerotium rolfsii at 1 % inoculum per 1 kilo soil leads to rotting all the seeds of Cicer arietinum in pots, while, S. rolfsii at concentration 0.125 % recorded the highest percentage of survival plants. S. rolfsii at concentrations (0.5 %, 0.25 % and 0.125 %) disintegrate the seeds of Cicer arietinum as it secrets amylase, pectinolyase and lyase enzymes that lead to the maceration and rotting of seeds tissue. Also, S. rolfsii at these concentrations stunt the seedlings as it

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Discussion decreases the mitotic division in root and shoot apex, which leads to preventing seedlings elongation and stunting of the plant.

Seventeen isolates of Sclerotium rolfsii from various vegetables, peanut and wheat were evaluated for their pathogenicity on Okrun area, an S. rolfsii - susceptible peanut cultivar, and for oxalic acid production in liquid culture. All isolates, except wheat isolate from Oklahoma, were pathogenic to peanut. All isolates produced significant amounts of oxalic acid. Oxalic acid is not the sole factor determining pathogenicity (Saude et al., 2003). On the other hand, Gemma et al., (2004) found that penetration of the urediniospores of the bean rust Uromyces appendiculatus occurred either enzymatically and/or mechanically, through appressorium or infection cushion structures, from which a thin penetrating hypha was generated. Enzyme production by the mycoparasite Cladosporium tenuissimum was suggested by the loosening of the matricial components of the spore wall, which sometimes left chitin fibrils visible. Mycoparasite hyphae grew within the host spore, emptied its content, and emerged profusely forming conidiophores and conidia.

Shrikant et al., (2005) studied effect of different amino acids and sugar nucleotides addition as metabolic precursors on the production of scleroglucan. A maximum yield of 20.00 g/l and 22.32 g/l was obtained with optimized media supplemented with L-lysine (1.1 mM) and uridine mono-phosphate, respectively as compared to 16.52 g/l scleroglucan achieved with the control in the absence of metabolic precursors.

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Discussion

In addition, Sarma et al., (2007) found that the chemicals zinc sulphate, oxalic acid, sodium malonate and sodium selenite reduced mortality of chickpea (Cicer arietinum) from Sclerotinia sclerotiorum the causal agent of stem rot in chickpea infection. Among them, zinc sulphate at 10 m mol gave the best result as only 13.6% mortality was recorded after 28 days compared to 100% in the control. Rekha and Pandey (2008) determined the relative pathogenicity of Sclerotium rolfsii on Parthenium hysterophorus weed. Sclerotium rolfsii isolate showed the maximum disease incidence (80%) against targeted host.

In the meantime, Remigijus et al., (2009) reported that four fungi caused symptomatic necroses of bark and cambium of F. excelsior crowns: Alternaria alternata, Epicoccum nigrum, Chalara fraxinea and Phomopsis sp. The most pathogenic was Chalara fraxinea, inducing symptoms on 50 % of inoculated trees, while three other fungi caused necroses on 3–17 % of inoculated trees.

4- Phytotoxicity of the tested compounds on Cicer arietinum seedlings in vitro. 4. a– Fungicide The choice of the fungicide depends on: . The persistence of the fungicide (fungicide which act on fungi loss its toxicity (deposited quickly) and has a short live time due to its rapid evaporation or oxidation on the air) is not effective under sunlight decomposing factors are considered to be of no toxic value.

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Discussion

. Good or succeeded fungicide has a low solubility for extending fungicide protection over a long period. . The solubility of fungicide must furnish the toxic ion in an a mount enough to kill the pathogen and not the host plant (other wise the fungicides must have no phytotoxicity)

Vitavax is a systemic fungicide, which means chemical that are absorbed by the plant and move through: a) The root b) The stem c) The leaves to protect the plant against fungal attack). Therefore, systemic fungicide inhibit different biosynthetic processes because they posses different mode of action.

Vitavax at concentration 50 ppm dehydrate the seed so, the seed is became dry. So that, the embryo of Cicer arietinum dies, because the fungicide vitavax inhibits the different biosynthetic processes of the seed and inhibit energy producing metabolic processes (inhibition of respiration and inhibition of oxidative phosphorylation). It also interfere with biosynthesis of cell wall material required for growth and maintenance of plant, disrupt internal cell structure, disrupt plasma membrane and result in loosing cell content. Vitavax at concentration 25 ppm decrease the mitotic division at root and shoot apex and lead to plant stunting, so the growth of the shoot system was 47.06 % and the growth of root system was 66.67 %. While, vitavax at concentration (0.1, 1.0, 5.0, and 10 ppm) show no phytotoxic effect on seedlings compared with control.

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Discussion

Alfieri and Knauss (1972) found that six fungicides proved phytotoxic to Peperomia obtusefolia that infected by stem and leaf rot incited by Sclerotium rolfsii as reflected by a reduction in top weight. Vitavax and MC-5077 were significantly more phytotoxic at the concentrations employed.

These results are in harmony with Ismail et al. (1996) who found that the fungicides Rizolex- T (tolclofos- methyl / thiram), Monceren- combi and Vitavax / captan were the most inhibitory fungicides to both root and shoot systems of cotton plants.

In the meantime, Zein et al., (1999) reported that root system of cotton; soybean and wheat seedlings were more sensitive to fungicides than shoot system. This phytotoxic effect might be due to the physiological properties and / or the direct contact between root system and the compound

However Kuo and Liu (2000) revealed that commercial fungicides mepronil, carbendazim, benomyl, cyproconazole, procymidone, flutolanil, flusiazole, tebuconazole, iprodione, mancozeb, hymexazol and polyxins) have no phytotoxicity on the growth of Lima bean (Phaseolus limensis) Benomyl proved to be an effective inhibitor of all three contaminants in concentrations as low as 2 ppm within the agar medium, and no evidence of phytotoxicity to arabidopsis seedlingswas observed until concentrations exceeded 20 ppm (Paul et al., 2001).

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Discussion

4. b – Oils Vital seeds of Cicer arietinum germinate and terminate the growth in two weeks from the germination. In case of mint oil the suspension of Tween 80 and the oil dehydrate the seeds of Cicer arietinum at concentrations (10, 25 and 50 ppm). Components of mint oil inhibit cell division at the apical meristems in the seedlings, toxify the embryo, inhibit the formation of spindle fibers during mitotic division and consequently inhibit cell division. Constitutes of mint oil inhibit cell elongation and lead to plant stunting. 0.1 ppm concentration of mint oil has a little effect on the growth of Cicer arietinum and has no phytotoxic effect on the seedlings.

The concentrations (5, 10, 25 and 50 ppm) of clove oil inhibit the growth of the seedlings gradually compared with the control. While 0.1 and 1.0 ppm concentrations of clove oil have no phytotoxic effect on Cicer arietinum seedlings, so they can be used in control of root rot; white rot and seed rot diseases. In the same time Shady and Ahmed (1999) reported that extracts of black pepper (Pipper nigrum) seeds and Cigar flower leaves had moderate phytotoxic effects on the of root system of cotton seedlings . Our results similar to Gbolade et al., (1999) assessed the effect of seed treatment and fumigation of artificially infested cowpea with volatile oil of air dried leaves of Ageratum conyzoides (Asteraceae) at concentrations of 2.5 to 10 Ml / 9.5 g bean and they found that no adverse physical effects on the bean at these concentrations.

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Discussion

Moreover, Kamara et al., (2000) mentioned that maize germination was significantly reduced by leaf extracts of Gliricidia sepium, Tetrapleura tetraptera, Lonchocarpus sireceus, Senna siamea and Leucaena leucocephala. Terminalia superba, Tetrapleura tetraptera, Pithecellobium dulce, Gliricidia sepium and Senna siamea significantly reduced maize root growth at the lowest extract concentration, while shoot length was most significantly reduced by Giricidia sepium. Leucaena leucocephala, Alchronea coordifolia, Pithecellobium dulce, Terminalia superba and Tetrapleura tetraptera at all concentrations.

Daizy et al., (2004) that volatile oil from Eucalyptus citriodora is phytotoxic and could be utilized as bioherbicide for Triticum aestivum, Zea mays, Raphanus sativus, Cassia occidentalis, Amaranthus viridis and Echinochloa crus-galli. In agreement with Emilia et al., (2009) reported that significant inhibitory activity of two Nepeta species collected in Lebanon on germination and initial radical elongation of Raphanus sativus L. (radish) and Lepidium sativum L. (garden cress).

The essential oils isolated from sweet fennel and sweet basil were the most phytotoxic on barnyardgrass, whereas those isolated from lacy phacelia and anise were the least phytotoxic (Kico et al., 2010)

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Discussion

4. c- Inducers Inducers are a group of chemical and synthetic compounds that applied in the field of agriculture to stimulate the growth of different plants, by inducing and enhancing the immune system of the plant.

Inducers induce the growth of the vegetative system and the root system as they increase the mitotic division at root and shoot apex, induce the formation of secondary meristem (secondary cambium) and increase the size, the weigh, the number of leaves. Inducers develop the length of plants, the number of buds, the availability, the branching of the root, the number of lateral roots, the numbers of root hairs and extend the absorptive surface of the root that lead to more growth. They also, increase the absorption of minerals necessary for the biosynthesis of different compounds in the plant (all these factors improve the resistance of the plant towards the invasion by different fungal and bacterial diseases, so that inducers increase the yield of the plant (fruits and vegetables).

Inducers have different mode of action in different plant cells. Inducers induce the formation of polysaccharide synthetase enzymes that are necessary for polysaccharides synthesis

Inducers stimulate the formation of protein that take part in the formation of plasma membrane and the enzymes necessary for synthesis of amino acids and protein units (polypeptides) that is specific for synthesizing the protein specific for plasma membrane. In addition, they induce the formation of nuclease enzymes and synthetases enzymes that synthesize

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Discussion

DNA and RNA nucleoprotein, nucleic acid that start cell division. Inducers induce the formation of spindle fibers, cell division, the formation of energy producing compounds ATP, ADP, NADH and NADH2 that is necessary for different anabolic and catabolic reactions in different plant cells as they enhance the growth of plant cells. Also, they induce the formation of different enzymes necessary for respiration involved in Krebs cycle.

Inducers induce the growth of root system in Cicer arietinum and stimulate the absorption of different minerals including Mg and N in the soil, these elements Mg and N share in the biosynthesis of chlorophyll molecules and increase biosynthesis of plastids that increase the availability of the plant

In addition, inducers increase production of enzymes that share in photosynthesis (proteases, amylases and carbohydratase). Finally, inducers increase cell division, cell differentiation, cell characterization, cell specification and cell elongation. All these processes leads to enhance the growth of the plant. In the present investigation, starner and copprus KZ at the concentrations (0.1, 1.0, 5.0, 10, 25 and 50 ppm.) induce the growth of shoot and root systems, so we recommended to use copprus KZ and starner in controlling root rot disease caused by Sclerotium rolfsii as it has no phyototoxic effect on Cicer arietinum plants.

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Discussion

5- Joint toxic effect of the tested compounds against pathogenic fungus Sclerotium rolfsii.

IC25 of vitavax fungicide and other treatments that control plant diseases added to each other to from mixtures. Each of these mixtures has a co – toxicity factor. According to the co – toxicity factor values of mixtures, they are classified into three main groups: when the mixtures inhibit the growth completely that mean it has a synergistic effect. Its C. T. F is more than 20, when the mixtures have a moderate effect and its C. T. F lies between 20 and - 20 the mixtures are described to have an additive effect and when the mixture have C. T. F. less than -20 that mean these mixtures have an antagonistic effect. These mixtures are new metabolites that have a new molecular weight, a new chemical formula, new physical and chemical characters that have a new side effect on fungi (pathogenic and antagonistic fungi) and on the seedlings in green house.

After previous, mixtures were tested on the infected plants that grown in pots under greenhouse conditions. By these experiments, it could be distinguished between mixtures, which be effective and could be used in field for controlling the disease.

The horse manure at high rates inhibited the growth of S. rolfsii, favored the development of T. harzianum and enhanced its antagonistic effect on S. rolfsii (Naima et al., 2004).

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Discussion

Singh (2006) reported that the essential oils that extracted of Chenopodium ambrosioides (CA), Lippia alba, Azadirachta indica and Eucalyptus globulus in combinations were found to be more fungitoxic than the individual oils against hyphal growth and sclerotia formation of Sclerotium rolfsii causing foot rot disease of barley (Hordeum vulgare).

In addition, chitinase showed synergistic effect with fungicides (cyprodinil+fludioxonil) and tebuconazole to inhibit fungal conidial germinations of phytopathogenic fungi, Alternaria brassicicola, Botrytis elliptica, and Colletotrichum gloeoporioides (Huang and Chen, 2008) .

Ainhoa et al., (2009) evaluated the interactions between four arbuscular mycorrhizal fungi (Glomus intraradices, Glomus mosseae, Glomus claroideum and Glomus constrictum) and Trichoderma harzianum for their effects on melon plant growth and biocontrol of Fusarium wilt in seedling nurseries.

From 11 Pseudomonas spp. isolated from rhizospheric soil, a soil bacterium identified as, Pseudomonas monteilii 9, showed highest antagonistic activity against Sclerotium rolfsii stem rot disease in groundnut (Rakh et al., 2011).

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Discussion

II- Green house experiments ( In vivo): 1- Effect of different antifungal treatments and their combinations on survival plants.

The pathogenic fungus infested all pots one week early before sowing the sees in order to allow the pathogenic fungus to multiply and spread in the soil to cause infection for Cicer arietinum seeds in the following experiment. Some pots which were designed to be treated by Gliocladium virens and Gliocladium deliquescens were treated by the same steps; each pot was sowed by 10 seeds of Cicer arietinum (the host plant). Data in tables (11 and 12) showed that infection appeared on the host plants in the form of:

1) Pre- emergence root rot that is number of plants infected during 15 days from beginning of sowing (cases of pre- emergence root rot were recorded in the present investigation through out the experiment period). The fungus may cause decaying the cells and tissues of cotyledon of seeds to damage seed embryo so that, it prevent germination.

2) Post- emergence root rot, which refers to the number of plants infected during 45 days from beginning of sowing. In this case, the S. rolfsii infest the transition zone, which located between the shoot and root regions of host plants (Cicer arietinum) to macerate the tissue at this zone, so it leads to death of plants and great lose in the yield. In the present investigation the percentage of post emergence damping off ranging from 0% to 30% compared by the healthy and infected control.

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Discussion

S. rolfsii enter and sporulate inside the xylem vessels. After that, it begin to secrete some secondary metabolites that blocks xylem vessels and prevent the water to reach to the upper branches of the plants, so that the plants dry and die. On the other hand, survival plants means plants that did not infect by the pathogenic fungus due to its high immunity. Some treatments raised the acquired immunity of the host plants compared by the healthy and infected control. On the other hand, Conway et al., (2001) revealed the effect of thiram+ carboxin (chemo- primed) and biological seed treatment (bio- primed Thrichoderma harzianum isolate. 1g suspended in 1 % carboxymethylcellulose). Chemo- primed seeds had a more uniform and faster emergence compared with untreated seeds.

Luiz and Rodrígo (2004) showed that high amounts of benzaldehyde (0.4 ml kg -1 of soil) and velvetbean (100 g kg -1) inhibited Sclerotium rolfsii, mycelial growth and sclerotium germination. However, low amounts of benzaldehyde (0.1 ml kg -1), kudzu (25 g kg -1), and pine-bark (25 g kg -1) stimulated mycelial growth and sclerotium germination. Kudzu (25-100 g kg -1) and velvetbean (25-100 g kg -1) inhibited the formation of sclerotia. Benzaldehyde at 0.2 and 0.4 ml kg-1 stimulated the formation of sclerotia. Kudzu (50 and 100 g kg -1) and pine-bark (50 g kg -1) favored the colonization of sclerotia by Trichoderma sp. Fouzia and Saleem (2005) Increase in inoculum density of Sclerotium rolfsii caused gradual reduction in growth parameters of sunflower and mungbean plants whereas there was a positive correlation between root colonization and population of S. rolfsii in soil.

- 182 -

Discussion

Shahid et al., (2006) aqueous plant leaf extracts of Datura alba and Calotropis procera were found effective 1.5 and 2% followed by C. sativus (2%) in reducing the mycelial growth of Sclerotium rolfsii over the non amended control.

2- Effect of different antifungal treatments and their combinations on different growth parameters of Cicer arietinum.

Our results recorded that the highest plant height in case of (copprus KZ, starner, vitavax + copprus KZ, vitavax + starner, copprus KZ + starner, copprus KZ + GV, copprus KZ + GD, starner + GV, starner + GD) as inducers induce cell division at root and shoot apex, cell elongation and cell differentiation. This leads to an increase in plant length, plant fresh weight and plant dry weight.

Ouf et al., (1999) mentioned that pine needles extract were effective in reducing damping off of cucumber seedlings after 28 days in soil infested with R. solani. Complete protection of cucumber was achieved after addition of Penecillum oxalicum to the amended soil.

However Mathivan et al., (2000) mentioned that coating seeds with the deferent doses of the biofungicides (Trichoderma viridi) increased shoot length in cotton, okra and sunflower.

- 183 -

Discussion

Our results are similar to El – Kafrawy (2000) as he recorded that the soil treatment with T. harzianum, T. hamatum, T. viridi and G. virens imoroved plant fresh weight of Phaseolus vulgaris.

Frank et al., (2005) reported the potential of Piriformospora indica to induce resistance to fungal diseases and tolerance to salt stress in the monocotyledonous plant barley in India. The systemically altered “defense readiness” is associated with an elevated antioxidative capacity due to an activation of the glutathione-ascorbate cycle and results in an overall increase in grain yield.

On the other hand, Shama (2006) reported that percentage of infected leaves of cucumber plants grown under greenhouse and cucumber downy mildew disease severity in inoculated plants were significantly reduced when nutrient solution amended with silicon or calcium. Best results were obtained in nutrient solution amended with 100 ppm silicon.

Abd EL-Ghany et al., (2009) found that the application of sodium silicate decreased severity of infection of leaf rust in woody plants caused by Salix tetrasperma in Egypt. The percentage of infected leaves was 51.53, 47.0, 46.44 and 22.88 respectively. On the other hand, application of Trichoderma harzianum and Saccharomyces cerevisiae gave reasonable control of leaf rust severity with infected leaves percentages of 10.69 and 19.18 % respectively compared with control.

- 184 -

Discussion

3- Effect of different antifungal treatments and their combinations on chlorophyll (a), chlorophyll (b) and total chlorophyll Cicer arietinum.

In case of plants treated by (copprus KZ, starner, vitavax + copprus KZ, vitavax + starner, copprus KZ + starner, copprus KZ + GV, copprus KZ + GD, starner + GV, starner + GD) inducers lead to an increase in the area of green leaves, a increase in the number of green leaves and an increase in the intensity of chlorophyll in each green leave and this leads to an increase in the amount of chlorophyll (a), chlorophyll (b), and total chlorophyll in our host plants (Cicer arietinum).

At the same time Alabi et al., (2005) found that Sclerotium rolfsii induced wilting on cowpea grown in Ago-Iwoye, South Western Nigeria between 4 and 12% of cowpea seedlings treated with plant extracts Vernonia amaygdalina, Bryophyllum. pinnatus, Ocimum gratissimum and Eucalyptna globules under field conditions while about 39.6% incidence of cowpea seedlings wilting under control experiment on the same experimental plot. The extracts increased significantly the plant height, shelf life, relative water content and chlorophyll contents of the cowpea seedlings. Furthermore, application of these extracts on the cowpea plants significantly enhanced the Leaf Area Index (LAI), number of branches and ponds per plant, total dry matter per plant, weight per pod, 100 grains weight and grain yield. In the mean time Akbudaka et al., (2006) reported that leaf chlorophyll values of the peppers (Capsicum annuum, Yalova Charleston

- 185 -

Discussion and Sari Sivri) exhibited significant decline in the plants subjected to Botrytis cinerea treatment in all cultivars. However, the chlorophyll content in the plants subjected to harpin protein + B. cinerea treatment was low.

On the other hand, Long-Fang et al., (2008) reported that post harvest yellowing in broccoli is known to result from chlorophyll degradation, with chlorophyllase enzyme, which degrade chlorophyll In broccoli.

Thamizhiniyan et al., (2009) found that the higher growth and biochemical content chlorophyll (a), chlorophyll (b), total chlorophyll, protein, starch and amino acid contents of Coleus aromaticus Benth plant was observed when the plants inoculated by Azospirillum + mycorrhizal fungi when compared with control plants.

4- Effect of different antifungal treatments and their combinations on enzyme activity (peroxidase and polyphenol oxidase).

Peroxidase and polyphenol oxidase enzymes were produced by the host plant (Cicer arietinum) as a defense mechanism against the invasion by Sclerotium rolfsii. As the amount of these enzymes increase in the host tissue, the pathogenecity of the fungus decreased. treatments (copprus KZ, starner, vitavax + copprus KZ, vitavax + starner, , copprus KZ + starner, copprus KZ + GV, copprus KZ + GD, starner + GV, starner + GD) enhance the acquired immunity of the host plant against the pathogenic fungus S.

- 186 -

Discussion rolfsii and as a result, the amount of (peroxidase and polyphenol oxidase enzymes) were increased compared to the healthy and infected control. The increase in peroxidase and polyphenol oxidase enzymes increases the percentage of survival plants in the recommended treatments.

In plants, polyphenol oxidase and peroxidase has a role in defense against pathogens. There is a positive correlation between levels of polyphenol oxidase and peroxidase enzymes and the resistance to pathogens

In agreement with Geraldo et al., (2006) investigated polyphenol oxidase (PPO) related to defense mechanism against pathogens in coffee trees regarding resistance against a leaf miner and coffee leaf rust disease. There were compatible and incompatible interactions of coffee with the fungus Hemileia vastatrix, the causal agent of the leaf orange rust disease and the insect Leucoptera coffeella (coffee leaf miner). The constitutive level of PPO activity observed for the 15 genotypes ranged from 3.8 to 88 units of activity/mg protein.

However, Laura and Drew (2007) reported that the enzymatic defense mechanisms of Gorgonia ventalina to the fungal pathogen Aspergillus sydowii play important roles in colony resistance to infection. Peroxidase activity was induced after an 8 day incubation period.

In addition, Rai et al., (2008) found that there was changes in the respiration and peroxidase activity of Bajra (Pennisetum typhoides) tissues affected with "Green ear" disease.

- 187 -

Discussion

Ghobrial et al., (2009) reported that application of some biofertilizers such as Rhizobium leguminosarum biovar viciae and prepared tea compost enriched with selected plant growth-promoting rhizobacteria (PGPR) increase the activities of peroxidase and polyphenol oxidase enzymes faba bean plants infected with bean mottle virus (BBMV).

III- Effect of gamma irradiation. 1- Effect of gamma irradiation on the antagonism between Gliocladium virens and Gliocladium deliquescence against S. rolfsii.

In the present work, the results showed that due to the increase of G. virens and G. deliquescence viability by increasing the dose levels of gamma radiation to dose 1.0 kGy of G. virens and 2.0 kGy of G. deliquescens, the antagonistic activity increased as a biological control mean against S. rolfsii. Several workers reported that, gamma irradiation could enhance mould growth and toxic metabolites biosynthesis (Applegate and Chipley, 1973). Furthermore, El-Zawahry and Mostafa (1987) investigated that gamma irradiation could be used to increase the production of cellulase enzyme by certain fungi as Trichoderma, Asperigillus, Penecillum and Fusarium. On the other hand, Khalaf, M.A. (1993) investigated that gamma irradiation decreased the antagonistic effect of Trichoderma lignorum in controlling damping- off in bean caused by Rhizoctonia solani. Neveen et al., (2006) who noticed that the lower doses of gamma irradiation increased total proteins and total soluble sugars of Alternaria

- 188 -

Discussion tenuissima, Botrytis cinerea, Penicillium expansum and Stemphylium botryosum but did not affect lipid synthesis. Abo-State et al., (2010) Aspergillus spp. that was isolated from agriculture wastes which exposed to 0.5 kGy produced higher than the parent strain.

2- Effect of gamma irradiation on Cicer arietinum seed germination.

Gamma irradiation of seed has been found to exert pronounced effects on plant growth. Any change in growth pattern will ultimately affect maturity and yield. The objective of this work was investigated whether pre-treatment of dry seeds of chickpea plants with different gamma irradiation doses before planting could act as a protective agent to the influence of root rot.

These results were detected by green house conditions which, soil treated with gamma irradiated bioagents increase percentage of survival plants, plant height, fresh weight and dry weight compared with unirradiated control. Our results show that decreased seed germination of Cicer arietinum by used gamma irradiation doses (5, 10, 15, 20, 25 and 30 Gy). As regard to different gamma irradiation doses it is clear that 30 Gy gave the lowest result regarding significant decrease than the unirradiated control.

Gamma rays belong to ionizing radiation and interact on atoms or molecules to produce free radicals in cells. These radicals can damage or

- 189 -

Discussion modify important components of plant cells and have been reported to affect differentially the morphology, anatomy, biochemistry and physiology of plants depending on the irradiation level. These effects include changes in the plant cellular structure and metabolism such as dilation of thylakoide membranes, alteration in photosynthesis, modulation of the antioxidative system and accumulation of phenolic compounds (Wi et al., 2005).

Several researches have studied effect of gamma irradiation on seed germination of plants. A number of morphological changes have been noted in the plants after exposure to gamma irradiation. The response varied considerably depending upon dose rate, duration of exposure, the particular species observed, the age of developmental stage of plant part irradiated and upon the physiological conditions of the plant (Gunckel et al., 1953, Gunckel, 1957).

Reduced growth has been attributed to auxin destruction, changes in ascorbic acid content and physiological and biochemical disturbance (Usuf and Nair, 1974). The reduction in the shoot length may be attributed to the damage to the process of cell division and cell elongation that generally result after mutagenic treatment (Iqbal, 1969).

Khalil et al., (1986) attributed decreased shoot and root lengths at gamma irradiation doses to reduced mitotic activity in meristematic tissues and reduce moisture content in seeds respectively.

- 190 -

Discussion

On the other hand, Habba (1989) reported that increasing dose of gamma irradiation up to 100 Gy gradually increased the germination percentage in Hyoscyamus muticus. The stimulating effects of gamma irradiation on germination may be attributed to the activation of RNA synthesis (Kuzin et al., 1975).

Cepero et al., (2001) observed maximum stimulating effect of gamma irradiation low doses (150 and 180 Gy) on seedling height of Leucaena leucocephala.

Dubey et al., (2007) showed an increase in plant height, number of leaves and branches per plant when okra seeds were irradiated with different doses of gamma irradiation.

3- Effect of different gamma irradiation doses on pathogenecity and infection process of S. rolfsii.

In this study, gamma irradiation at doses (0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 kGy) increase the pathogenecity of S. rolfsii on Cicer arietinum, while gamma irradiation at dose 5 kGy completely inhibited the growth of S. rolfsii. So, gamma irradiation at dose 5 kGy considered lethal dose.

These results due to an important advantage of gamma irradiation over most chemical treatments, which drives from its short wavelength, is its ability to penetrate into the tissues. This enables irradiation to reach not microorganisms which located within the host, as active infections (Barkai-

- 191 -

Discussion

Goldan, 2001). However, radiation doses required for direct suppression of postharvest pathogens are generally above the tolerance level of the fruit and result in radiation damage (Barkai - Golan, 1992). Some times, irradiation can not destroy pathogens completely but it may results in cell damage (Smith and Pillai, 2004), and directly harm the chromosomal DNA of living cell (Barkai- Goldan, 2001).

Complete inhibition of fungal growth has been reported that it results by gamma ray destroy DNA structure of cells and cells can not continue their function (Smith and Pillai, 2004), while incomplete inhibition may result from a little injury of cells (Aubrey, 2002).

Tauxe (2001) reported that the high energy rays of irradiation directly damage the DNA of living organisms, including growth linkages and other changes that make an organism unable to grow or reproduce. When these rays interact with water molecules in an organism, they generate transient free radicals that can cause additional direct damage to DNA.

In the present study, at dose 0.25 kGy the infected plants was 80 % and the survival plants was 20 %. The pathogenecity of S. rolfsii increased by increasing of gamma radiation dose to give 100 % of infected plants at dose 3 kGy. On the other hand, at 5 kGy the there was no infected plants and the survival plants was 100 % due to the death of the pathogenic fungus. These attribute to changes or non changes of pathogenecity of fungi after irradiated treatment deepened on type of fungi, a level of tolerance, their characteristic and the doses of ray.

- 192 -

Discussion

As the study of Gherbawy (1998) showed that the lowest dose of gamma irradiation enhance virulence of Aspergillus niger by producing more polygalacturonase, cellulose and protease, while the higher doses were inhibitory to the growth of fungi.

4- Purification of polygalacturonase produced by gamma irradiated and unirradiated isolates of S. rolfsii.

It is known that many micro-organisms are able to produce pectic enzymes and research has shown that the degradation of pectin is due to the action of a complex of enzymes, including polygalacturonase, pectinesterase and pectin lyase (Konno et al., 1986). The action patterns of polygalacturonase may be either of the endo-type (random cleavage of the polymer), or exo-type (terminal cleavage) (Rexova and Markovico, 1976).

We have already shown that S. rolfsii produced an extracellular enzyme with a polygalacturonate depolymerizing activity. The affinity of the enzyme to polygalacturonate, and the acidic pH optimum suggest that this enzyme may be characterized as a polygalacturonase (Ried and Colmer, 1986).

In our study, the polygalacturonase of unirradiated isolate of S. rolfsii (control) was purified to the yield of 52.8% with the specific activity of 82.2 U/mg, on the other hand The polygalacturonase of irradiated isolates of S. rolfsii at doses (0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3 kGy) was purified to the

- 193 -

Discussion yields of (50.6, 52.3, 48.6, 50.9, 44.26, 43.6, 41.8%), respectively, with the specific activity of (67.5, 117.7, 105, 622, 1328, 99.7, 301.4 U/mg), respectively.

The enzyme activity of polygalacturonase at dose 5 kGy was not detected because it is considered as the lethal dose of gamma radiation to the isolate. The homogeneity of the purified enzymes was tested by polyacrylamide gel electrophoresis (SDS−PAGE). Each of them had a single band visualized when stained with Coomassie Brilliant Blue which has a molecular weigh of 72 kDa so they were homogeneous.

In the same work El- Batal and Abo- State (2006) produced cellulases, xylanase, and alpha amylase by Bacillus sp. And their mixed cultures with Candida tropicalis and Rhodotorula glutinis under solid state fermentation. Also, Basil (1984) produced cellulase and α-Glucosidase from a mutant of Alternaria alternate.

On the other hand, Sema et al., (2010) recorded that as the radiation dose increased, the concentrations of iron, copper, zinc, soluble protein, and malondialdehyde in soybean seeds increased, but the activities of superoxide dismutase and peroxidase enzymes activities were significantly decreased. Production of pectinases increases by gamma irradiated inter specific hybrids of Aspergillus sp. using agro- industrial wastes recorded by (El- Batal and Khalaf, 2002).

- 194 -

Discussion

El- Batal and Khalaf (2003) use wheat bran as a substrate for enhanced thermostable alpha- amylase production by gamma irradiated Bacillus megaterium in solid-state fermentation. Also, in this context the enhancement of phenylalanine ammonium lyase enzymeby gamma irradiation of Rhodotorula glutinisyeast was recorded by (El- Batal et al., 2000).

- 195 - Summary

Summary

In this investigation Sclerotium rolfsii was grown on PDA medium. It was found that it contains white knobs varying in size and shape until they reach to the brown and mature sclerotia after 3 days from inoculation. Sclerotia of S. rolfsii was found to consist of two main zones, the external zone or pseudoparanchymatus zone and the internal zone (the zone of microsclerotia), after that an artificial experiment was carried out on Cicer arietinum seedlings and seeds to cause Cicer arietinum seeds rot disease and show different developments of the fungus in Cicer arietinum seeds .

In the present investigation the fungus was detected inside the cortex and xylem tissues and it was found that the fungus secrets secondary metabolites that block xylem vessels and prevent the passage of water to the whole plants. The fungus macerate the tissues of the plant and cause damping off disease to Cicer arietinum seedlings, after that, the fungus was detected and photographed inside the root system of the Cicer arietinumm host during the experiment period. Antagonism between (Gliocladium virens against S. rolfsii and Gliocladium deliquescens against S. rolfsii) was recorded. It was found that G. deliquescens and G. virens hyphae lyse the fungus cell wall of Sclerotium rolfsii to prevent its growth. On the other hand, Trichoderma hamatum has no antagonistic effect on S. rolfsii. The culture filtrate of G. virens and G. deliquescens after 1, 2, 3 weeks of inoculation was found to have great antimicrobial activity against the growth of S. rolfsii in vitro as it contain the following antibiotics (gluoveriens, gluotoxin and sidrofore compounds).

- 196 - Summary

Antimicrobial activity of fungicides vitavax and monceren / thiram

was determined. It was found to have ICR50R 0.9 and 1.2, respectively. Monceren / thiram has antifungal effect on bioagents G. virens and G. deliquescens higher than vitavax for that we used fungicide vitavax in the following experiments. In this investigation, it was found that mint oil and clove oil have a great antimicrobial activity on the growth of S. rolfsii and no effect on the growth of G. virens and G. deliquescens.

Starner and copprus KZ inducers have no antifungal effect on the growth of the pathogenic fungus and the antagonistic fungi. In the present investigation it was fount that joint toxic effect of some compounds and their combinations were tested on the growth of S. rolfsii. These compounds have (an additive, synergstic or antagonistic effect) on the growth of pathogenic fungus (S. rolfsii).

Vitavax, mint oil, clove oil, starner and copprus KZ have no phytotoxic effect at low concentrations on the growth of Cicer arietinum seedlings. The pathogenecity test of S. rolfsii was measured in vivo during the period from years 2007- 2008. November was the best suitable month for the growth of Cicer arietinum seeds in green house and sterilization by autoclave was the best sterilization for soil. 1/2 clay and 1/2 sandy soil was the best soil for cultivation of Cicer arietinum seeds, 0.25% for 1 kg of sterilized soil was the best inoculum for the experiment. Different combinations (vitavax, clove oil, mint oil, copprus KZ, starner, Gliocladium virens, Gliocladium deliquescens, vitavax + clove oil, vitavax + mint oil, vitavax + copprus KZ, vitavax + starner, vitavax + G.

- 197 - Summary

virens, vitavax + G. deliquescens, clove oil + mint oil, copprus KZ + starner, G. virens + G. deliquescens, clove oil + G. virens, clove oil + G. deliquescens , mint oil + G. virens, mint oil + G. deliquescens, copprus KZ + G. virens, copprus KZ + G. deliquescens, starner + G. virens, starner + G. deliquescens) were tested in green house, but (copprus KZ, starner, vitavax + copprus KZ, vitavax + starner, copprus KZ + starner, copprus KZ + G. virens, copprus KZ + G. deliquescens, starner + G. virens, starner + G. deliquescens) achieved the best survival plants, plant height, plant dry weight, plant fresh weight, chlorophyll (a, b, and total chlorophyll) and enzyme activity (peroxidase and polyphenol oxidase enzymes).

The effect of gamma irradiation was studied where the effect of gamma irradiation at doses (0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 kGy) on the antagonistic effect of Gliocladium virens and Gliocladium deliquescens against Sclerotium rolfsii was demonstrated. It was found that the antagonistic effect of GV and GD increase by increasing gamma irradiation to give maximum antagonistic effect at doses 1.0 kGy for GV and 2.0 kGy for GD. Irradiation of Cicer arietinum seeds at gamma irradiation doses (5, 10, 15, 20, 25 and 30 Gy) decrease seed germination, plant height, fresh weight and dry weight of Cicer arietinum seeds.

In green house, gamma irradiated GV and gamma irradiated GD increase survival plant percentage compared by unirradiated (control). Gamma irradiation affects on the external morphology of the pathogenic fungus at gamma irradiation doses (0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 kGy).

- 198 - Summary

Gamma irradiation increases the pathogenecity of the pathogenic fungus on Cicer arietinum at radiation doses (0.25 up to 3.0 kGy), on the other hand gamma radiation at dose 5 kGy inhibit the growth of S. rolfsii.

Polygalacturonase was found to be the most important and effective enzyme of S. rolfsii facilities the fungus invasion into plant tissues. This study includes purification of polygalacturonase of irradiated and unirradiated isolates of S. rolfsii at doses (0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 kGy). This technique was achieved by precipitation by ammonium sulphate (saturation 70%) as primary step in purification technique, and then the enzyme was purified by Sephadex G- 75).

The activity of polygalacturonase produced of irradiated isolates increased with the increasing of irradiation dose to give maximum enzyme activity at gamma irradiation dose 3.0 kGy compared to unirradiated (control).

Purification of polygalacturonase was approved by determination molecular weight of enzyme isolates by using SDS- poly acrylamide gel electrophoresis and it was found to be (72 kDa).

- 199 - Conclusion

Conclusion In the present study, root- rot disease of chickpea which caused by the pathogenic fungus Sclerotium rolfsii the most important disease that causing great losses in chickpea crop. It was found that November was the best suitable month for the growth of Cicer arietinum seeds and 2.5% for 1 kg of sterilized soil was the best inoculum for the experiment in green house. The combinations (vitavax + copprus KZ, vitavax + starner, copprus KZ + starner, copprus KZ + Gliocladium virens, copprus KZ + G. deliquescens, starner + G. virens, starner + G. deliquescens) achieved the best survival plants, plant height, plant dry weight, plant fresh weight, chlorophyll (a, b, and total chlorophyll) and enzyme activity (peroxidase and polyphenol oxidase enzymes) in green house. Gamma irradiation of G. virens and G. deliquscens increase the antagonistic effect of them against S. rolfsii in vitro and in vivo. Gamma irradiation of chickpea seeds at doses (5, 10, 15, 20, 25 and 30Gy) has no effect in controlling root rot disease of chickpea. The effect of gamma irradiation on S. rolfsii was studied. Polygalacturonase was found to be the most important offensive force of isolated fungi which facilities the fungus invasion into plant tissues. It was purified, and characterized for unirradiated and irradiated S. rolfsii isolates. It was found that the activity of polygalacturonase produced of irradiated isolates increased with the increasing of irradiation dose to give maximum enzyme activity at gamma irradiation dose 3.0 kGy compared to unirradiated control. These results were detected by determination molecular weight of enzyme isolates by SDS- poly acrylamide gel electrophoresis to be (72 kDa).

- 200 - References

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- 234 - Arabic summary

ﺍﻟﻤﻠﺨﺺ ﺍﻟﻌﺮﺑﻰ

ﻣﻜﺎﻓﺤﺔ ﻣﺮﺽ ﺍﻟﻌﻔﻦ ﺍﻟﺠﺬﺭﻯ ﻟﻨﺒﺎﺕ ﺍﻟﺤﻤﺺ ﺍﻟﻤﺴﺒﺐ ﺑﻮﺍﺳﻄﺔ ﺍﻹﺳﻜﻠﻮﺭﻭﺷﻴﻢ ﺭﻭﻟﻔﺴﺎﻯ ﺑﺎﺳﺘﺨﺪﺍﻡ ﻋﻮﺍﻣﻞ ﻣﺨﺘﻠﻔﺔ ﻭﺃﺷﻌﺔ ﺟﺎﻣﺎ

. ﻓﻲ ﻫﺬﺍ ﺍﻟﺒﺤﺚ ﺗﻢ ﺯﺭﺍﻋﺔ ﻓﻄﺮ ﺍﻹﺳﻜﻠﻮﺭﻭ ﺷ � ﻴ ﻢ ﺭﻭﻟﻔ ﺴ � ﺎﻯ ﻋﻠ�ﻰ ﺑﻴﺌ�ﺔ PDA ﻭﺑﻌ�ﺪ ﺃﺧ�ﺬ ﻗﻄﺎﻋ�ﺎﺕ ﻛﺎﻣﻠﺔ ﺑﻪ ﻭﺟﺪ ﺃﻧﻪ ﻳﺘﻜﻮﻥ ﻣ�ﻦ ﻋﻘ�ﺪ ﺧﻄﻴ�ﺔ ﺑﻴﻀ�ﺎء ﺍﻟﻠ�ﻮﻥ ﺗﺨﺘﻠ�ﻒ ﻓ�ﻲ ﺍﻟﺤﺠ�ﻢ ﺣﺘ�ﻲ ﻳ�ﺘﻢ ﺍﻟﻨﻀ�ﺞ ﺍﻟﻜﺎﻣ�ﻞ ﻟﻺﺳﻜﻠﻮﺭﺷ��ﻴﺎ ﻭﺗﺼ��ﺒﺢ ﻋﻠ��ﻲ ﺷ��ﻜﻞ ﺳﻜﻠﻮﺭﺷ��ﻴﺎ ﺑﻨﻴ��ﺔ ﺍﻟﻠ��ﻮﻥ ﺑﻌ��ﺪ ﺛﻼﺛ��ﺔ ﺃﻳ��ﺎﻡ ﻛﻤ��ﺎ ﺗ��ﻢ ﺃﺧ��ﺬ ﻗﻄﺎﻋ��ﺎﺕ ﻓ��ﻲ ﺍﻹﺳﻜﻠﻮﺭﺷﻴﺎ ﺍﻟﻨﺎﻣﻴﺔ ﺣﻴﺚ ﻭﺟﺪ ﺃﻧﻬﺎ ﺗﺘﻜﻮﻥ ﻣﻦ ﻃﺒﻘﺘﻴﻦ ﺍﻟﻄﺒﻘﺔ ﺍﻟﺨﺎﺭﺟﻴﺔ ﻋﺒ�ﺎﺭﺓ ﻋ�ﻦ ﺧﻼﻳ�ﺎ ﺑﺮﻧﺸ�ﻴﻤﺔ ﻛﺎﺫﺑﺔﻭﻣﻦ ﺍﻟﺪﺍﺧﻞ ﻋﺒﺎﺭﺓ ﻋﻦ ﺇﺳﻜﻠﻮﺭﺷﻴﺎﺕ ﺻﻐﻴﺮﺓ ﺍﻟﺤﺠﻢ (ﻣﻴﻜﺮﻭ ﺳﻜﻠﺮﻭﺷﻴﻮﻝ) .

. ﺑﻌﺪ ﺫﻟﻚ ﺗﻢ ﻋﻤﻞ ﻋﺪﻭﻱ ﺻﻨﺎﻋﻴﺔ ﻓﻲ ﺑﺬﻭﺭ ﻧﺒﺎﺕ ﺍﻟﺤﻤﺺ ﺑﻤﺮﺽ ﻋﻔﻦ ﺍﻟﺒﺬﻭﺭ ﻭﺗﻢ ﺗﺼ�ﻮﻳﺮﻩ ﻭﺃﺧ��ﺬ ﻗﻄﺎﻋ��ﺎﺕ ﺑ��ﻪ ﺑﻌ��ﺪ ﺷ��ﻬﺮ ﻭﺑﻌ��ﺪ ﺷ��ﻬﺮﻳﻦ ﻭﺑﻌ��ﺪ ﺛﻼﺛ��ﺔ ﺃﺷ��ﻬﺮ ﺃﺛﻨ��ﺎء ﻣﺮﺍﺣ��ﻞ ﺍﻟﻌ��ﺪﻭﻯ ﺍﻟﻤﺨﺘﻠﻔ��ﺔ ﻭﺗ��ﻢ ﺗﺼ��ﻮﻳﺮ ﻣﺤﺘﻮﻳ��ﺎﺕ ﺑ��ﺬﻭﺭ ﻧﺒ��ﺎﺕ ﺍﻟﺤﻤ��ﺺ ﺃﺛﻨ��ﺎء ﺗﺤﻠﻠﻬ��ﺎ ﺧ��ﻼﻝ ﻣﺮﺍﺣ��ﻞ ﺍﻟﻌ��ﺪﻭﻯ ﺍﻟﻤﺨﺘﻠﻔ��ﺔ ﻭﻣﻘﺎﺭﻧﺘﻬ��ﺎ ﺑﺎﻟﻜﻨﺘﺮﻭﻝ ﺣﺘ�ﻲ ﺗﺤﻮﻟ�ﺖ ﻛ�ﻞ ﻣﺤﺘﻮﻳ�ﺎﺕ ﺍﻟﺒ�ﺬﻭﺭ ﺇﻟ�ﻲ ﺳ�ﺎﺋﻞ ﺑﻨ�ﻲ ﻭﺗﺤﻠﻠ�ﺖ ﻛ�ﻞ ﻣﺤﺘﻮﻳ�ﺎﺕ ﺍﻟﻨﺸ�ﺎ ﺍﻟﺪﺍﺧﻠﻴ�ﺔ ﻭﺃﺻﺒﺢ ﺍﻟﻔﻄﺮ ﻳﺘﻮﺍﺟﺪ ﻓﻲ ﺃﻧﺴﺠﺔ ﺍﻟﺒﺬﺭﺓ ﺍﻟﺪﺍﺧﻠﻴﺔ ﻋﻠﻰ ﺷﻜﻞ ﺍﻟﻌﺪﻳﺪ ﻣﻦ ﺍﻹﺳﻜﻠﻮﺭﺷﻴﺎﺕ .

. ﻛﻤ��ﺎ ﺗ��ﻢ ﺇﺣ��ﺪﺍﺙ ﻋ��ﺪﻭﻯ ﺻ��ﻨﺎﻋﻴﺔ ﻟﻠﺴ��ﺎﻕ (ﺍﻟﻤﻨﻄﻘ��ﺔ ﺍﻻﻧﺘﻘﺎﻟﻴ��ﺔ ﻣ��ﻦ ﺍﻟﺠ��ﺬﻭﺭ ﻭﺍﻟﺴ��ﺎﻕ ﻟﻨﺒ��ﺎﺕ ﺍﻟﺤﻤﺺ) ﺑﻔﻄﺮ ﺍﻹﺳﻜﻠﻮﺭﻭﺷﻴﻢ ﺭﻭﻟﻔﺴﺎﻯ ﻛﻤﺎ ﺗﻢ ﺃﺧﺬ ﻗﻄﺎﻋﺎﺕ ﻓﻲ ﺍﻟﻤﻨﻄﻘ�ﺔ ﺍﻻﻧﺘﻘﺎﻟﻴ�ﺔ ﻭﺃﻭﺿ�ﺤﺖ ﻫ�ﺬﻩ ﺍﻟﻘﻄﺎﻋ��ﺎﺕ ﻃﺮﻳﻘ��ﺔ ﺩﺧ��ﻮﻝ ﺍﻟﻤﻴﻜﺮﻭﺳﻜﻠﻮﺭﺷ��ﻴﺎﺕ ﺇﻟ��ﻲ ﺍﻷﻧﺴ��ﺠﺔ ﺍﻟﺪﺍﺧﻠﻴ��ﺔ ﻟﻠﺴ��ﺎﻕ ﻭﺍﻓﺮﺍﺯﻫ��ﺎ ﺑﻌ��ﺾ ﺍﻹﻧﺰﻳﻤﺎﺕ ﺍﻟﺘﻲ ﻳﺤﻠﻞ ﺑﻬﺎ ﺍﻟﻔﻄﺮ ﻃﺒﻘﺔ ﺍﻟﺒﺸﺮﺓ ﻭﺍﻟﻘﺸﺮﺓ ﻟﻨﺒﺎﺕ ﺍﻟﺤﻤﺺ ﺩﺍﺧ�ﻞ ﺃﻧﺴ�ﺠﺔ ﺍﻟﻨﺒ�ﺎﺕ ﺍﻟﺪﺍﺧﻠﻴ�ﺔ. ﻛﻤﺎ ﺗﻢ ﺗﺼﻮﻳﺮ ﺍﻟﻔﻄﺮ ﻓ�ﻲ ﻧﺴ�ﻴﺞ ﺍﻟﻘﺸ�ﺮﺓ ﻭﻧﺴ�ﻴﺞ ﺍﻟﺨﺸ�ﺐ ﻭﻭﺟ�ﺪ ﺃﻧ�ﻪ ﻳﻔ�ﺮﺯ ﻧ�ﻮﺍﺗﺞ ﺃﻳﻀ�ﻴﺔ ﺗﻌﻤ�ﻞ ﻋﻠ�ﻰ ﺍﻧﺴﺪﺍﺩ ﺃﻭﻋﻴﺔ ﺍﻟﺨﺸﺐ ﻭﺗﻤﻨﻊ ﻣﺮﻭﺭ ﺍﻟﻤﻴﺎﻩ ﺑﻬﺎ ﻛﻤﺎ ﻭﺟﺪ ﺃﻥ ﺍﻟﻔﻄﺮ ﻳﺤﻠﻞ ﻛﻞ ﺃﻧﺴﺠﺔ ﺍﻟﺴﺎﻕ ﺍﻟﻤﺨﺘﻠﻔﺔ ﻭﻳﺘﻮﺍﺟﺪ ﺑﻌﺪ ﺫﻟﻚ ﻓﻲ ﺻﻮﺭﺓ ﺍﻟﻌﺪﻳﺪ ﻣﻦ ﺍﻹﺳﻜﻠﻮﺭﺷﻴﺎﺕ ﻭﻳﻈﻞ ﻓﻲ ﺍﻟﺘﺮﺑﺔ .

. ﻛﻤﺎ ﺗﻢ ﺇﺣﺪﺍﺙ ﻋ�ﺪﻭﻯ ﺻ�ﻨﺎﻋﻴﺔ ﻟﻠﺠ�ﺬﺭ ﺑﻔﻄ�ﺮ ﺍﻹﺳﻜﻠﻮﺭﻭﺷ�ﻴﻢ ﺭﻭﻟﻔﺴ�ﺎﻯ ﻭ ﺗ � ﻢ ﺗﺼ�ﻮﻳﺮ ﺍﻟﻔﻄ�ﺮ ﻭﻫﻮ ﻳﺴﺘﻌﻤﺮ ﺍﻟﻄﺒﻘﺔ ﺍﻟﺨﺎﺭﺟﻴ�ﺔ ﻟﺠ�ﺬﻭﺭ ﻧﺒ�ﺎﺕ ﺍﻟﺤﻤ�ﺺ، ﻛﻤ�ﺎ ﺗ�ﻢ ﺗﺼ�ﻮﻳﺮ ﺍﻹﺳﻜﻠﻮﺭﺷ�ﻴﺎ ﻭﻫ�ﻲ ﻣﻠﺘﺼ�ﻘﺔ

- ١ - Arabic summary

ﺑﺎﻟﻄﺒﻘﺔ ﺍﻟﺨﺎﺭﺟﻴﺔ ﻟﺠﺬﺭ ﻧﺒﺎﺕ ﺍﻟﺤﻤﺺ ﻭﺗﺤﻠﻴﻠﻬﺎ ﻟﻠﻄﺒﻘ�ﺎﺕ ﺍﻟﻤﺨﺘﻠﻔ�ﺔ ﺃﻳﻀ�ﺎ ﺗ�ﻢ ﺗﺼ�ﻮﻳﺮ ﺍﻹﺳﻜﻠﻮﺭﺷ�ﻴﺎﺕ ﺍﻟﻌﺼﻮﻳﺔ ﺩﺍﺧﻞ ﻧﺴﻴﺞ ﺍﻟﺨﺸﺐ ﻓﻲ ﻧﺒ�ﺎﺕ ﺍﻟﺤﻤ�ﺺ ﻭﺗ�ﻢ ﺗﺼ�ﻮﻳﺮ ﻃﺒﻘ�ﺎﺕ ﺍﻟﺠ�ﺬﺭ ﺍﻟﻤﺨﺘﻠﻔ�ﺔ ﻭﻫ�ﻲ ﻣﺘﺤﻠﻠ�ﺔ ﺗﺤﻠ�ﻼً ﻛ�ﺎﻣﻼً ﻛﻤ�ﺎ ﺗ��ﻢ ﺗﺼ�ﻮﻳﺮ ﺍﻟﻔﻄ�ﺮ ﻭﻫ��ﻮ ﻣﺘﻮﺍﺟ�ﺪ ﻓ�ﻲ ﺻ��ﻮﺭ ﺇﺳﻜﻠﻮﺭﺷ�ﻴﺎ ﻭﻳﺒﻘ�ﻲ ﻓ��ﻲ ﺍﻟﺘﺮﺑ�ﺔ ﻓ�ﻲ ﻫ��ﺬﻩ ﺍﻟﺼﻮﺭﺓ ﻭﺍﻟﺘﻰ ﺗﻤﻜﻦ ﺍﻟﻔﻄﺮ ﻣﻦ ﺇﺣﺪﺍﺙ ﻋﺪﻭﻯ ﺟﺪﻳﺪﺓ ﻟﻨﻔﺲ ﺍﻟﻤﺤﺼﻮﻝ ﻓﻰ ﺳﻨﻮﺍﺕ ﻣﺘﺘﺎﻟﻴﺔ.

. ﻓ��ﻲ ﻫ��ﺬﻩ ﺍﻟﺪﺭﺍﺳ��ﺔ ﺗ��ﻢ ﻣﻌﺮﻓ��ﺔ ﺍﻟﺘﻀ��ﺎﺩ ﺍﻟﻔﻄ��ﺮﻯ ﺑ��ﻴﻦ ﻓﻄ��ﺮﻯ ﺍﻟﺠﻠﻴ��ﻮﻛﻼﺩﻳﻢ ﺩﻟﻴﻜﻮﻳﺴ��ﻨﺲ ﻭ ﺍﻟﺠﻠﻴﻮﻛﻼﺩﻳﻢ ﻓﺮﻳﻨﺲ ﺿﺪ ﻧﻤﻮ ﻓﻄﺮ ﺍﻹﺳﻜﻠﻮﺭﺷﻴﻢ ﺭﻭﻟﻔﺴﺎﻯ ﺣﻴﺚ ﻭﺟ�ﺪ ﺃﻥ ﻫ�ﺬﻩ ﺍﻟﻔﻄﺮﻳ�ﺎﺕ ﺗﻤﻨ�ﻊ ﻧﻤ�ﻮ ﺍﻟﻔﻄ��ﺮ ﺍﻟﻤﻤ��ﺮﺽ (ﺇﺳﻜﻠﻮﺭﺷ��ﻴﻢ ﺭﻭﻟﻔﺴ��ﺎﻯ) ﻭﺗﺴ��ﺒﺐ ﻟ��ﻪ ﻣﻴﻜﻮﺑﺎﺭﺍﺳ��ﻴﺘﺰﻡ ﺣﻴ��ﺚ ﺗﻠﺘ��ﻒ ﻫﻴﻔ��ﺎﺕ ﻓﻄ��ﺮﻯ ﺍﻟﺠﻠﻴﻮﻛﻼﺩﻳﻢ ﺩﻟﻴﻜﻮﻳﺴﻨﺲ ﻭ ﺍﻟﺠﻠﻴﻮﻛﻼﺩﻳﻢ ﻓﺮﻳﻨﺲ ﻋﻠﻰ ﺧﻴﻮﻁ ﺍﻟﻔﻄﺮ ﺍﻟﻤﻤﺮﺽ ﻭﺗﺒﺪﺃ ﻓﻲ ﺗﺤﻠ ﻴ � ﻞ ﺟ�ﺪﺍﺭ ﻓﻄ��ﺮ ﺍﻹﺳﻜﻠﻮﺭﺳ��ﻴﻢ ﺭﻭﻟﻔﺴ��ﺎﻯ ، ﻛﻤ��ﺎ ﻭﺟ��ﺪ ﺃﻥ ﻓﻄ��ﺮ ﺗﺮﻳﻜﻮﺩﺭﻣ��ﺎ ﻫﺎﻣ��ﺎﺗﻢ ﻟ��ﻴﺲ ﻟ��ﻪ ﻧﺸ��ﺎﻁ ﺇﺑ��ﺎﺩﻱ ﻟﻔﻄ��ﺮ ﺍﻹﺳﻜﻠﻮﺭﺷﻴﻢ ﺭﻭﻟﻔﺴﺎﻯ ﻭﻗﺪ ﺗﻢ ﺍﺳﺘﺨﺪﺍﻡ ﺍﻟﺮﺍﺷﺢ ﺍﻟﻔﻄ�ﺮﻯ ﺍﻟﺠﻠﻴ�ﻮﻛﻼﺩﻳﻢ ﺩﻟﻴﻜﻮﻳﺴ�ﻨﺲ ﻭ ﺍﻟﺠﻠﻴ�ﻮﻛﻼﺩﻳﻢ ﻓﺮﻳﻨﺲ ﺑﻌﺪ ﺃﺳ�ﺒﻮﻉ ﻭﺃﺳ�ﺒﻮﻋﻴﻦ ﻭﺛﻼﺛ�ﺔ ﺃﺳ�ﺎﺑﻴﻊ ﻭﻭﺟ�ﺪ ﺃﻥ ﻫ�ﺬﺍ ﺍﻟﻨﺸ�ﺎﻁ ﺍﻹﺑ�ﺎﺩﻯ ﻟﻔﻄ�ﺮﻯ ﺍﻟﺠﻠﻴ�ﻮﻛﻼﺩﻳﻢ ﺿﺪ ﺍﻟﻔﻄﺮ ﺍﻟﻤﻤﺮﺽ ﻳﺰﺩﺍﺩ ﺑﺰﻳﺎﺩﺓ ﻓﺘﺮﺓ ﺗﺤﻀﻴﻦ ﺍﻟﺮﺍﺷﺢ. . ﻛﻤ��ﺎ ﺗ��ﻢ ﻓ��ﻲ ﻫ��ﺬﻩ ﺍﻟﺪﺭﺍﺳ��ﺔ ﺗﻘ��ﺪﻳﺮ ﺍﻟﻨﺸ��ﺎﻁ ﺍﻹﺑ��ﺎﺩﻱ ﻟﻤﺒﻴ��ﺪﻯ ﺍﻟﻔﻴﺘﺎﻓ��ﺎﻛﺲ ﻭﺍﻟﻤﻮﻧﺴ��ﺮﻳﻦ ﺛﻴ��ﺮﺍﻡ ﻭﻭﺟﺪ ﺃﻥ ﻟﻬﻤﺎ ﻧﺸﺎﻁ ﺇﺑﺎﺩﻱ ﻋﺎﻟﻰ ﻋﻠﻰ ﻓ ﻄ � ﺮ ﺍﻹﺳﻜﻠﻮﺭﺷ�ﻴﻢ ﺭﻭﻟﻔﺴ�ﺎﻯ (ﺍﻟﺘﺮﻛﻴ�ﺰ ﺍﻟﻤﺜ�ﻴﻂ ﻟ�ـ ٥٠% ﻣ�ﻦ ﻧﻤﻮ ﺍﻟﻔﻄﺮ ﺍﻟﻤﻤﺮﺽ ﻟﻬﻢ ﻛﺎﻥ ﻋﻠ�ﻰ ﺍﻟﻨﺤ�ﻮ ﺍﻟﺘ�ﺎﻟﻲ ٠.٩ ـ ١.٢) ﻛﻤ�ﺎ ﻭﺟ�ﺪ ﺃﻥ ﻟﻠﻤﻮﻧﺴ�ﺮﻳﻦ ﺛﻴ�ﺮﺍﻡ ﺗ�ﺄﺛﻴﺮﺍ ﺇﺑﺎﺩﻳ��ﺎً ﻋﻠ��ﻰ ﺍﻟﻔﻄﺮﻳ��ﺎﺕ ﺍﻟﻤﻀ��ﺎﺩﺓ ﺃﻋﻠ��ﻰ ﻣ��ﻦ ﺍﻟﻔﻴﺘﺎﻓ��ﺎﻛﺲ ﻟ��ﺬﻟﻚ ﺗ��ﻢ ﺍﺳ��ﺘﺨﺪﺍﻡ ﻣﺒﻴ��ﺪ ﺍﻟﻔﻴﺘﺎﻓ��ﺎﻛﺲ ﻓ��ﻰ ﺑ��ﺎﻗﻰ ﺍﻟﺘﺠﺎﺭﺏ (ﺍﻟﻤﻘﺎﻭﻣﺔ ﺍﻟﺤﻴﻮﻳﺔ) .

. ﻛﻤﺎ ﺗﻢ ﺍﺧﺘﺒﺎﺭ ﺗﺄﺛﻴﺮ ﺯﻳﺖ ﺃﻭﺭﺍﻕ ﻧﺒﺎﺕ ﺍﻟﻨﻌﻨﺎﻉ ﻭﺯﻳﺖ ﺑﺮﺍﻋﻢ ﻧﺒﺎﺕ ﺍﻟﻔﺮﻧﻔﻞ ﻋﻠﻰ ﺍﻟﻨﺸﺎﻁ ﺍﻹﺑ�ﺎﺩﻱ ﻟﻔﻄﺮ ﺍﻹﺳﻜﻠﻮﺭﺷ�ﻴﻢ ﺭﻭﻟﻔ ﺴ � ﺎﻯ ﻭﻛ�ﻞ ﻣ�ﻦ ﺍﻟﻔﻄﺮﻳ�ﺎﺕ ﺍﻟﻤﻀ�ﺎﺩﺓ ﻭﻛ�ﺎﻥ ﻟﻬ�ﻢ ﺗ�ﺄﺛﻴﺮ ﺇﺑ�ﺎﺩﻱ ﻋ�ﺎﻟﻲ ﻋﻠ�ﻲ ﺍﻟﻔﻄ�ﺮ ﺍﻟﻤﻤﺮﺽ (ﺇﺳﻜﻠﻮﺭﺷﻴﻢ ﺭﻭﻟﻔﺴﺎﻯ) ﻭﻟﻴﺴﺖ ﻟﻬﻤﺎ ﺃﻯ ﺗﺄﺛﻴﺮ ﻋﻠﻰ ﺍﻟﻔﻄﺮﻳﺎﺕ ﺍﻟﻤﻀ�ﺎﺩﺓ ﻛﻤ�ﺎ ﺗ�ﻢ ﺍﺧﺘﻴ�ﺎﺭ ﺗ�ﺄﺛﻴﺮ ﺍ ﻟ ﻤ ﻨ ﺸ � ﻄ ﺎ ﺕ ( ﻛ � ﻮ ﺑ ﺮ ﺱ ﻛ � ﻰ ﺯ ﺩ ـ ﺳ � ﺘ ﺎ ﺭ ﻧ ﺮ) ﻋﻠ�ﻰ ﺍﻟﻨﺸ�ﺎﻁ ﺍﻹﺑ�ﺎﺩﻱ ﻟﻺﺳﻜﻠﻮﺭﺷ�ﻴﻢ ﺭﻭﻟﻔ ﺴ � ﺎﻱ ﻭﻭﺟ�ﺪ ﺃﻥ ﻫ�ﺬﻩ ﺍﻟﻤﻨﺸﻄﺎﺕ ﻟﻴﺲ ﻟﻬﺎ ﺃﻯ ﺗﺄﺛﻴﺮ ﺇﺑﺎﺩﻱ ﻋﻠﻰ ﺍﻟﻔﻄﺮ ﺍﻟﻤﻤﺮﺽ ﺃﻭ ﺍﻟﻔﻄﺮﻳﺎﺕ ﺍﻟﻤﻀﺎﺩﺓ .

- ٢ - Arabic summary

. ﻛﻤ��ﺎ ﺗ��ﻢ ﺍﺧﺘﺒ��ﺎﺭ ﺍﻟﺘ��ﺄﺛﻴﺮ ﺍﻟﺴ��ﺎﻡ ﺍﻟﻤﺸ��ﺘﺮﻙ ﻟ��ﺒﻌﺾ ﺍﻟﻤﺮﻛﺒ��ﺎﺕ ﻭﻭﺟ��ﺪ ﺃﻥ ﺑﻌ��ﺾ ﻫ��ﺬﻩ ﺍﻟﻤﺮﻛﺒ��ﺎﺕ ﺍﻷﻳﻀ��ﻴﺔ ﺍﻟﺠﺪﻳ��ﺪﺓ ﻟﻬ��ﺎ ﺗ��ﺄﺛﻴﺮ ﻣﻨﺸ��ﻂ ﻭﻟﺒﻌﻀ��ﻬﺎ ﺗ��ﺄﺛﻴﺮ ﻣﻀ��ﺎﺩ ﻭﻟﺒﻌﻀ��ﻬﺎ ﺗ��ﺄﺛﻴﺮ ﺿ��ﻌﻴﻒ ﻋﻠ��ﻰ ﻧﻤ��ﻮ ﺍﻟﻔﻄ��ﺮ ﺍﻟﻤﻤﺮﺽ (ﺇﺳﻜﻠﻮﺭﺷﻴﻢ ﺭﻭﻟﻔﺴ ﺎ ﻯ ) . ﻛﻤ��ﺎ ﺗ��ﻢ ﻗﻴ��ﺎﺱ ﺍﻟﺴ��ﻤﻴﺔ ﺍﻟﻨﺒﺎﺗﻴ��ﺔ ﻟﻤﺒﻴ��ﺪ ﺍﻟﻔﻴﺘﺎﻓ��ﺎﻛﺲ ﻭﻟﺰﻳ��ﺖ ﺃﻭﺭﺍﻕ ﺍﻟﻨﻌﻨ��ﺎﻉ ﻭﻟﺰﻳ��ﺖ ﺑ��ﺮﺍﻋﻢ ﺍﻟﻘﺮﻧﻔ��ﻞ ﻭﺍﻟﻤﻨﺸ��ﻄﺎﺕ ﻛ��ﻮﺑﺮﺱ ﻛ��ﻰ ﺯﺩ ﻭﺍﺳ��ﺘﺮﻧﺮ ﻭﻭﺟ��ﺪ ﺃﻥ ﻟ��ﻴﺲ ﻟﻬ��ﺬﻩ ﺍﻟﻤﺮﻛﺒ��ﺎﺕ ﻋﻨ��ﺪ ﺍﻟﺘﺮﻛﻴ��ﺰﺍﺕ ﺍﻟﻤﻨﺨﻔﻀﺔ ﺃﻯ ﻧﺸﺎﻁ ﺳﺎﻡ ﻋﻠﻰ ﺍﻟﻨﺒﺎﺕ ﻛﻤﺎ ﺗﻢ ﺍﺧﺘﺒﺎﺭ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻤﺮﺿﻴﺔ ﻟﻔﻄ�ﺮ ﺍﻹﺳﻜﻠﻮﺭﺷ�ﻴﻢ ﺭﻭﻟﻔ ﺴ � ﻴﺎﻯ ﺑﺎﺳﺘﺨﺪﺍﻡ ﺗﺮﻛﻴﺰﺍﺕ ﻣﺨﺘﻠﻔ�ﺔ ﻣ�ﻦ ﺍﻟﻔﻄ�ﺮ ﺍﻟﻨ�ﺎﻣﻲ ﻋﻠ�ﻰ ﻟﻘ�ﺎﺡ ﻧﺨﺎﻟ�ﺔ ﺍﻟﻘﻤ�ﺢ ﻭﻭﺟ�ﺪ ﺃﻥ ﺗﺮﻛﻴ�ﺰ ٠.١٢٥% ﺃﻋﻄ��ﻲ ﺃﻓﻀ��ﻞ ﻧﺘ��ﺎﺋﺞ ﻛﻤ��ﺎ ﺗ��ﻢ ـ ﺯﺭﺍﻋ��ﺔ ﺍﻟﺤﻤ��ﺺ ﻋﻠ��ﻰ ﻣ��ﺪﺍﺭ ﻋ��ﺎﻣﻰ ٢٠٠٧ – ٢٠٠٨ ﻭﻭﺟ��ﺪ ﺃﻥ ﺷ � ﻬ ﺮ ﻧﻮﻓﻤﺒﺮ ﻛ��ﺎﻥ ﺃﻓﻀ�ﻞ ﺍﻟﺸ��ﻬﻮﺭ ﻟﺰﺭﺍﻋ�ﺔ ﻧﺒ��ﺎﺕ ﺍﻟﺤﻤ�ﺺ ﻭﺃﻥ ﺍﻟﺘﻌﻘ��ﻴﻢ ﺑ�ﺎﻷﺗﻮﻛﻼﻑ ﻛ��ﺎﻥ ﺃﻓﻀ�ﻞ ﺗﻌﻘ��ﻴﻢ ﻟﻠﺘﺮﺑ��ﺔ، ﻛﻤ��ﺎ ﻭﺟ��ﺪ ﺃﻥ ﺍﻟﺘﺮﺑ��ﺔ ﺑﺘﻜ��ﻮﻳﻦ ١/٢ ﻃﻤ��ﻲ ﻭ ١/٢ ﺭﻣ��ﻞ ﻛ��ﺎﻥ ﺃﻓﻀ��ﻞ ﺍﻷﻧ��ﻮﺍﻉ ﻟﺰﺭﺍﻋ��ﺔ ﻧﺒ��ﺎﺕ ﺍﻟﺤﻤﺺ.

. ﻓﻲ ﺍﻟﺼﻮﺑﺔ ﺗﻢ ﺍﺧﺘﺒﺎﺭ ﺗﺄﺛﻴﺮ(ﺍﻟﻔﻴﺘﺎﻓﺎﻛﺲ ﻭﺃﻭﺭﺍﻕ ﺍﻟﻨﻌﻨﺎﻉ ﻭﺯﻳﺖ ﺑﺮﺍﻋﻢ ﺍﻟﻘﺮﻧﻔ�ﻞ ﻭﺍﻟﻤﻨﺸ�ﻄﺎﺕ ﻛ��ﻮﺑﺮﺱ ﻛ��ﻰ ﺯﺩ ﻭﺍﺳ��ﺘﺮﻧﺮﻭﻓﻄﺮﻯ ﺍﻟﺠﻠﻴ��ﻮﻛﻼﺩﻳﻢ ﺩﻟﻴﻜﻮﻳﺴ��ﻨﺲ ﻭ ﺍﻟﺠﻠﻴ��ﻮﻛﻼﺩﻳﻢ ﻓ��ﺮﻳﻨﺲ) ﻭﻣﺮﻛﺒ��ﺎﺗﻬﻢ ﻟﻠﺤﺼﻮﻝ ﻋﻠ�ﻰ ﺃﺣﺴ�ﻦ ﻋ�ﻼﺝ ﻟﻨﺒ�ﺎﺕ ﺍﻟﺤﻤ�ﺺ ﻣ�ﻦ ﻓﻄ�ﺮ ﺍﻹﺳﻜﻠﻮﺭﺷ�ﻴﻢ ﺭﻭﻟﻔ ﺴ � ﺎﻯ ﻭﻭﺟ�ﺪ ﺃﻥ ( ﺍﻟﻔﻴﺘﺎﻓ�ﺎﻛﺲ ﻭﻛ�ﻮﺑﺮﺱ ﻛ��ﻰ ﺯﺩ، ﺍﻟﻔﻴﺘﺎﻓ�ﺎﻛﺲ ﻭﺍﺳ��ﺘﺮﻧﺮ، ﻛ��ﻮﺑﺮﺱ ﻛ�ﻰ ﺯﺩ ﻭﺍﺳ��ﺘﺮﻧﺮ، ﻛ��ﻮﺑﺮﺱ ﻛ�ﻰ ﺯﺩ ﻭﺍﻟﻔﻄ��ﺮ ﺍﻟﻤﻀ��ﺎﺩ ﺍﻟﺠﻠﻴ��ﻮﻛﻼﺩﻳﻢ ﺩﻟﻴﻜﻮﻳﺴ��ﻨﺲ، ﻛ��ﻮﺑﺮﺱ ﻛ��ﻰ ﺯﺩ ﻭﺍﻟﻔﻄ��ﺮ ﺍﻟﻤﻀ��ﺎﺩ ﺍﻟﺠﻠﻴ��ﻮﻛﻼﺩﻳﻢ ﻓ��ﺮﻳﻨﺲ، ﺍﺳ��ﺘﺮﻧﺮ ﻭﺍﻟﻔﻄ��ﺮ ﺍﻟﻤﻀ��ﺎﺩ ﺍﻟﺠﻠﻴ��ﻮﻛﻼﺩﻳﻢ ﺩﻟﻴﻜﻮﻳﺴ��ﻨﺲ، ﺍﺳ��ﺘﺮﻧﺮ ﻭﺍﻟﻔﻄ��ﺮ ﺍﻟﻤﻀ��ﺎﺩ ﺍﻟﺠﻠﻴ��ﻮﻛﻼﺩﻳﻢ ﻓ��ﺮﻳﻨﺲ) ﺃﺣﺴ��ﻦ ﻣﻌ��ﺎﻣﻼﺕ ﺃﻋﻄﺖ ﺃﺣﺴﻦ ﻧﺴﺒﺔ ﻧﻤﻮ ﻟﻠﻨﺒﺎﺕ ؛ ﻛﻤﺎ ﻭﺟﺪ ﺃﻥ ﻫﺬﻩ ﺍﻟﻤﻌﺎﻣﻼﺕ ﺃﻋﻄﺖ ﺃﺣﺴﻦ ﻧﻤﻮ ﻟﺴ�ﻴﻘﺎﻥ ﻧﺒ�ﺎﺕ ﺍﻟﺤﻤ�ﺺ ﻭﺃﻋﻠﻲ ﻭﺯﻥ ﻏﺾ ﻭﺃﻋﻠﻲ ﻭﺯﻥ ﺟﺎﻑ ﻛﻤﺎ ﺃﻋﻄﺖ ﺃﻋﻠﻲ ﻧﺴﺒﺔ ﻣﻦ ﺍﻟﻜﻠﻮﺭﻭﻓﻴ�ﻞ (ﺃ ، ﺏ) ﻭﺍﻟﻤﺤﺘ�ﻮﻱ ﺍﻟﻜﻠ�ﻰ ﻟﻠﻜﻠﻮﺭﻭﻓﻴﻞ ﻭﺍﻹﻧﺰﻳﻤﺎﺕ (ﺑﻴﺮﻭﻛﺴﻴﺪﺍﺯ ﻭﺍﻟﺒﻮﻟﻰ ﻓﻴﻨﻮﻝ ﺃﻭﻛﺴﻴﺪﺍﺯ) .

. ﺗ��ﻢ ﺩﺭﺍﺳ��ﺔ ﺗ��ﺄﺛﻴﺮ ﺃﺷ��ﻌﺔ ﺟﺎﻣ��ﺎ ﻋﻨ��ﺪ ﺍﻟﺠﺮﻋ��ﺎﺕ (٠.٢٥، ٠.٥، ١، ١.٥، ٢، ٢.٥، ٣ ﻛﻴﻠ��ﻮ ﺟ��ﺮﺍﻯ) ﻋﻠ��ﻰ ﺍﻟﺘﻀ��ﺎﺩ ﺍﻟﻔﻄ��ﺮﻯ ﻟﻔﻄ��ﺮﻯ ﺍﻟﺠﻠﻴ��ﻮﻛﻼﺩﻳﻢ ﻓ��ﺮﻳﻨﺲ ﻭ ﺍﻟﺠﻠﻴ��ﻮﻛﻼﺩﻳﻢ ﺩﻟﻴﻜﻮﻳﺴ��ﻨﺲ ﺿ��ﺪ ﺍﻟﻔﻄ��ﺮ ﺍﻟﻤﻤ��ﺮﺽ ﺍﻹﺳﻜﻠﻮﺭﺷ��ﻴﻢ ﺭﻭﻟﻔﺴ��ﺎﻯ ﺣﻴ��ﺚ ﻭﺟ��ﺪ ﺃﻥ ﻗ��ﺪﺭﺓ ﻓﻄ��ﺮﻯ ﺍﻟﺠﻠﻴ��ﻮﻛﻼﺩﻳﻢ ﺿ��ﺪ ﻓﻄ��ﺮ ﺍﻹﺳﻜﻠﻮﺭﺷﻴﻢ ﺭﻭﻟﻔﺴﺎﻯ ﺗﺰﻳﺪ ﺑﺰﻳﺎﺩﺓ ﺟﺮﻋﺎﺕ ﺃﺷﻌﺔ ﺟﺎﻣﺎ ﻟﺘﻌﻄﻰ ﺃﻋﻠﻰ ﺗﺄﺛﻴﺮ ﻋﻨ�ﺪ ﺍﻟﺠﺮﻋ�ﺔ ١ ﻛﻴﻠ�ﻮ ﺟﺮﺍﻯ ﻟﻔﻄﺮ ﺍﻟﺠﻠﻴﻮﻛﻼﺩﻳﻢ ﻓﺮﻳﻨﺲ ﻭﺍﻟﺠﺮﻋﺔ ٢ ﻛﻴﻠﻮ ﺟﺮﺍﻯ ﻟﻔﻄﺮ ﺍﻟﺠﻠﻴﻮﻛﻼﺩﻳﻢ ﺩﻟﻴﻜﻮﻳﺴﻨﺲ.

- ٣ - Arabic summary

. ﺗﺸﻌﻴﻊ ﺑﺬﻭﺭ ﻧﺒﺎﺕ ﺍﻟﺤﻤﺺ ﻋﻨﺪ ﺍﻟﺠﺮﻋﺎﺕ (٥، ١٠، ١٥، ٢٠، ٢٥، ٣٠ ﺟ�ﺮﺍﻯ) ﺃﺩﻯ ﺇﻝ ﺗﻘﻠﻴ�ﻞ ﻣﻌﺪﻝ ﺇﻧﺒﺎﺕ ﺍﻟﺒﺬﻭﺭﻭﻃﻮﻝ ﺍﻟﻨﺒﺎﺕ ﻭﺍﻟﻮﺯﻥ ﺍﻟﺠﺎﻑ ﻭﺍﻟﻮﺯﻥ ﺍﻟﻐﺾ ﻟﻨﺒﺎﺕ ﺍﻟﺤﻤﺺ.

. ﻓﻰ ﺍﻟﺼﻮﺑﺔ ﻭﺟﺪ ﺃﻥ ﺇﺳ�ﺘﺨﺪﺍﻡ ﻓﻄ�ﺮﻯ ﺍﻟﺠﻠﻴ�ﻮﻛﻼﺩﻳﻢ ﻓ�ﺮﻳﻨﺲ ﻭﻓﻄﺮﺍﻟﺠﻠﻴ�ﻮﻛﻼﺩﻳﻢ ﺩﻟﻴﻜﻮﻳﺴ�ﻨﺲ ﻓﻰ ﺍﻟﺘﺮﺑﺔ ﺍﻟﻤﺼﺎﺑﺔ ﺑﺎﻟﻔﻄﺮ ﺍﻟﻤﻤﺮﺽ ﺍﻹﺳﻜﻠﻮﺭﺷﻴﻢ ﺭﻭﻟﻔﺴ�ﺎﻯ ﺃﺩﻯ ﺫﻟ�ﻚ ﺇﻟ�ﻰ ﺯﻳ�ﺎﺩﺓ ﻧﺴ�ﺒﺔ ﺍﻧﺒ�ﺎﺕ ﻧﺒﺎﺕ ﺍﻟﺤﻤﺺ ﺑﺎﻟﻤﻘﺎﺭﻧﺔ ﺑﻤﻌﺪﻝ ﺇﻧﺒﺎﺕ ﺍﻟﺤﻤﺺ ﺍﻟﻤﻌﺎﻟﺞ ﺑﺎﻟﻔﻄﺮ ﻏﻴﺮ ﺍﻟﻤﺸﻌﻊ.

. ﻛﻤﺎ ﺗﻢ ﺩﺭﺍﺳﺔ ﺗﺄﺛﻴﺮ ﺍﺷﻌﺔ ﺟﺎﻣﺎ ﻋﻠﻰ ﺍﻟﺸﻜﻞ ﺍﻟﺪﺍﺧﻠﻰ ﻟﻔﻄﺮ ﺍﻹﺳﻜﻠﻮﺭﺷ�ﻴﻢ ﺭﻭﻟﻔﺴ�ﺎﻯ ﺣ ﻴ � ﺚ ﻭ ﺟ � ﺪ

ﺃﻥ ﺃﺷ��ﻌﺔ ﺟﺎﻣ��ﺎ ﻋﻨ��ﺪ ﺍﻟﺠﺮﻋ��ﺎﺕ ﺗﺤ��ﺖ ﺍﻟﻤﻤﻴﺘ��ﺔ (٠.٢٥، ٠.٥، ١، ١.٥، ٢، ٢.٥، ٣ ﻛﻴﻠ��ﻮ ﺟ��ﺮﺍﻯ)

ﺃﺣﺪﺛﺖ ﺍﺧﺘﻼﻑ ﻓﻰ ﺍﻟﺸﻜﻞ ﺍﻟﺨﺎﺭﺟﻰ ﻟﻠﻔﻄﺮ ﺑﺎﻟﻤﻘﺎﺭﻧﺔ ﺑﺎﻟﻜﻨﺘﺮﻭﻝ ﺍﻟﻐﻴﺮ ﻣﺸﻌﻊ ﻭﻋﻨﺪ ﺍﻟﺠﺮﻋ�ﺔ ﺍﻟﻤﻤﻴﺘ�ﺔ

(٥ ﻛﻴﻠﻮ ﺟﺮﺍﻯ) ﻓﺈﻥ ﺃﺷﻌﺔ ﺟﺎﻣﺎ ﺃﺩﺕ ﺇﻟﻰ ﺇﺑﺎﺩﺓ ﺍﻟﻔﻄﺮ ﺇﺑﺎﺩﺓ ﺗﺎﻣﺔ.

. ﻭﻗﺪ ﺗﻢ ﺩﺭﺍﺳﺔ ﺗﺄﺛﻴﺮ ﺃﺷﻌﺔ ﺟﺎﻣﺎ ﻋﻠﻰ ﺣﻴﻮﻳﺔ ﺍﻟﻔﻄﺮ ﻭﻗﺪﺭﺗﻪ ﻋﻠ�ﻰ ﺇﺣ�ﺪﺍﺙ ﺍﻟﻌ�ﺪﻭﻯ ﻟﻨﺒ�ﺎﺕ ﺍﻟﺤﻤ�ﺺ

ﺣﻴﺚ ﻭﺟﺪ ﺃﻧﻪ ﺑﺰﻳﺎﺩﺓ ﺟﺮﻋﺎﺕ ﺃﺷﻌﺔ ﺟﺎﻣﺎ ﺗﺤﺖ ﺍﻟﻤﻤﻴﺘﺔ (٠.٢٥ ﺗﺼﻞ ﺇﻟﻰ ٣ ﻛﻴﻠﻮ ﺟ�ﺮﺍﻯ) ﺯﺍﺩﺕ ﻗ�ﺪﺭﺓ

ﺍﻟﻔﻄﺮ ﻋﻠﻰ ﺇﺣﺪﺍﺙ ﺍﻟﻌﺪﻭﻯ ﻓﻰ ﻧﺒﺎﺕ ﺍﻟﺤﻤﺺ ﺣﺘﻰ ﻭﺻﻠﺖ ﺇﻟ�ﻰ ﺇﺣ�ﺪﺍﺙ ﺍﻟﻌ�ﺪﻭﻯ ﺑﻨﺴ�ﺒﺔ ١٠٠ % ﻋﻨ�ﺪ

ﺍﻟﺠﺮﻋﺔ ٣ ﻛﻴﻠﻮ ﺟﺮﺍﻯ ؛ ﻭﻋﻠﻰ ﺍﻟﺠﺎﻧﺐ ﺍﻷﺧﺮ ﻓﺈﻥ ﺃﺷﻌﺔ ﺟﺎﻣﺎ ﻋﻨﺪ ﺍﻟﺠﺮﻋ�ﺔ ﺍﻟﻤﻤﻴﺘ�ﺔ (٥ ﻛﻴﻠ�ﻮ ﺟ�ﺮﺍﻯ)

ﺃﺩﺕ ﺇﻟﻰ ﻭﻗﻒ ﻗﺪﺭﺓ ﺍﻟﻔﻄﺮ ﻋﻠﻰ ﺇﺣﺪﺍﺙ ﺍﻟﻌﺪﻭﻯ ﻭﺫﻟﻚ ﺑﺴﺒﺐ ﻣﻮﺕ ﺍﻟﻔﻄﺮ ﻋﻨﺪ ﻫﺬﻩ ﺍﻟﺠﺮﻋﺔ.

. ﺍﺷ��ﺘﻤﻠﺖ ﻫ��ﺬﻩ ﺍﻟﺪﺭﺍﺳ��ﺔ ﻋﻠ��ﻰ ﻋﻤ��ﻞ ﺗﻨﻘﻴ��ﺔ ﻹﻧ��ﺰﻳﻢ ﺍﻟﺒ��ﻮﻟﻰ ﺟ��ﺎﻻﻛﺘﻴﺮﻭﻧﻴﺰ ﻣ��ﻦ ﺭﺷ��ﻴﺢ ﻣ��ﺰﺍﺭﻉ ﻓﻄ��ﺮ ﺍﻹﺳﻜﻠﻮﺭﺷﻴﻢ ﺭﻭﻟﻔﺴﺎﻯ ﺍﻟﺴﺎﺋﻠﺔ ﻭﺫﻟﻚ ﻟﻠﻌﺰﻻﺕ ﺍﻟﻤﺸﻌﻌﺔ ﻋﻨﺪ ﺍﻟﺠﺮﻋﺎﺕ (٠.٢٥ ﺣ ﺘ � ﻰ ٣ ﻛﻴﻠ�ﻮ ﺟ�ﺮﺍﻯ) ﻭﺍﻟﻌﺰﻟﺔ ﻏﻴﺮ ﺍﻟﻤﺸﻌﻌﺔ. ﻭﺗﻤﺖ ﻫﺬﻩ ﺍﻟﺘﻨﻘﻴﺔ ﺑﺎﻟﺘﺮﺳ�ﻴﺐ ﺑﺈﺳ�ﺘﺨﺪﺍﻡ ﻛﺒﺮﻳﺘ�ﺎﺕ ﺍﻷﻣﻮﻧﻴ�ﻮﻡ (٧٠ % ﺗﺸ�ﺒﻊ) ﻛﺨﻄ��ﻮﺓ ﺍﺑﺘﺪﺍﺋﻴ��ﺔ ﻓ��ﻰ ﻣﺮﺍﺣ��ﻞ ﺗﻨﻘﻴ��ﺔ ﻫ��ﺬﺍ ﺍﻹﻧ��ﺰﻳﻢ؛ ﺛ��ﻢ ﺑﻌ��ﺪ ﺫﻟ��ﻚ ﺗ��ﻢ ﺗﻨﻘﻴ��ﺔ ﺍﻹﻧ��ﺰﻳﻢ ﺑﺎﻟﺘﺮﺷ��ﻴﺢ ﺍﻟﻬﻼﻣ��ﻰ ﺑﺈﺳﺘﺨﺪﺍﻡ ﻣﺎﺩﺓ ﺍﻟﺴ�ﻴﻔﺎﺩﻛﺲ ﺟ�ﻰ- ٧٥ ؛ ﺣﻴ�ﺚ ﻭﺟ�ﺪ ﺃﻥ ﻧﺸ�ﺎﻁ ﺍﻹﻧ�ﺰﻳﻢ ﻳ�ﺰﺩﺍﺩ ﺑﺼ�ﻮﺭﺓ ﻭﺍﺿ�ﺤﺔ ﻟﻴﻌﻄ�ﻰ ﻗﻴﻤﺔ ﻋﻈﻤﻰ ﻋﻨﺪ ﺍﻟﻌﺰﻟ�ﺔ ﺍﻟﻤﺸ�ﻌﻌﺔ ﻋﻨ�ﺪ ﺍﻟﺠﺮﻋ�ﺔ ٣ ﻛﻴﻠ�ﻮ ﺟ�ﺮﺍﻯ. ﺗ�ﻢ ﺗﺄﻛﻴ�ﺪ ﻫ�ﺬﻩ ﺍﻟﻨﺘ�ﺎﺋﺞ ﺑﺎﻟﺘﺤﻠﻴ�ﻞ ﻣ�ﻦ

- ٤ - Arabic summary

ﺧ��ﻼﻝ ﺟﻬ��ﺎﺯ ﺍﻟﺘﻐﺮﻳ��ﺮ ﺍﻟﻜﻬﺮﺑ��ﺎﺋﻰ ﻟﻠﺒﺮﻭﺗﻴﻨ��ﺎﺕ ﻋﻠ��ﻰ ﺍﻟﻬ��ﻼﻡ ﻋﺪﻳ��ﺪ ﺍﻷﻛﺮﻳﻼﻣﻴ��ﺪ ﺣﻴ��ﺚ ﺗ��ﻢ ﺗﺼ��ﻮﻳﺮ ﺇﻧ��ﺰﻳﻢ ﺍﻟﺒﻮﻟﻰ ﺟﺎﻻﻛﺘﻴﺮﻭﻧﻴﺰ ﻓﻰ ﺣﺰﻣﺔ ﺑﺮﻭﺗﻴﻨﻴﺔ ﻣﻔﺮﺩﺓ ﻋﻨﺪ ﻭﺯﻥ ﺟﺰﻳﺌﻰ ٧٢ ﻛﻴﻠﻮ ﺩﺍﻟﺘﻮﻥ.

- ٥ - ﻟﺠﻨـﺔ ﺍﻟﺤﻜـﻢ ﻭﺍﻟﻤﻨـﺎﻗﺸــــﺔ

ﺍﻷﺳﺘﺎﺫ ﺍﻟﺪﻛﺘﻮﺭ: ﻋﻄﻴﺔ ﻳﻮﺳﻒ ﻗﺮﻳﻄﻢ ﺍﺳﺘﺎﺫ ﻛﻴﻤﻴﺎء ﻭﺳﻤﻴﺔ ﺍﻟﻤﺒﻴﺪﺍﺕ ﺑﻜﻠﻴﺔ ﺍﻟﺰﺭﺍﻋﺔ – ﺟﺎﻣﻌﺔ ﻛﻔﺮ ﺍﻟﺸﻴﺦ (ﻡ. ﺧﺎﺭﺟﻰ)

ﺍﻷﺳﺘﺎﺫ ﺍﻟﺪﻛﺘﻮﺭ:ﺇﻟﻬﺎﻡ ﻣﺴﻌﺪ ﺍﻟﺮﻓﺎﻋﻰ ﺍﺳﺘﺎﺫ ﺍﻟﻔﻄﺮﻳﺎﺕ ﻭﺃﻣﺮﺍﺽ ﺍﻟﻨﺒﺎﺕ ﺑﻘﺴﻢ ﺍﻟﻨﺒﺎﺕ – ﻛﻠﻴﺔﺍﻟﻌﻠﻮﻡ- ﺟﺎﻣﻌﺔ ﻃﻨﻄﺎ (ﻡ. ﺧﺎﺭﺟﻰ . ﺭﺋﻴﺴﺎ )

ﺍﻷﺳﺘﺎﺫ ﺍﻟﺪﻛﺘﻮﺭ: ﻋﺒﺪ ﺍﻟﻮﻫﺎﺏ ﻋﻨﺘﺮ ﺇﺳﻤﺎﻋﻴﻞ ﺭﺋﻴﺲ ﻗﺴﻢ ﺍﻟﻤﻜﺎﻓﺤﺔ ﺍﻟﻤﺘﻜﺎﻣﻠﺔ- ﻣﻌﻬﺪ ﺍﻣﺮﺍﺽ ﺍﻟﻨﺒﺎﺕ- ﻣﺮﻛﺰ ﺑﺤﻮﺙ ﺍﻟﺠﻴﺰﺓ (ﻡ. ﺩﺍﺧﻠﻰ)

ﺍﻷﺳﺘﺎﺫ ﺍﻟﺪﻛﺘﻮﺭ: ﺣﻨﺎﻥ ﻣﺤﻤﻮﺩ ﻣﺒﺎﺭﻙ ﺍﺳﺘﺎﺫ ﻣﺴﺎﻋﺪ ﺍﻟﻔﻄﺮﻳﺎﺕ ﻭﺃﻣﺮﺍﺽ ﺍﻟﻨﺒﺎﺕ ﺑﻘﺴﻢ ﺍﻟﻨﺒﺎﺕ – ﻛﻠﻴﺔﺍﻟﻌﻠﻮﻡ- ﺟﺎﻣﻌﺔ ﻃﻨﻄﺎ (ﻡ. ﺩﺍﺧﻠﻰ )

ﺭﺋﻴﺲ ﻣﺠﻠﺲ ﻗﺴﻢ ﺍﻟﻨﺒﺎﺕ

ﺍﻷﺳﺘﺎﺫ ﺍﻟﺪﻛﺘﻮﺭ / ﺣﺴﻦ ﻓﺮﻳﺪ ﺍﻟﻘﺎﺿﻰ

ﺍﻟﻤﺸـﺮﻓـــﻮﻥ

ﺍﻷﺳﺘﺎﺫ ﺍﻟﺪﻛﺘﻮﺭ: ﻋﺒﺪ ﺍﻟﻮﻫﺎﺏ ﻋﻨﺘﺮ ﺇﺳﻤﺎﻋﻴﻞ ﺭﺋﻴﺲ ﻗﺴﻢ ﺍﻟﻤﻜﺎﻓﺤﺔ ﺍﻟﻤﺘﻜﺎﻣﻠﺔ ﺑﻤﻌﻬﺪ ﺑﺤﻮﺙ ﺃﻣﺮﺍﺽ ﺍﻟﻨﺒﺎﺗﺎﺕ ﺑﺎﻟﺠﻴﺰﺓ.

ﺍﻷﺳﺘﺎﺫ ﺍﻟﺪﻛﺘﻮﺭ: ﺃﺣﻤﺪ ﺇﺑﺮﺍﻫﻴﻢ ﺍﻟﺒﻄﻞ ﺃﺳﺘﺎﺫ ﺍﻟﻤﻴﻜﺮﻭﺑﻴﻮﻟﻮﺟﻴﺎ ﺍﻟﺘﻄﺒﻴﻘﻴﺔ ﻭ ﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴﺎ ﺍﻟﺤﻴﻮﻳﺔ ﺑﺎﻟﻤﺮﻛﺰ ﺍﻟﻘﻮﻣﻰ ﻟﺒﺤﻮﺙ ﻭﺗﻜﻨﻮﻟﻮﺟﻴﺎ ﺍﻹﺷﻌﺎﻉ – ﻫﻴﺌﺔ ﺍﻟﻄﺎﻗﺔ ﺍﻟﺬﺭﻳﺔ.

ﺍﻷﺳﺘﺎﺫ ﺍﻟﺪﻛﺘﻮﺭ: ﺣﻨﺎﻥ ﻣﺤﻤﻮﺩ ﻣﺒﺎﺭﻙ ﺃﺳﺘﺎﺫ ﺍﻟﻔﻄﺮﻳﺎﺕ ﺍﻟﻤﺴﺎﻋﺪ – ﻗﺴﻢ ﺍﻟﻨﺒﺎﺕ – ﻛﻠﻴﺔ ﺍﻟﻌﻠﻮﻡ – ﺟﺎﻣﻌﺔ ﻃﻨﻄﺎ.

ﺭﺋﻴﺲ ﻣﺠﻠﺲ ﻗﺴﻢ ﺍﻟﻨﺒﺎﺕ

ﺍﻷﺳﺘﺎﺫ ﺍﻟﺪﻛﺘﻮﺭ / ﺣﺴﻦ ﻓﺮﻳﺪ ﺍﻟﻘﺎﺿﻰ

ﻛﻠﻴﺔ ﺍﻟﻌﻠﻮﻡ ﺟﺎﻣﻌﺔ ﻃﻨﻄﺎ ﻗﺴﻢ ﺍﻟﻨﺒﺎﺕ

ﺭﺳﺎﻟﺔ ﻣﻘﺪﻣﺔ ﺇﻟﻰ ﻛﻠﻴﺔ ﺍﻟﻌﻠﻮﻡ- ﺟﺎﻣﻌﺔ ﻃﻨﻄﺎ ﻛﺠﺰء ﻣﺘﻤﻢ ﻟﻠﺤﺼﻮﻝ ﻋﻠﻰ ﺩﺭﺟﺔ ﻣﺎﺟﺴﺘﻴﺮ ﺍﻟﻌﻠﻮﻡ ﻓﻰ ﺍﻟﻤﻴﻜﺮﻭﺑﻴﻮﻟﻮﺟﻰ (ﻓﻄﺮﻳﺎﺕ). ﻣﻘﺪﻣﺔ ﻣﻦ ﺭﺷــﺎ ﻣﺤـﻤــﺪ ﻓﺘـﺤﻰ ﺍﻟﺴـــﻴﺪ ﺑﻜﺎﻟﻮﺭﻳﻮﺱ ﻋﻠﻮﻡ ﻣﻴﻜﺮﻭﺑﻴﻮﻟﻮﺟﻰ- ٢٠٠٤- ﺟﺎﻣﻌﺔ ﺍﻷﺯﻫـــﺮ. ٢٠١٢ ﺍﻟﻤﺸـﺮﻓـــﻮﻥ ﺍﻷﺳﺘﺎﺫ ﺍﻟﺪﻛﺘﻮﺭ: ﻋﺒﺪ ﺍﻟﻮﻫﺎﺏ ﻋﻨﺘﺮ ﺇﺳﻤﺎﻋﻴﻞ ﺭﺋﻴﺲ ﻗﺴﻢ ﺍﻟﻤﻜﺎﻓﺤﺔ ﺍﻟﻤﺘﻜﺎﻣﻠﺔ ﺑﻤﻌﻬﺪ ﺑﺤﻮﺙ ﺃﻣﺮﺍﺽ ﺍﻟﻨﺒﺎﺗﺎﺕ ﺑﺎﻟﺠﻴﺰﺓ.