in vitro control of fusarium oxysporum by soil isolates of

In Vitro Control of Fusarium oxysporum by Soil Isolates of Bacillus species

Prepared by

Nidal Abed-Algader Fayyad

Supervisor

Dr. Abboud El Kichaoui

Submitted in partial fulfillment of the requirements for the degree of master of biological sciences in microbiology.

December, 2006

الجامعة الإسلامیة غزة عمادة الدراسات العلیا

كلیة العلوم ةقسم العلوم الحیاتی

Islamic University –Gaza Deanery of Higher Education Faculty of Science Biological Science

I

بسم االله الرحمن الرحيم

قالوا سبحانك لا علم لنا إلا ما علمتنا انك أنت العليم الحكيم

٣٢ البقرة

II

DEDICATION

Dedicated to my father and my mother's soul

To my wife

To my sons Abed and Asem

To my daughter Dana and to all my family members

III

Declaration

I hereby declare that this submission is my own work and that, to the best of my knowledge and belief, it contains no material previously puplished or written by another person nor material which to a substantial extent has been accepted for the award of any other degree of the university or other institutes, except where due acknowledgment has been made in the text. Signature

Nidal Abed-Algader Fayyad

Copyright

All rights reserved: No part of this work can be copied, translated or stored in retrival system, without prior permission of the author.

IV

In vitro control of Fusarium oxysporum by soil isolates of Bacillus species Nidal Abed-Algader Fayyad

Faculty of Science, Department of Biology

Abstract

Fusarium wilt is caused by Fusarium oxysporum, which leads to high losses of

crops in many countries. Chemical fungicides are the primary means to control

Fusarium wilt disease. Biological control has emerged as one of the most

promising alternatives because it reduces the risks of chemicals on the

environment and health.

The aim of this study was to isolate bacteria from soil that have antagonistic

fungal (F. oxysporum) activity and hence a biocontrol potential. Bacterial isolates

were isolated from soil, and were then morphologically and biochemically

characterized. The isolates were identified according to standard methods as

Bacillus subtilis. Antifungal effect of the bacterial isolates was tested by

measuring the growth inhibition zone of Fusarium oxysporum on Petri dishes.

Pot trials with Bacillus subtilis were also carried out in order to see if it works in

soil. Here we had four groups of pots, one of which has had F. oxysporum and

treated by carbendazim as chemical antifungal ( chemical treated group ). The

second group has had F. oxysporum and treated with Bacillus subtilis ( biological

treated group ). The third group has had no pathogen added (the negative control

group). The last group has had F. oxysporum without treatment ( positive control

group ). The effect of the various treatments was measured in terms of the dry

weight, stem length and root length of the tested plants.

The results of this study have shown that dry weight and stem length of

watermelon, cucumber and muskmelon were significantly increases in the

chemically and biologically treated groups as compared to the positive control

V

group. It was also observed that the root length was significantly increased in the

biologically treated group in all the three plants but not in the chemically treated

group again in comparison to the positive control group.

In conclusion, we have been able to isolate Bacillus subtilis strains that have a

clear antifungal activity against Fusarium oxysporum. Therefore, we suggest, that

our isolates can be used in the control of Fusarium oxysporum in agriculture, in

particular in the protection of watermelon, cucumber and muskmelon.

VI

سابتیلس المعزولة من التربة باسیلس باستخدام بكتیریا فطر فیوزاریوم اكسیسبوراممكافحة

الملخص خ سائر كبی رة ف ي المحاص یل الزراعی ة إلىمرض الذبول الوعائي یسببھ فطر فیوزاریوم اكسیسبورام و الذي یؤدي

ال رئیس وال ذي ق د ی سبب أض رارا ج سیمة للبیئ ة المكافحة الكیمیائیة لھ ذا الم رض ھ و ال سبیل ف ،البلدانفي العدید من

.الأضرار قوي وھو المكافحة البیولوجیة للتقلیل من تلك بدا یظھر بدیل لذلك الإنسان،وصحة

.ھدفت ھذه الدراسة إلى عزل بكتیریا من التربة لھا القدرة على مكافحة فطر فیوزاریوم اكسیسبورام

ث م و ك ذلك والمجھری ة ةالم ز رعی ب الطرق تشخی صھ ت م باتات م صابة و تم عزل الفطر المسبب لھذا المرض من ن

اكسی سبورام فط ر فیوزاری وم ق درتھا عل ى مكافح ة ودراس ة ب الطرق المتبع ة عزل البكتیریا م ن الترب ة وتشخی صھا

.غذائي على وسط المحتویة بتري النمو لھذا الفطر في أطباق ط تثبیمقداروذلك بقیاس

بتك وین أرب ع مجموع ات م ن نبات ات البط یخ أوعی ة بلاس تیكیة ف ي عل ى الفط ر بكتیری ا المعزول ة ت أثیر الت م تقی یم

فالمجموع ة الأول ى زرع ت بالنبات ات وت م إمراض ھا بواس طة الفط ر وعلاجھ ا بع د ذل ك كیمیائی ا وال شمام،والخی ار

وع ة الثانی ة م ن النبات ات ت م إمراض ھا المجمأم ا ) المجموع ة المعالج ة كیمیائی ا (باس تخدام مبی د فط ري الكاربن دازیم

الثالث ة المجموع ة أم ا )المجموعة المعالج ة بیولوجی ا ( بواسطة الفطر وعلاجھا بیولوجیا بواسطة البكتیریا المعزولة

والمجموع ة الأخی رة ت م إمراض ھا بواس طة ) المجموع ة ال ضابطة ال سلبیة ( فھي تحتوي على نباتات بدون إمراضھا

تم قیاس تأثیر العلاج ات الم ستخدمة بواس طة ال وزن الج اف ) المجموعة الضابطة الإیجابیة ( علاج الفطر وبدون أي

.للنباتاتوطول الساق والجذر

في الوزن الجاف وطول ال ساق ف ي نب ات البط یخ والخی ار وال شمام ذات دلالة إحصائیة أظھرت النتائج وجود زیادة

كم ا بین ت .الإیجابی ةلمعالج ة بیولوجی ا بالمقارن ة م ع المجموع ة ال ضابطة للمجموع ة المعالج ة كیمیائی ا والمجموع ة ا

في ط ول الج ذر ف ي المجموع ة المعالج ة بیولوجی ا ف ي ال ثلاث نبات ات الت ي ذات دلالة إحصائیة وجود زیادة النتائج

رن ة م ع المجموع ة ف ي المجموع ة المعالج ة كیمیائی ا مقا ذات دلال ة إح صائیة زی ادة دولا یوج تم ت علیھ ا الدراس ة

.الإیجابیةالضابطة

واض ح عل ى فط ر فیوزاری وم ریت أث لھا تم عزلھا من التربة المحلیةسابتیلس التيبكتیریا باسیلس نستنتج من ذلك أن

مكافحة مرض الذبول الوع ائي ف ي الزراع ة وخاص ة ف ي النبات ات الت ي تم ت علیھ ا ل اكسیسبورام و یمكن استخدامھا

.والشمامخ والخیار البطی أي الدراسة

VII

Acknowledgement

I wish to express my deepest gratitude to Dr. Abboud El Kichaoui, Associated

Professor of Mycology, Biology Department, Faculty of Science, Islamic

University- Gaza, the supervisor of this work, for initiating and planning this

study, enlightening supervision, useful assistance, valuable advice and

continuous support during the course of this study.

I would like also to express my great thanks to Prof. Dr. Fadel Sharif. Professor

of Molecular biology, Medical Technology Department, Faculty of Science,

Islamic University- Gaza.

We thank Dr. Kamal Elnabris for assistance with statistical analysis. Assistant

professor of Marine Biology. Biology department, Faculty of Science, , Islamic

University- Gaza.

The author is very grateful to Dr. Abdalla Abed. Assistant professor of Human

Genetics. Biology department, Faculty of Science, Islamic University- Gaza.

I would like also to thank all my friends and colleagues for their fruitful

discussions and support.

VIII

CONTENTS DEDICATION II DECLARATION III ABSTRACT IV

ARABIC ABSTRACT VII ACKNOWLEDGMENT VIII CONTENTS XIII LIST OF TABLES XIII

LIST OF FIGURES XIV

CHAPTER I INTRODUCTION

1

AIM OF THE STUDY

4

CHAPTER II

LITERATURE REVIEW 2.1. Fusarium oxysporum 5

2.1.1. Distribution of Fusarium 5

2.1.2. Host specificity and formae speciales 5

2.1.3. Taxonomical Position 6

2.1.4. Mycelium 6

2.1.5. Reproduction 6

2.1.6. Disease Cycle 7

2.1.7. Transmission of Fusarium oxysporum 8

2.1.8. Biological control Fusarium strains 8

2.2 Plant diseases cuased by Fusarium oxysporum 8

2.2.1. Fusarium wilt 9

2.2.2. Fusarium crown and root rot 10

IX

2.2.3. Fusarium Root Rot 10

2.3. Control of Fusarium incited diseases 11

2.3.1. Chemical Control 11

2.3.2. Biological Control (Biocontrol) 13

2.3.2.1.Biocontrol Approaches 14

a- Resistant cultivars 14

b- Non- pathogenic fungi 15

Fusarium 15

Trichoderma 15

c- Bacteria 15

Pseudomonas 17

Rhizobacteria 18

Myxobacteria 18

Streptomyces 18

Bacillus 19

2.4. Bacillus subtilis 19

2.4.1. Competition through colonization of the rhizosphere and the

rhizoplane by Bacillus subtilis 20

2.4.2. Formation of antibiotic metabolites 21 2.4.3. Plant resistance induced by Bacillus subtilis 23 2.4.4. Promotion of root growth 24

2.4.5. Effect on plant growth and yield 25

2.5. Integrated control (chemical and biological) 27 CHAPTER III

MATERIALS AND METHODS

3.1. Materials 28

3.1.1. Apparatus and special equipments 28

3.1.2. Stains 29

3.1.3. Reagents 30

3.1.4. Culture media 30

3.2. METHODOLOGY 32

3.2.1. Isolation of Fusarium oxysporum 32

X

3.2.2. Isolation of Bacillus spp from soil 33

3.2.3. In vitro inhibition of Fusarium oxysporum by isolated soil

bacteria

34

3.2.4. Application of Bacillus species as biocontrol against Fusarium

oxysporum

34

3.2.5. Pot experiment 34

3.2.6. Statistical analysis 36

CHAPTER IV

RESULTS

4.1. Microscopic identification of F. oxysporum 37

4.1.1. Characteristics of F. oxysporum 38

4.1.1.1. On PDA 38

4.1.1.2. On Komada’s Medium 39

4.1.1.3. On SDA Medium 40

4.2. Identification of Bacillus subtilis 41

4.2.1. Biochemical characteristics of Bacillus 42

4.3. In vitro inhibition of F. oxysporum by soil bacterial isolates 42

4.4. Application of carbendazim as chemical control agent against Fusarium

oxysporum 44

4.4.1. Carbendazim 44 4.5. Application of Bacillus subtilis as a biocontrol agent against Fusarium

oxysporum 45

4.5.1. Application of Bacillus subtilis and F. oxysporum together 45 4.5.2. Application of Bacillus subtilis on one day old F. oxysporum culture 46 4.5.3. Application of Bacillus subtilis on two day’s old F. oxysporum culture 47

4.5.4. Application of Bacillus subtilis on three days old F .oxysporum culture. 48

4.6. Symptoms observed on infected plants 49

4.7. Pot trials 50

4.7. Statistical analysis 53

XI

4.7.1. Effect of different treatments on watermelon. 53

4.7.1.1. Dry weight 53

4.7.1.2. Stem length 53

4.7.1.3. Root length 54

4.7.2. Effect of different treatments on cucumber 55




4.7.3. Effect of different treatments on Muskmelon 56




CHAPTER V

DISCUSSION 5.1. Characteristics of Fusarium oxysporum 59

5.2. In vitro inhibition of Fusarium oxysporum by soil bacterial isolates 60

5.3. Application of carbendazim as chemical control agent against

Fusarium oxysporum

61

5.3.1. Carbendazim 61

5.4. Application of Bacillus subtilis as a biocontrol agent against

Fusarium oxysporum

61

5.5. Symptoms observed on infected plants 61

5.6. Effect of different treatments in pot trials 63

.

CHAPTER VI CONCLUSION AND RECOMMENDATIONS 64

CHAPTER VII

REFERENCES 65

XII

LIST OF TABLES

Table 3.1. Illustrates the preparation of nutrient stock solutions for preparing the

irrigation solution.

36

Table 4.1. Examined biochemical and microscopical characteristics of

Bacillus isolates.

42

Table 4.2. The average inhibition zones diameter after application of

carbendazim

44

Table 4.3. The average diameter of the inhibition zones after

application of different dilutions of Bacillus subtilis

45

Table 4.4. The average diameter of the inhibition zones after one day

application of different dilutions of Bacillus subtilis

46

Table 4.5. The average diameter of the inhibition zones old F.

oxysporum culture of different dilutions of B. subtilis

47

Table 4.6. The average diameter of the inhibition zones three days

old culture F. oxysporum of different dilutions of B. subtilis

48

Table 4.7. Effect of biological and chemical control on dry weight of

watermelon

53

Table 4.8. Effect of biological and chemical control on stem length of

watermelon

53

Table 4.9. Effect of biological and chemical control on root length of

watermelon

54


cucumber

55


cucumber

55


cucumber

56

XIII


muskmelon

57


muskmelon

57


muskmelon.

57

LIST OF FIGURES

Figure 4.1. Characteristics of Fusarium oxysporum: (a)

macroconidia, (b) microconidia, and (c) chlamydospores

37

Figure 4.2. A representative photograph of F. oxysporum grown on

PDA

38

Figure 4.3. Growth rate (25-40 mm in diameter) of F. oxysporum on

PDA at 30 0C after three days in complete

39

Figure 4.4. A microphotograph of F. oxysporum normal 39

Figure 4.5. A photograph of F.oxysporum colony on Komada’s

Medium.

40

Figure 4.6. A photograph of F. oxysporum colony on SDA media 40

Figure 4.7. Appearance of Bacillus subtilis colonies on a nutrient

agar plate

41

Figure 4.8. Gram stained Bacillus subtilis cells. 41

Figure 4.9. A photograph of Bacillus endospores 41

Figure 4.10. Photographs of different Bacillus isolates tested for their

ability to inhibit the growth of F. oxysporum. Two inhibition zones are

indicated by arrows

43

Figure 4.11. A photograph of F. oxysporum hyphae damaged by

Bacillus isolates. Swelling is indicated by arrow.

43

Figure 4.12. A photograph shows that inhibition zones by different

dilutions of carbendazim.

44

Figure 4.13. A photograph illustrating the inhibition zones produced 45

XIV

by different dilutions of Bacillus subtilis

Figure 4.14. A photographic illustrating the inhibition zones produced

by different dilutions of Bacillus subtilis one day old F.oxysporum

culture

46

Figure 4.15. A photograph illustrating the inhibition zones by different

dilutions of Bacillus two day old F. oxysporum culture.

47

Figure 4.16. A photograph illustrating the inhibition zones by different

dilutions of Bacillus subtilis

48

Figure 4.17. Necrotic lesion of watermelon stem 49

Figure 4.18 Vascular area discoloration as viewed internally 50

Figure 4.19. A photograph of watermelon grown under different

treatments

51

Figure 4.20. A photograph of cucumber grown under different

treatments

52

Figure 4.21. A photograph of muskmelon grown under different

treatments

52

Figure 4.22. The Means of dry weight, stem length and root length in

the three different groups ( chemically treated, biologically treated and

positive control ) of watermelon.

54



positive control ) of cucumber.

54



positive control ) of muskmelon.

58

1

CHAPTER I

INTRODUCTION

Fungal plant diseases are one of the major concerns to agriculture worldwide. It

has been estimated that total losses as a consequence of plant diseases reach

25% of the yield in Western countries and almost 50% in the developing

countries. Of these diseases, one third is due to fungal infections (1).

Fusarium species are among the most important plant pathogens in the world.

Fusarium oxysporum is responsible for wilt and cortical rot diseases of more than

100 economically important plants (2).

Potential means of pathogen control and disease management are: regulation of

production and transfer of propagation material, seed disinfestation, suppressive

growing media, sanitation of the greenhouse by heating or chemicals, crop

rotation, soil and substrate disinfestation using methyl bromide or solarization,

biocontrol agents, and fungicides (3).

So there is a pressing need to control fungal diseases that reduce the crop yield

so as to ensure a steady and constant food supply to ever increasing world

population. Conventional practice to overcome this problem has been the use of

chemical fungicides which have adverse environmental effects causing health

hazards to humans and other non-target organisms, including beneficial life

forms (4).

Carbendazim is a fungicide used to control a broad range of fungal diseases on

arable crops, fruits, and vegetables in Gaza strip. It could be harmful to human

health and the environment. Carbendazim works by inhibiting the development of

fungi probably by interfering with spindle formation at mitotic cell division.

2

The wide use of synthetic chemicals has caused the development of resistant

pathogenic populations (5).

The spiraling up cost of fungicides, pollution to soil, water and air due to

continuous use of fungicides and development of resistant strains of pathogen to

these chemicals are therefore now forcing scientists to look for biological

methods which are eco-friendly safe and more specific to pathogens (6).

Biological control is a nonchemical measure that has been reported in several

cases to be as effective as chemical control (7). Biological control involves the

use of beneficial microorganisms, such as specialized fungi and bacteria, to

attack and control pathogens offers an environmentally friendly approach to the

managment of plant disease and can be incorporated into cultural and physical

controls and limited chemical usage for an effective integrated pest managment

(IPM) system. Biological control can be a major component in the development of

more sustainable agriculture system.

IPM strategy, the established view of biological control is that, even though it is

safer than chemical control. It is less efficient and less reliable. To be realistic, we

should not expect a very broad range of pest or disease control from biological

agents, or that they control major pests or pest complexes in major crops in a

wide range of environments (8).

The general mechanism of biological control can be divided into direct and

indirect effects of the biocontrol agent (BCA) on the plant pathogen. Direct effects

include competition for nutrients or space, production of antibiotic and lytic

enzymes, inactivation of the pathogen’s enzymes and parasitism. Indirect effects

include all those aspects that produce morphological and biochemical changes in

the host plant, such as tolerance to stress through enhanced root and plant

development, solubilization or sequestration of inorganic nutrients, and induced

resistance (9).

3

When testing bacterial and fungal isolates from the environment for biocontrol

activities, between 1 and 10% show at least some capacity to inhibit the growth

of pathogens in vitro. However, fewer isolates can suppress plant diseases under

diverse growing conditions and fewer still have broad-spectrum activity against

multiple pathogenic taxa. Nonetheless, intensive screens have yielded numerous

candidate organisms for commercial development. Some of the microbial taxa

that have been successfully commercialized and are currently marketed as

Environmental Protection Agency EPA-registered biopesticides include bacteria

belonging to the genera Agrobacterium, Bacillus, Pseudomonas, and

Streptomyces (10).

Screening is a critical step in the development of biocontrol agents. The success

of all subsequent stages depends on the ability of a screening procedure to

identify an appropriate candidate. Many useful bacterial biocontrol agents have

been found by observing zones of inhibition in Petri plates (11).

Fusarium wilt disease is caused by pathogenic forms of the soil – inhabiting

fungus Fusarium oxysporum. Fusarium oxysporum is a major disease problem

on many crops. Currently, control of this pathogen is generally by chemical

control (fungicides) which create potential length and environmental effects.

Thus, alternative control measure are needed.

This research explores the potential of biological control for the management of

this disease. In this study Fusarium oxysporum strains were collected from

infected plants in the field. As a biological agent. Bacillus spp was collected from

a fusarium wilt – suppresive soil were tested for their efficacy in controling

fusarium wilt.

4

AIM OF THE STUDY

The control of fungi-caused plant diseases relies on the heavy use of chemical

fungicides especially methyl bromide gas. Generally fungicides are known to be

one of the major sources of ground water pollution. This comprises a major

problem for Gaza strip because of its overall limited area with a high density of

cultivation and scarcity of water.

Numerous chemical and non-chemical pesticides are available now which

effectively control many of the pests for which methyl bromide is used. One such

alternative is biocontrol which relies on the use of natural safe organisms for the

control of plant soil pathogenic fungi. This technique proved to be more efficient

than many other chemicals for the control of soil pathogenic fungi in many

countries such as Canada and Belgium.

The following specific objectives will be achieved: 1. Isolation of Bacillus subtilis from soil that have antagonistic fungal (Fusarium

oxysporum ) activity and hence a biocontrol potential.

2. Investigate the effect of various Bacillus subtilis isolated from soil on Fusarium

oxysporum both on plates and on plants grown in pots.

3. The efficacy of the Bacillus subtilis will also be compared to the chemical

antifungal agent carbendazim.

5

CHAPTER II

LITERATURE REVIEW 2.1. Fusarium oxysporum Fusarium species are among the most important fungal plant pathogens in the

world. Fusarium oxysporum is responsible for wilt and cortical rot diseases of

more than 100 economically important plants (2).

2.1.1. Distribution of Fusarium

Fusarium oxysporum is the most widely dispersed of the Fusarium spp and can

be recovered from most soils arctic, tropical or desert and cultivated or not .It

also can be dispersed by insects and recovered from marine algae. Fusarium

oxysporum also is without a doubt the most economically important species in

the Fusarium genus given its numerous hosts and the level of loss that can result

when it infects a plant. The Fusarium oxysporum species is clearly

heterogeneous and composed of at least dozens of species that need to be

clearly defined and separated in a usable manner (12).

2.1.2. Host specificity and formae speciales Hosts of Fusarium oxysporum include: potato, sugarcane, garden bean, cowpea,

prickly pear, cultivated zinnia, pansy, Assam rattlebox, Baby's breath, Asparagus,

Banana, Carnation, Cotton, Cucurbits, squash, melons, and cucumbers (13).

Like various other plant pathogens, Fusarium oxysporum has several specialized

forms - known as formae specialis (f.sp.) - that infect a variety of hosts causing

various diseases (13). Over 100 formae speciales of Fusarium oxysporum have

been described (12). These include: Fusarium oxysporum f.sp. asparagi

(fusarium yellows on asparagus); f.sp. callistephi (wilt on China aster); f.sp.

cubense (Panama disease/wilt on banana); f.sp. dianthi (wilt on carnation); f.sp.

koae (on koa); f.sp. lycopersici (wilt on tomato); f.sp. melonis (fusarium wilt on

muskmelon); f.sp. niveum (fusarium wilt on watermelon); f.sp. pisi (on edible-

6

podded pea); f.sp. tracheiphilum (wilt on Glycine max); and f.sp. zingiberi

(fusarium yellows on ginger) (13).

2.1.3. Taxonomical position Fusarium is classified as follows:

Division................ Eumycota

Subdivision ...........Deutromycotina

Form- Class ..........Hyphomycetes

Form-Order ............Moniliales

Form-Family ...........Tuberculariaceae

Form-Genus ............Fusarium

2.1.4. Mycelium The aerial mycelium first appears white, and then may change to a variety of

colors - ranging from violet to dark purple - according to the strain (or special

form) of F. oxysporum. If sporodochia are abundant, the culture may appear

cream or orange in color (14).

2.1.5. Reproduction

F. oxysporum produces three types of asexual spores: microconidia,

macroconidia, and chlamydospores. Microconidia are one or two celled, and are

the type of spore most abundantly and freqeuntly produced by the fungus under

all conditions. It is also the type of spore most frequently produced within the

vessels of infected plants. Micrconidia produced from simple phialides or from

branched or unbranched conidiophores. Macroconidia are long, multicellular

(three to five celled), crescent-shaped or sickle-shaped bodies, gradually pointed

and curved toward the ends. These spores are commonly found on the surface

of plants killed by this pathogen as well as in sporodochia like groups.

Chlamydospores are round or oval, thick-walled spores, produced either

terminally or intercalary on older mycelium or in macroconidia. These spores are

either one or two celled. They get detached and germinate by means of germ

7

tube, if the conditions are favourable. The chlamydospores are very durable and

remain viable for a long time (15).

2.1.6. Disease cycle Fusarium oxysporum includes many representatives that are pathogenic to

plants often causing vascular wilt disease, damping-off problems, and crown and

root rots (12). F. oxysporum is an abundant and active saprophyte in soil and

organic matter, with some specific forms that are pathogenic to plants (14). Its

saprophytic ability enables it to survive in the soil between crop cycles in infected

plant debris. The fungus can survive either as mycelium, or as any of its three

different spore types. Healthy plants can become infected by F. oxysporum if the

soil in which they are growing is contaminated with the fungus. The fungus can

invade a plant either with its sporangial germ tube or mycelium by invading the

plant's roots. The roots can be infected directly through the root tips, through

wounds in the roots, or at the formation point of lateral roots . Once inside the

plant, the mycelium grows through the root cortex intercellulary. When the

mycelium reaches the xylem, it invades the vessels through the xylem's pits. At

this point, the mycelium remains in the vessels, where it usually advances

upwards toward the stem and crown of the plant. As it grows, the mycelium

branches and produces microconidia, which are carried upward within the vessel

by way of the plant's sap stream. When the microconidia germinate, the

mycelium can penetrate the upper wall of the xylem vessel, enabling more

microconidia to be produced in the next vessel. The fungus can also advance

laterally as the mycelium penetrates the adjacent xylem vessels through the

xylem pits (15).

Due to the growth of the fungus within the plant's vascular tissue, the plant's

water supply is greatly affected. This lack of water induces the leaves' stomata to

close, the leaves wilt, and the plant eventually dies. It is at this point that the

fungus invades the plant's parenchymatous tissue, until it finally reaches the

surface of the dead tissue, where it sporulates abundantly (15). The resulting

8

spores can then be used as new inoculum for further spread of the fungus. The

mycelium produces toxic secretion in the vessels, which ultimately blocks the

vascular bundle and results into wilting of the host plant (15).

2.1.7. Transmission of Fusarium oxysporum F. oxysporum is primarily spread over short distances by irrigation water and

contaminated farm equipment. The fungus can also be spread over long

distances either in infected transplants or in soil. Although the fungus can

sometimes infect the fruit and contaminate its seed, the spread of the fungus by

way of the seed is very rare (15). It also can be dispersed by insects (68) and

recovered from marine algae (17).

2.1.8. Biological control by Fusarium strains Isolates of F. oxysporum have often been proposed for use in biological control

programs. In some cases the F. oxysporum are mycoparasites (18), or are

parasites of a weed or other undesirable plants (19). Strains that are better

pathogens of target weeds have been constructed following transformation with

genes that produce plant hormones (20). There is also a report that F.

oxysporum may be pathogenic towards some insects (21) and sea turtles (22). In

other cases apparently non-pathogenic strains are an important component of a

suppressive soil in which a disease is either sharply reduced or completely

eliminated (23), perhaps through the induction of systemic resistance (18). Non-

pathogenic mutants of known pathogenic strains also have been shown to be

effective in controlling disease of cucurbits (24). Competition between pathogenic

and biocontrol strains for nutrients or space on the roots of the hosts may be an

important factor in determining the efficacy of some biocontrol strains (25).

2.2 Plant diseases caused by Fusarium oxysporum Fusarium oxysporum and its various formae specials have been characterized as

causing the following symptoms: vascular wilt, yellows, root rot, and damping-off.

The most important of these is vascular wilt. Of the vascular wilt-causing Fusaria,

9

Fusarium oxysporum is the most important species (15). Strains that are rather

poorly specialized may induce yellows, rot, and damping-off, rather than the

more severe vascular wilt (14).

2.2.1. Fusarium wilt Fusarium wilts first appear as slight vein clearing on the outer portion of the

younger leaves, followed by epinasty (downward drooping) of the older leaves. At

the seedling stage, plants infected by F. oxysporum may wilt and die soon after

symptoms appear. In older plants, vein clearing and leaf epinasty are often

followed by stunting, yellowing of the lower leaves, formation of adventitious

roots, wilting of leaves and young stems, defoliation, marginal necrosis of

remaining leaves, and finally death of the entire plant (15).

Browning of the vascular tissue is a strong evidence of fusarium wilt. Further, on

older plants, symptoms generally become more apparent during the period

between blossoming and fruit maturation (14)

Fusarium wilt of muskmelon and watermelon caused by Fusarium oxysporum f.

sp. melonis and F. oxysporum f. sp. niveum, respectively, inflicts considerable

yield loss worldwide (26). Due to the persistent nature of these pathogens in soil,

once the disease is established in the field, the pathogen is likely to remain

indefinitely because subsequent crops of susceptible melon and watermelon

cultivars increase pathogenic populations. Fusarium spp. of muskmelon and

watermelon cause plant wilting by colonizing the hosts’ vascular system,

eventually resulting in seedling or mature plant mortality. Due to the persistent

nature of these wilt pathogens; the diseases are best managed with wilt-resistant

cultivars. However, as resistant cultivars are utilized, new virulent populations

(physiological races) may develop in specific locations.

In F. oxysporum f. sp. melonis, four common races exist worldwide and have

been designated 0, 1, 2, and 1, 2 (27). Race 1, 2 is virulent to muskmelon

10

cultivars possessing resistance genes FOM-1 and FOM-2 (26) and, to date, no

known field resistant cultivars are commercially available to this pathogen. With

F. oxysporum f. sp. niveum, a similar phenomenon of resistance is found.

Whereas resistant watermelon cultivars have been developed to races 0 and 1,

no commercially resistant cultivars are currently available to race 2 (5).

2.2.2. Fusarium crown and root rot Fusarium crown and root rot, caused by the fungus Fusarium oxysporum f. sp.

radicis-lycopersici. The fungus invades susceptible plants through wounds and

natural openings created by newly emerging roots. Early symptoms caused by

Fusarium oxysporum in tomato seedlings include stunting, yellowing, and

premature loss of cotyledons and lower leaves. A pronounced brown lesion that

girdles the hypocotyls (root/shoot junction), root rot, wilting, and death.

Infected plants in the field may be stunted, and as they begin to heavily bear fruit,

their lower leaves turn yellow and wilt. Wilting first occurs during the warmest part

of the day, and plants appear to recover at night. Infected plants may either

totally wilt and die, or persist in a weakened state, producing reduced numbers of

inferior fruit.

The tap root of infected plants often rots entirely, and chocolate brown cankers

appear at the soil line. When diseased plants are sectioned lengthwise, extensive

brown discoloration and rot are evident in the cortex of the crown and roots (28).

2.2.3. Fusarium Root Rot Fusarium root rot, caused by several species of Fusarium, including Fusarium

roseum. Fusarium solani, and Fusarium oxysporum, affects seedlings of most

conifer species. Douglas-fir and pines are most susceptible to the disease,

spruces are less susceptible, cypresses and cedars are relatively resistant (29).

11

The disease is common throughout North America and in many other parts of the

world. It especially has been a problem in Western Canada and the Western

United States and in the North Central and Southern States (30).

Fusarium root rot causes seedling mortality in the nursery, increased numbers of

culls, and reduced survival after outplanting. Mortality generally is confined to the

first growing season. Losses vary greatly depending on tree species, seed lot,

geographic area, and soil type (31).

Root rot, also known as late damping-off, occurs on seedlings 1 to 3 months old.

Yellowing or purpling and wilting of terminal needles. Needles on the entire plant

eventually turn brown and dry out, appearing to be drought damaged. A

constriction may form at the base of the stem, but seedlings remain erect (31).

Diseased root systems have few laterals, and existing roots are often dark,

swollen, and lack actively growing tips. Although the disease usually is fatal, it

may sometimes destroy only the primary root, resulting in a deformed root

system and stunted seedling. The bark and cortex can be stripped away easily

from the cambium (31).

In the absence of host plants, most species of Fusarium remain dormant as

thick-walled resting spores called chlamydospores, either in the soil or on

decaying organic matter like dead roots. As a result, roots of diseased seedlings

from previous crops are suspected as major sources of inoculum for new

infections (31).

The chlamydospores germinate when root exudates are present and when soil

temperature is warm. When conditions are favorable, the fungus grows over the

root surface, penetrates the epidermis, and spreads through the cortex and

xylem of infected roots, resulting in death and decay of these tissues (31).

2.3. Control of Fusarium Incited Diseases 2.3.1. Chemical control

12

Fungicides such as benomyl or captafol have been demonstrated to be effective,

they have some drawbacks. They are expensive, cause environmental pollution

and may induce pathogen resistance (32).

Fumigation with methyl bromide and chloropicrin (MBC) is currently used in many

cropping systems to control soil-borne plant pathogens. Pathogens as well as

beneficial organisms are killed when they are in contact with the fumigant. It is

unknown if current fumigation techniques destroy resistant myxospores (33).

Disadvantages of soil fumigation include, possible over estimation of the

contribution of soil-borne pathogens to yield suppression because it also controls

weeds, nematodes and other pests. It is not on economical control measure for

soil-borne pathogens (34). Despite these major advantages, the use of methyl

bromide has been associated with major problems, including the depletion of the

ozone layer (35).

Benzimidazoles include benomyl, thiabendazole, carbendazim and thiophate.

They are not toxic to fungi in their original state, but must be converted to their

ester metabolites, which are known to be toxic entities. Carbendazim is the active

moiety of benomyl. Benomyl is transformed to methyl-2-benzimidazole

carbamate, which was later named carbendazim. These metabolites cause

morphological distortion of germinating spores and are thought to upset cell

division by inhibiting β-tubulin assembly during mitosis.

The wide use of synthetic chemicals has caused the development of resistant

pathogenic populations. Resistance to biocontrol microorganisms of fungal

pathogens has not been reported and is considered an environmental-friendly

approach. The use of saprophytic or nonpathogenic isolates of Fusarium spp. for

biological control of pathogenic Fusarium spp. in various crops has been

extensively studied and applied (36). Likewise, certain pathogenic races of

Fusarium spp. are able to protect plants from specific pathogens (4). The

proposed mechanisms involved in Fusarium spp. biological control include (i)

13

competition for infection sites, (ii) competition for nutrients, and (iii) local and

systemic induced resistance (37, 36, and 25).

2.3.2. Biological control (Biocontrol) Biological control (BC) can be defined as the use of natural enemies to reduce

the damage caused by a pest population. According to most biological control

practitioners, biological control differs from "natural control." Natural control is

what occurs much of the time, natural enemies keeping populations of potential

pests in check without intervention. Biological control, on the other hand, requires

intervention, rather than simply letting nature take its course. Biological control is

an approach that fits into an overall pest management program, and represents

an alternative to continued reliance on pesticides.

In an effort to introduce new biocontrol approaches, it has been attempted to

develop biocontrol relying on non-pathogenic mutant strains derived from the

wild-type pathogens themselves. The non-pathogenic mutant strain (path-1) of

Colletotrichum magna retained the wild-type phenotype, colonized cucurbit and

other host tissues, and protected plants against the wild-type and F. oxysporum f.

sp. Niveum in watermelon by priming the host defense response (38).

Several isolates of non-pathogenic Fusarium. spp. (F. oxysporum and F. solani)

that effectively controlled Fusarium wilt of tomato, watermelon, and muskmelon

in greenhouse tests. The mechanism of action for selected isolates was shown to

involve induced systemic resistance. These isolates were effective in significantly

reducing wilt incidence at low antagonist inoculum densities, at high pathogen

densities, and under varying environmental conditions, including in a variety of

soil types, over a range of temperatures, against multiple races, isolates, and

formae specials of the pathogen, and on several different tomato cultivars.

Research with the best biocontrol isolates (CS-20 and CS-1) was conducted to

determine their efficacy under field conditions as well as to evaluate some

potential (11).

14

2.3.2.1.Biocontrol Approaches a- Resistant cultivars Fusarium wilt is best controlled by the use of race-specific resistant cultivars, the

efficacy of which against virulent races of pathogen might be enhanced by the

use of antagonistic microorganisms. Rhizosphere bacteria have proved to be

effective biocontrol agents against root diseases of many crop plants. Their

antibiotic production now recognized as an important factor in disease

suppression. So far, antibiotic production has not been shown to be involved in

the suppression of Fusarium wilt diseases. Also, in most cases the antagonistic

ability of the putative biocontrol rhizosphere bacteria is determined against just

one or a few isolates of the target pathogen, and neither the variability in the

pathogen population nor the beneficial rhizosphere bacteria are usually

considered in terms of antagonistic activity (39).

In general, methods for control of Fusarium wilt, root, and stem rots on green-

house crops have emphasized development of resistant cultivars and avoidance

of primary inoculums. Breeding for resistance, however, can be difficult if

resistance genes have not been identified, as is the case for Fusarium root and

stem rot of cucumber. Pavlou et al. (40) recently identified disease-resistant

cucurbit root stocks onto which susceptible cucumber varieties could be grafted.

Reduction of primary inoculum (e.g., on seed, planting stock, and in growing

media) is an alternative option, through the use of fungicides, heat or chemical

treatments, disease-free planting stock, sanitation, and fumigation (41). Several

reports have also demonstrated the successful use of biological control agents

(mostly bacteria and fungi) against diseases caused by various formae speciales

of F. oxysporum on a range of hosts (42) grown under greenhouse or field

15

conditions. Similarly, composts and chitin amendments have been reported to

reduce the impact of diseases caused by F. oxysporum on several hosts (43).

b- Non- pathogenic fungi

Several fungi produce hydrolytic enzymes that degrade the cell walls of

phytopathogenic fungi including Fusarium (44). Examples of fungal biocontrol

agents include:

Fusarium

Non-pathogenic isolates of F. oxysporum and F. solani colonize the surface and

cortex of plant roots without causing disease symptoms and may play an

important role in the ecology and control of Fusarium-incited diseases. Several

diseases caused by formae specials of F. oxysporum have been controlled, with

varying degrees of success, by prior inoculation of the plant with (i) pathogenic or

nonpathogenic fungi of genera different from Fusarium (45), (ii) non-pathogenic

and mildly pathogenic Fusarium species such as F. solani (46), (iii) non-

pathogenic formae specials of F. oxysporum (47), (iv) nonpathogenic races of the

same forma specials (48), and (v) nonpathogenic F. oxysporum isolates (49).

Komada (50) demonstrated that sweet potato plants were protected against

Fusarium wilt, caused by F. oxysporum f. sp. batatas, in naturally infested soil by

prior inoculation with non-pathogenic F. oxysporum isolates, which were often

found in the vessels of healthy sweet potato plants and natural soils. It was

concluded that the control effect is due to the cross protection associated with

the host response, because no antagonistic interaction was observed between

nonpathogenic and pathogenic isolates in vitro. This biological control method

gave nearly the same effect as chemical control with benomyl.

Trichoderma

16

Various species of Trichoderma spp have received the most attention.

Trichoderma harzianum is a fungal biocontrol agent that attacks a range of

phytopathogenic fungi. T. harzianum alone or in combination with other

Trichoderma species can be used in biological control of several plant diseases

(51). It has been also shown to be effective in controlling Fusarium crown and

root rot under greenhouse and field conditions. Although Trichoderma spp is

ubiquitous, the type of the soil can affect growth, proliferation and effectiveness

as biocontrol agent. Because soil ecology is complex, and with year-to-year

fluctuations in climate conditions, treatments with microbials are often

inconsistent (52).

Research has demonstrated that biological control of Fusarium crown root rot

(FCRR) has been successful in some instances under greenhouse and field

conditions. The fungus Trichoderma, a natural soil-inhabiting genus, has been

used successfully to control Fusarium crown rot and root rot of tomatoes (53).

Trichoderma harzianum and Phytophthora macerans significantly reduced

severity of FCRR. Trichoderma harzianum reduced the severity of FCRR by 12%

and P. macerans 9% in comparison to the untreated control in the nonfumigated

treatments. No differences were observed between the biologicals and the

untreated control in the MBr treated plots (42)

c- Bacteria Bacteria are the most abundant microorganisms in the soil, with an average of 6

x 108 cells per gram of soil and a weight of approximately 10,000 kg/ha. Bacterial

mass thus accounts for approximately 5% of the total organic dry weight of soils.

The number of bacteria depends strongly on the season, the type of soil, the

moisture content, the oxygen supply in the soil, as well as the tillage and

fertilization of the soil, and also on the penetration of the soil by plant roots and

the depth from which the soil samples were taken. The micro-organism

population density and the make-up of the population in terms of species can

17

vary by up to a factor of 50 only as a result of soil tillage or organic fertilization

(32).

Next to the genera Pseudomonas, Arthrobacter, Clostridium, Achromobacter,

Micrococcus, and Flavobacterium, Bacillus species are the most common types

of bacteria isolated from soil samples (58) and can account for up to 36% of the

bacterial populations. Like the total number of microorganisms, this amount

varies according to environmental factors, plantation, type of fertilization, and the

other factors mentioned above (32).

Bacteria having the ability to form antifungal metabolites can be isolated easily

from soil samples. However, there have been only little systematic studies of the

abundance of such micro-organisms as a percentage of the total population.

Leyns et al. (74) and Lievens et al. (88) found that about 30% of all bacteria

isolated from soils were able to produce antifungal inhibition zones in vitro. About

3% of these isolates were assigned taxonomically to the genus Bacillus.

The rhizosphere, which comprises the region close to the surfaces of roots, and

the root surface itself, the rhizoplane, are colonized more intensively by

microorganisms than the other regions of the soil. Rhizobium bacteria,

Pseudomonads, and mycorrhiza fungi are among the best-known colonizers of

this region. Many micro-organisms from the rhizosphere can influence plant

growth and plant health positively, and are therefore often referred to as “plant

growth promoting rhizobacteria”. However, their effects must be seen as the

complex and also cumulative result of various interactions between plant,

pathogen, antagonists, and environmental factors (112).

Pseudomonas

Pseudomonas fluorescens have been studied extensively as biocontrol agents of

plant disease (101). Some strains particularly effective for controlling several soil-

borne pathogens (102). Because these bacteria possess many desirable

attributes, their potential for use in disease control strategies is substantial (101).

18

Combining the nonpathogenic F. oxysporum strain Fo47 with the bacterial strain

Pseudomonas putida Wc5358, two different disease- suppressive mechanisms

acted together to enhance suppression of fusarium wilt of carnation (103)

Rhizobacteria Chao (106) demonstrated that Rhizobium leguminosarum biovar phaseoli was

variably effective on the inhibition of the Pythium, Fusarium and Rhizoctonia

species.

Myxobacteria Six myxobacterial species belonging to the genus Myxococcus were tested in

vitro against 9 soil-borne plant pathogenic fungi (Cylindrocarpon spp, Fusarium

oxysporum f. sp. apii, Phytopthora capsici, Pythium ultimum, Rhizoctonia spp,

Sclerotinia minor, S. sclerotiorum, Verticillium albo-atrum, and V. dahliae)

Myxobacteria exhibited a strong inhibitory effect on both pathogenic and

beneficial fungal growth in vitro, and the degree of inhibition varied with the

species tested. The ability to produce certain antibiotics by the bacterial BC

agents had an effect on their interaction with myxobacteria. Antibiotic production

by BC agents may protect them from lyses by myxobacteria. Studies are

beginning to test the biological control efficacy of myxobacteria against soilborne

diseases of strawberry greenhouse studies (110).

Streptomyces

Streptomyces griseoviridis has been reported as an antagonist to the plant

pathogens Alternaria brassicicola, Botrytis cinerea, Fusarium avenaceum, F.

culmorum, F. oxysporum f.sp. dianthi, Pythium debaryanum, Phomopsis

sclerotioides, Rhizoctonia solani and Sclerotinia sclerotiorum (111).

19

Production of chitinolytic enzymes is hypothesized to be responsible for

suppression of fusarium wilt of cucumber by streptomyces (54).

Bacillus

Bacillus subtilis is a focal point in this study and will be handled in a separate

section

2.4. Bacillus subtilis

The genus Bacillus consists of a large number of diverse, rod-shaped Gram

positive (or positive only in early stages of growth) bacteria that are motile by

peritrichous flagella and are aerobic. Members of the genus are capable of

producing endospores that are highly resistant to unfavorable environment

conditions (55). The genus consists of a diverse group of organisms as

evidenced by the wide range of DNA base ratios of approximately 32 to 69 mol %

G + C (55), which is far wider than that usually considered reasonable for a

genus (56).

B. subtilis is a common saprophytic inhabitant of soils and is thought to

contribute to nutrient cycling due to the variety of proteases and other enzymes

members of the species are capable of producing. Growth normally occurs under

aerobic conditions, but in complex media in the presence of nitrate, anaerobic

growth can occur. Under adverse environmental conditions, B. subtilis produces

endospores that are resistant to heat and desiccation (55).

B. subtilis has been shown to produce a wide variety of antibacterial and

antifungal compounds (57). It produces novel antibiotics such as difficidin and

oxydifficidin that have activity against a wide spectrum of aerobic and anaerobic

bacteria (58) as well as more common antibiotics such as bacitracin, bacillin, and

20

bacillomycin B (59). The use of B. subtilis as a biocontrol agent of fungal plant

pathogens is being investigated because of the effects of antifungal compounds

on Monilinia fructicola (60), Aspergillus flavus and A. parasiticus and Rhizoctonia

(61).

Bacillus strains have been the most frequently exploited bacteria for commercial

development of biocontrol agents because of their resistant endospores, which

may remain viable for long periods and are tolerant to extreme temperatures and

pH (62).

The development of biological products based on beneficial microorganisms can

extend the range of options for maintaining the health and yield of crops.

Targeted research into the principles of biological control of microbial pathogens

began in the early twentieth century (63).

The product was based on a Bacillus species now known by the taxonomic name

Bacillus subtilis. This was the first major commercial success in the use of an

antagonist. In Germany, FZB24® Bacillus subtilis has been on the market since

1999 and is used mainly as a seed dressing for potatoes (64).

2.4.1. Competition through colonization of the rhizosphere and the rhizoplane by Bacillus subtilis

Micro-organisms in the rhizosphere and the rhizoplane live on discarded and

dead epidermis cells and root hairs and as well from metabolites such as

assimilates and amino acids excreted by the roots. In total, up to 20% of the

energy gained through assimilation in the leaves of a plant may be lost again via

its roots (43). Especially the so-called border cells which are eliminated into the

surroundings from the periphery of the root above the root cap are significant for

plant – microbe interactions (65). Under controlled conditions, border cells and

the metabolites formed by them accounted for 98% of the carbohydrate rich

material released by the plant as an exudates (66).

They have chemotactic effects on microorganisms and stimulate their sporulation

and growth. Though Bacillus subtilis is generally characterized as less

21

competitive in the rhizosphere than e.g. Pseudomonads, colonization of the root

by various strains of this species has been found. Relatively high population

densities have been isolated from root surfaces and from the rhizosphere (67).

As for other bacteria that colonize the rhizosphere, for Bacillus subtilis,

colonization of the roots and “co-growth” during their further development

appears to require the presence of a thin film of water on the root surface (68).

Antagonists for the control of plant diseases are also selected according to their

ability to colonize the rhizosphere (69). As a result of the colonization by the

applied antagonist, the naturally occurring micro-organisms are faced with a

competition situation, both for space that offers favorable possibilities for

development, such as attachment sites or regions into which plant exudates

emerge, and for nutrients and essential growth factors. Roots evidently have only

a limited capacity to provide a certain population size and a certain species of

microorganisms (70).

2.4.2. Formation of antibiotic metabolites

Various antibiotics can be produced by Bacillus subtilis; of these, bacilysin is

regarded as taxonomically relevant for the group because of the regularity of its

occurrence (71). In liquid cultures, FZB24® Bacillus subtilis also produces iturin-

like lipopeptides such as those described by Krebs et al. (72).

The efficacy of purified lipopeptides of this type against various phytopathogenic

fungi is in the range of 5–100 µg/ml, which is similar to that of fungicidal agents.

The formation of secondary metabolites by micro-organisms in synthetic culture

media and the quantity and composition of these secondary metabolites depends

strongly on the culture conditions and the growth phase of the culture. which is

also true in the case of Bacillus subtilis. For the production of large quantities of

these metabolites it is therefore necessary to optimize the growth conditions and

culture media during the fermentation process. Additionally the production of

such metabolites also depends on the stage of development of the bacteria. The

lipopeptides formed by Bacillus subtilis are released into the medium only at the

22

time of endogenous spore formation during the stationary phase of the culture

(71).

Investigations have been carried out to determine the significance of the

metabolites being effective against fungi in vitro, especially of the lipopeptides,

for the efficacy of FZB24® Bacillus subtilis. Maize seedlings were planted in

sterile quartz sand in small pots and drenched with 107 spores per ml of

substrate, corresponding to the recommended application rate for horticultural

crops. Applications of lipopeptides added directly to the substrate were used for

comparison. Whereas the added lipopeptides could be partly re-extracted and

quantitatively determined, no lipopeptides were detected in the substrates and

roots treated with FZB24® Bacillus subtilis. The definite proof of the formation of

these antibiotically active metabolites in non-sterile humous substrates was

impossible because of the complex matrix and the metabolic activity of the

accompanying microflora. The assignment of the lipopeptides found to a distinct

organism is hindered by the fact that organisms that produce lipopeptides of this

type occur very widely in soil and plant samples under natural conditions, as was

shown by Lievens et al. (88).

Moreover, it is probable that due to the competition between the micro-organisms

in the soil, only very small amounts of free nutrients are present, so that

secondary metabolites of the type in question are formed only in extremely small

quantities. Where lipopeptides were actually detectable, their concentrations

were less than the minimum inhibitory concentration for phytopathogenic soil

fungi.

Further confirmation that the formation of antifungal metabolites does not

contribute significantly to the effect emerges from a comparison of different

isolates of Bacillus subtilis. No correlation is found between e.g. the ability to

form metabolites that are effective against Fusarium oxysporum on various

media in vitro and the observed effects on the course of the Fusarium wilt

disease in greenhouse experiments with ornamentals (73).

23

It is therefore difficult to judge whether the lipopeptides play any part in the

reduction of the incidence and severity of plant diseases achieved by the

application of FZB24® Bacillus subtilis. In contrast the formation of compounds

possessing antibiotic activity appears to be a more basic factor for the affectivity

of Pseudomonads.The importance of the antibiotics, like phenazine, formed by

these micro-organisms, for the suppression of plant diseases has been

demonstrated with mutants deficient for phenazine- production (74). Moreover,

the antibiotic was detected on roots in the soil (75), and a quantitative dose-effect

relationship between antibiotic formation and the disease-suppressing effect of

Pseudomonas fluorescens against Pythium sp. has been demonstrated in

cucumbers (76).

2.4.3. Plant resistance induced by Bacillus subtilis All plants have evolved defense mechanisms against pathogens. The efficacy of

these resistance reactions is modified as a function of the ontogenetic

development of the plants and the influence of biotic and a biotic environmental

factors. Thus, contact with non-pathogenic micro-organisms or limited infections

leads to a decrease in the susceptibility of the plants. This increased resistance

due to exogenous factors with no alteration of the plant genome is known as

induced resistance. Induced resistance can be triggered both by pre-inoculation

with non-pathogens, pathogens, symbionts, and saprophytes and by application

of so-called abiotic inducers such as salicylic acid or microbial metabolites (77).

The induction of resistance has frequently been described and discussed in the

literature as an ability of micro-organisms. Established phytopathological tests for

induced resistance are based on the separation in space and/or in time between

the application of the inducing agent and the inoculation of the plants. Biotrophic

fungal pathogens such as powdery and downy mildews or Phytophthora

infestans are better controlled with resistance inducers.

24

It is assumed that the enhanced resistance of the plants is due to altered gene

expression. In many cases the induction of resistance is accompanied by

induction of various so-called PR proteins (pathogenesis-related proteins). Some

of these are 1,3-glucanases and chitinases having the ability to lyses fungal cell

walls. Other PR proteins are less well characterized or exhibit antimicrobial

activities. On the one hand, PR proteins are regarded as markers of induced

resistance, while on the other; these proteins themselves appear to be involved

in the increased resistance of the plants (78).

2.4.4. Promotion of root growth

A larger and healthier root system, such as has been observed in a number of

greenhouse and field experiments with FZB24® Bacillus subtilis, also leads to

improved uptake of water and nutrients. In a greenhouse experiment with

kohlrabi plants, the soil was drenched immediately after sowing and again 4

weeks later with 0.2 g FZB24®WG/l water at a rate of 2 l/m2. The treatment led to

a 5% increase in the dry root weight. In addition to an improved germination of

the seeds, the yield of the plants at the end of the cultivation time was up to 12%

higher, depending on the variety. The root development of potato plants was

determined in a field trial in 1998. The potatoes were planted in mid-May and

treated during planting with a liquid seed treatment in the recommended dosage

of 10g of FZB24® WG/100 kg of seed potatoes. With the beginning of tuber

formation in early August, the root fresh weight of the plants treated with FZB24®

was 6% higher than that of untreated plants.

The yield of the plants after treatment with FZB24® in this experiment was

increased at harvest time in September by about 8%. Enhanced root formation of

infected plants has been described by Garett (79) as a disease escape

mechanism. Increased root growth enables the plant to grow out of contaminated

regions of the substrate and to replace infected root sections more easily, and at

the same time enables the plant to reach earlier growth stages in which it is less

susceptible.

25

The intensified root formation after application of FZB24® may therefore also be

a reason for a reduction of plant damage due to infections of Rhizoctonia solani

or Fusarium oxysporum.

2.4.5. Effect on plant growth and yield

Another reason that has been proposed for the promotion of plant growth by

bacteria that colonize the rhizosphere is the production of phytohormones and

phytohormonally active metabolites (80). Dolej (81) was able to show that the

growth-promoting effect of culture filtrates of FZB24® Bacillus subtilis is not due

to lipopeptides having an antibiotic action. This is supported by investigations

with Bacillus subtilis mutants that no longer had the ability to form antibiotics, but

still led to increased yields from peanut plants (64).

The phytohormonal activity of the metabolites formed by FZB24® Bacillus subtilis

in liquid cultures has been demonstrated by biological test methods. Complex

culture filtrates and fractions derived from them led, like cytokinines, to enhanced

growth of radish cotyledons, and like auxins, increased the elongation of the cells

of wheat coleoptiles. These effects appear to be initiated by mixtures of several

proteins, while further separation and purification of the culture filtrates led to the

loss of the effects (82). According to Tang (82), a number of Bacillus subtilis

isolates have the ability to form phytohormones such as zeatin, gibberellic acid,

and abscisic acid.

A culture filtrate of a Bacillus subtilis isolate that was used as a resistance

inducer against biotrophic fungal plant pathogens has also been found to contain

the cytokinins zeatin and zeatin riboside. The senescence-delaying effect of them

could be a cause of the reduced damaging effect of pathogens and the increased

yield of plants in which resistance has been induced (83).

26

Enhanced root growth is often accompanied by increased branching and a higher

number of root tips. Their meristems are the most important sites for the

synthesis of free cytokinins (84).

These are presumably transported into the shoot via the xylem. Intensified and

prolonged synthesis of these phytohormones may be regarded as a cause of

delayed senescence and improved yields (85). Since the application of Bacillus

subtilis leads to stronger root growth, there may also be an increased synthesis

of plant cytokinins, which also cause delayed senescence and higher yields, as

described above. These effects on the phytohormone balance of the plants can

explain why increased yields are found even for plants that show no visible attack

by soil-borne or root diseases.

An increase in the yield was also achieved by leaf applications of FZB24®. In

three field trials in 1998 with potatoes, a leaf treatment was carried out in

combination with the application of leaf fungicides to control P. infestans. Four

applications of FZB24® with a dosage of 0.4% were carried out at intervals of 10

to 14 days beginning in mid-June. The effects were compared with a dry seed

treatment with FZB24®. Because of the fungicide treatment, the plants were

largely protected against attack by P. infestans. The application of FZB24® WG

to the leaf did not produce any visible improvement in the protection against the

leaf pathogen, but led to an increase in the yield of 8.5%, which was higher than

the yield increase achieved with the dry seed treatment in these experiments

In addition to the effects on the phytohormone balance of the plants, an

improvement of the tolerance of the plants may also contribute to increased

yields. Tolerance is defined as the ability of the plant to survive attack by

pathogens or the action of a biotic stress factors with smaller losses of viability

and productivity than another plant subjected to the same exposure intensity

(86). Possible factors that lead to tolerance of plants towards pathogens

according to Clarke (87) are:

27

• Reduced sensitivity of the plants towards toxic metabolites produced

by the pathogens.

• Ability of infected and uninfected parts of the plants to compensate

through increased metabolic activity, e.g. photosynthesis.

2.5. Integrated control (chemical and biological)

Bacillus megaterium and Burkholderia cepacia, demonstrated to be antagonistic

against Fusarium oxysporum f.sp. radicis-lycopersici, the causal organism of

fusarium crown and root rot of tomato, was evaluated as biocontrol agents alone

and when integrated with the fungicide carbendazim. In an initial screening,

these isolates reduced disease incidence by 75 and 88%, respectively.

In vitro, both biocontrol agents were highly tolerant to the fungicide carbendazim,

commonly used to control fusarium diseases. Carbendazim reduced disease

symptoms by over 50% when used at > 50 μ g mL-1, but had little effect at lower

concentrations. Combination of the bacterial isolates and carbendazim gave

significant (P ≤ 0·05) control of the disease when plants were artificially

inoculated with the pathogen. Application of carbendazim at a low concentration

(1 μ g mL-1) in combination with B. Cepacia c91 reduced disease symptoms by

46%, compared with a reduction of 20% obtained with the bacterium alone and

no control with the chemical treatment alone. A combination of B. Megaterium

c96 with an increased application rate of 10 μ g mL -1 carbendazim significantly

reduced disease symptoms by 84% compared with inoculated controls and by

77% compared with carbendazim treatment alone. The integrated treatment also

slightly outperformed application of 100 μ g mL-1 carbendazim, and bacteria

applied without fungicide also provided good disease control (28).

28

CHAPTER III

MATERIALS AND METHODS

3.1. Materials 3.1.1. Apparatus and special equipments

• Water bath

• Incubator

• Mixer, Vortex

• Autoclave

• Hot air oven

• Refrigerator

• Hot plate and stirrer

• pH meter

• Computer

• Magnetic stirrer

• Thermometer

• Table lamp

• Light microscope

• Digital camera

• Balance (top loader or pan)

• Inoculating wire loop

• Inoculating needle

• Automatic pipetts

• Aluminum paper

• Cotton

• Filter paper

• Tissue paper

• Parafilm

29

• Tips

• Plastic droppers

• Plastic Petri plates

• Screw cap culture tubes

• Glassware

• Sterile cotton swab

• Sterile cotton

• Plastic pots

3.1.2. Stains

• Methylene blue stain

Methylene blue - 0.3 g

95% Ethanol - 30 mL

Distilled water - 100 mL

• Safranin stain

• Malachite green stain 5%

• Congo red stain

Congo Red - 1 gm

Alcohol 50% - 100 mL

• Gram stain kit

• Crystal Violet Solution

0.2% Crystal Violet in 2.5% Acetic acid

• Crystal Violet Solution for Gram's stain

Crystal Violet - 2 gm

95% alcohol - 20 mL

30

Ammonium oxalate - 0.8 gm

Distilled water - 80 mL

Dissolve crystal violet in alcohol and the ammonium oxalate in water. Allow the

Ammonium oxalate solution to stand overnight or heat gently until dissolved. Mix

the two solutions together and filter.

• Lactophenol Cotton blue

Stock Solution: Cotton blue - 1 g - dissolve in 100 mL of Lacto phenol

Working Solution:

5 mL of stock solution in 100 mL of lactophenol

3.1.3. Reagents

• Sterile distilled water

• Normal saline

• Hydrogen peroxide (3%)

• Sodium hypochlorite (2%)

• Glycerol

• Tartaric acid

• Lactic acid

• 95% Ethanol

• Streptomycin sulfate

3.1.4. Culture media

• Sulfur Indole Motility medium

• Nutrient Agar Medium

Nutrient agar plates were (7.5 cm diameter) prepared having

0.5 cm thick layer of medium.

31

• Nutrient Broth with 10% NaCl

• Muller Hinton Agar

• Simmon,s Citrate Agar Medium

• Methyl Red-Voges Proskauer Medium

• Starch Agar Medium

• Nitrate Broth

• Nutrient Gelatin Medium

• Triple Sugar Iron Agar Medium

• Fermentation sugars (D glucose, L arabinose, D-xylose, D-mannitol,

lactose, salicin, sucrose and maltose

• Skim Milk Agar

15 g Skim Milk

1000 ml dH2O

15 g agar

Dispense 4 ml to each test tube. Autoclave.

Cool, store in the media refrigerator.

• Potato Dextrose Agar Medium

Suspend 39 g of dehydrated media in 1000 ml of purified filtered water. Heat with

frequent agitation and boil for one minute. Sterilize at 121º C for 15 minutes. Cool

to 45-50º C. Mix gently and dispense into sterile Petri dishes. To adjust pH at 3,5,

add 10 ml of sterile Tartaric Acid Solution at 10%

• Komada’s Selective Medium

The basal medium contains the following constituents in 1L distilled water and is

autoclaved (121°C for 15 min) and cooled to 55°C before antimicrobial agents

are added.

32

K2HPO4 1.0g

KCl 0.5g

MgSO4.7H2O 0.5g

F3Na EDTA 0.01g

L Asparagine 2.0g

D Galactose 20.0g

Agar 15.0g

To the basal medium is added in 10ml sterile distilled water:

PCNB as Terrachlor® 1.0g

Oxgall 0.5g

Na2B4O7.10H2O 1.0g

Streptomycin sulfate 0.3g

The pH is adjusted to 3.8±0.2 with 10% phosphoric acid.

• Sabrouad Dextrose Medium

3.2. METHODOLOGY 3.2.1. Isolation of Fusarium oxysporum Tissue selected for isolation should be typical of the diseased material. Ideally,

freshly infected tissue or tissue at the advancing edge of a necrotic area should

be selected (89).

Necrotic tissues of plants were washed and cut into small pieces (2 mm).

Sections of necrotic tissues were surface disinfested in 1 % NaClO for 2 min,

rinsed in distilled water, and damp-dried on absorbent paper towels before being

plated on the media (90).

This procedure will reduce the chance of bacterial contamination. Removal of

cortex will facilitate isolation of Fusarium oxysporum from vascular tissue (89).

33

Then placed on Komada’s Fusarium-selective medium (91) and acidified potato-

dextrose agar (APDA). Many saprophytic fungi and bacteria also can grow on the

medium and interfere with the recovery of the Fusarium present. If PDA is used

for the recovery of fungi from plant material, then the concentration of potato and

dextrose should be reduced by 50-75%, and broad-spectrum antibiotics included

inhibiting bacterial growth (89).

Plates were incubated at room temperature (25-28 °C). Fungi developing from

the plated seedling segments were transferred to APDA and APDA-WA (water

agar) to be identified. Fungal colonies were identified by microscopic observation

of conidium and microconidiophore morphology, along with the cultural

characteristics on APDA, such as pigmentation and colony morphology (90).

The APDA-WA technique (92) was used for conidium, conidiophore morphology,

and chlamydospore production by placing a small APDA plug (about 1 cm2) with

the fungus on a 2 % WA plate. A glass cover slip was placed on the WA to

facilitate microscopic observations. Fungal colonies were subcultured on PDA

slants. Maintenance of cultures was done by transferring active cultures on PDA

slants twice a year. After suitable growth and sporulation, the slants were kept at

4-5 °C.

3.2.2. Isolation of Bacillus spp from soil Different soil samples were taken from Beit Hanoun and Biet Lahia. The

grassland soil used in all experiments was collected from the 0-15 cm layer.

Each gram of sample was suspended in 99 ml of sterile distilled water and

shaken vigorously for 2 min by vortex mixer. The samples were heated at 60 0C

for 60 min in a water bath. Then the soil suspensions were serially diluted in

sterile distilled water, and the dilutions from 10-1 to 10-6 were plated on nutrient

agar medium. The plates were incubated at 28- 37 0C for 24-48 hour (93+94).

Antagonistic isolates of bacteria were identified by biochemical (according to PIB

win software).

34

3.2.3. In vitro inhibition of Fusarium oxysporum by isolated soil bacteria Soil bacteria isolates were tested for their ability to inhibit Fusarium oxysporum in

vitro, on agar plates as described by Weller and Cook (95) and Wong and Baker

(96). The pathogenic Fusarium oxysporum was transferred to regular Petri

dishes containing fresh PDA to produce fungal mycelium plugs. Each bacterial

isolate was streaked at opposite ends of agar plates near the edge and

incubated at 27±1°C for 48 h. An agar plug (5 mm in diameter) containing fungal

mycelium, taken from the radiating edge of a culture grown on PDA was placed

in the center of each plate. Plates were incubated for about five days more or

until the leading edge of the fungus reached the edge of the plate. The size of the

zone of inhibition of fungal growth around each bacterial species was used as a

measure of the ability of those bacteria to inhibit Fusarium oxysporum and was

scored as described by Weller and Cook (95).

3.2.4. Application of Bacillus species as biocontrol against Fusarium oxysporum Serial dilutions from pure cultures of the isolated bacteria were loaded into wells

opened at the periphery of nutrient agar at equal distance from the center as

shown in the Figure (4.13).

Pathogenic Fusarium spp isolates from diseased plants were inoculated at the

center of the plates 0, 1, 2, and 3 days after the loading of bacteria. Plates were

incubated for several days at ambient temperature. Diameters of inhibition zones

of fungal growth were measured.

Commercial antifungal chemical (carbendazim) was used as a control and its

activity was compared with the bacterial inhibition zones of fungal growth. The

concentrations of carbendazim used were 0.4, 0.8, 1.0, 2.0 g/L

3.2.5. Pot experiment Ten centimeter plastic pots with perforated bases were washed in detergent,

soaked in Biocide overnight, and then rinsed and stacked back on the shelves in

the preparation room. Soil was put into autoclavable bags and sterilized in the

35

autoclave at 1210C, 15 min. Each pot was filled with 500 g soil .The soil was

taken from the same field on which the pathogenic F. oxysporum was isolated

(97).

The bacterial suspension was adjusted turbidimetrically (McFarland standard) to

about 108 colony forming units (CFU/ mL) for each experiment. The bacterial

suspensions were used for seed coating (98) as described below.

Seeds were immersed in a slurry solution (10% sucrose) to allow bacteria to

adhere on the seeds.

Seeds of three plant species: watermelon, muskmelon, and cucumber, were

planted in the mentioned pots and were irrigated with 50 ml daily, these pots

were distributed into four groups:

Positive control group, negative control group, chemically treated group, and

biologically treated group.

Conidial suspensions (10 6 conidia/ ml) were prepared by washing the 7 to 14

day old PDA slants with sterile distilled water and filtering the suspension through

cheesecloth. Conidial densities in the suspension were determined by use of a

hemcytometer under a compound microscope (99). Conidial suspensions were

inserted in all the groups except the negative control group.

The experimental groups, positive control group, biological treated group, and the

carbendazim treated group were injected by macroconidia of F. oxysporum (1 ml

of suspension which had 106 macroconidia). The injections were done by a

syringe nearly 5 cm subsurface of soil around the plantlets root system.

By planting rooted cuttings in soil for two weeks, wounding roots by passing a

metal spatula through the soil around each plant, and then pouring water

containing conidia of the pathogen onto the soil (100). In the chemically treated

group carbendazim was sprayed (28).

36

Table 3.1. Illustrates the preparation of nutrient stock solutions for preparing the irrigation

solution.

Solution symbol

Substrate

Molarity Grams per liter

A Ca(NO3)2 1 236.1 B KNO3 1 101.1 C MgSO4 1 246.4 D KH2PO4 1 136.1

E

Microelements: MnCL2 H3BO3 ZnSO4 CuSO4 H2MoO4

1.81 2.86 0.22 0.08 0.09

F Fe-EDTA 32.8 The required volume (in ml) to prepare four liters of nutrient solution from the

stock solution described above are as shown below.

A B C D E F Complete nutrient solution 20 20 8 4 4 4

3.2.6. Statistical analysis Statistical analysis was conducted using the SPSS version 11.0. Data were

analyzed using standard analysis of variance (ANOVA) with interactions. The

significance was evaluated at P< 0.05 for all tests.

37

CHAPTER IV

RESULTS

4.1. Microscopic identification of F. oxysporum

All isolates of F. oxysporum produced abundant, single-celled, oval or oblong

microconidia as shown in (Figure 4.1b.). Short and unbranched

microconidiophores, and long, four to five septate, slightly falcate macroconidia

as shown in (Figure 4.1a). These were the most important morphological

characteristics to identify F. oxysporum. Chlamydoconidia are round hyaline,

smooth walled, borne singly or in pairs on short lateral branches as shown in

(Figure 4.1 c).

(c) Figure 4.1. Characteristics of Fusarium oxysporum: (a) macroconidia, (b) microconidia, and (c)

chlamydospores

(a) (b)

38

4.1.1. Characteristics of F. oxysporum 4.1.1.1. On PDA Colony morphology on PDA varies widely. Mycelia may be floccose, spare or

abundant and range in color from white to pale violet. F. oxysporum usually

produces a pale to dark violet or dark magenta pigment in the agar. A

representative example is shown in (Figure 4.2)

Colony morphology, pigmentation and growth rates (Figure 4.3) of cultures of

most Fusarium species on PDA are reasonably consistent if the medium is

prepared in a consistent manner, and if the cultures are initiated from standard

inocula and incubated under standard conditions. These colony characteristics

often are useful secondary criteria for identification.

The isolates had different colony characteristics and pigmentation on PDA. The

aerial mycelium of F. oxysporum isolates ranged from white to purple and the

undersurface of colonies ranged from white to dark purple. The aerial mycelium

and the undersurface of colonies for F. solani isolates ranged from cream to tan.

Conidia formed on PDA are usually variable in shape and size and so are less

reliable for use in identification (Figure 4.1.)

Figure 4.2. A representative photograph of F .oxysporum grown on PDA

39

4.1.1.2. On Komada’s Medium The colonies of F. oxysporum on Komada’s Medium are pigmented with a

reddish purple color and surmounted by a pinkish white aerial mycelium as

shown in Figure 4.5. Other Fusarium species are suppressed.

Figure 4.4. A microphotograph of F. oxysporum normal haphae

Figure 4.3. Growth rate (25-40 mm in diameter) of F. oxysporum on PDA at 300C after three days in

complete darkness.

40

4.1.1.3. On SDA Medium F. oxysporum grows rapidly on Sabouraud dextrose agar at 25°C and produces

woolly to cottony, flat, spreading colonies as shown in Figure 4.6.

Figure 4.6. A photograph of F. oxysporum colony on SDA media

Figure 4.5. A photograph of F .oxysporum colony on Komada’s Medium.

41

4.2. Identification of Bacillus subtilis Bacillus colonies are typically white and dry or pasty looking as shown in Figure

4.7but some form very mucoid colonies (that can drip onto the lid of the plate).

Bacillus cells are typically fairly rectangular rods. Often occurring in pairs or

chains as shown in Figure 4.8.

Figure 4.8. Gram stained Bacillus

subtilis cells. Figure 4.7. Appearance of Bacillus subtilis colonies on a nutrient agar

plate

Figure 4.9. A photograph of Bacillus endospores

42

4.2.1. Characteristics of Bacillus The following table (4.1) illustrates the examined microscopically and biochemical characteristics of Bacillus isolates. The results of the tested parameters were according to PIB win software (88). Table 4.1. Examined biochemical and microscopic characteristics Bacillus isolates.

Test Result Gram stain Gram positive Spores shape Oval Spores position Central Spores bulging negative Casein hydrolysis positive Hippurate hydrolysis positive Starch hydrolysis positive Urease positive Chloramphenicol resistance S Nalidixic acid S Polymyxin B R Streptomycin S Fructose acid positive Galactose acid negative Lactose acid negative Xylose acid Positive Citrate utilisation positive Growth at 50 0C positive Growth in 10% NaCl positive Anaerobic growth negative Nitrate reduction positive Oxidase reaction positive Voges-Proskauer test positive 4.3. In vitro inhibition of F. oxysporum by soil bacterial isolates The size of the zone of inhibition of fungal growth around each bacterial species

was used as a measure of the ability of those bacteria to inhibit F. oxysporum.

Zones of growth inhibition were detected around bacterial strains placed at three

positions on the plate as illustrated in Figure 4.10.

43

The damage caused by the bacterium to the fungal mycelium was studied

microscopically. The mycelium along with the agar disc present in the inhibition

zone and control mycelium were taken, stained with lactophenol cotton blue and

observed under microscope. Light microscope study revealed the presence of

abnormal hyphae, with condensation and deformation as shown in Figure 4.11.

There were swellings of mycelial tips and cells in between.

Figure 4.10. Photographs of different Bacillus isolates tested for their ability to inhibit the growth of F. oxysporum. Two inhibition

zones are indicated by arrows

Figure 4.11. A photograph of F. oxysporum hyphae damaged by

Bacillus isolates. Swelling is indicated by arrow.

Bacillus isolates

44

4.4. Application of carbendazim as chemical control agent against Fusarium oxysporum

4.4.1. Carbendazim Figure 4.13: illustrates the effect of carbendazim on F. oxysporum and table 4.2. Indicates the concentrations of carbendazim and the inhibition zones obtained. It is worth mentioning here that the concentration of carbendazim used by farmers is 0.8 g/L.

Table 4.2. The average of inhibition zones diameter after application of carbendazim

Diameter of inhibition zone

(mm)


(mm) 0.4 10 0.8 9 1.0 10 2.0 11

Figure 4.12. A photograph shows that inhibition zones by different dilutions of carbendazim.

45

4.5. Application of Bacillus subtilis as a biocontrol agent against Fusarium oxysporum 4.5.1. Application of Bacillus subtilis and F. oxysporum together

Figure 4.13. Shows the inhibitory effect of various dilutions of B. subtilis isolate

on F. oxysporum. Table 4.3. Illustrates the average diameter of inhibition zones

produced by serial dilutions of B. subtilis.

Table 4.3. Average inhibition zones after application of different dilutions of Bacillus subtilis

Bacterial dilution


(mm) Stock culture

(2.6x107) CFU/ml 9

10-1 7 10-2 8 10-3 7 10-4 4 10-5 0

Figure 4.13. A photograph illustrating the inhibition zones produced by applying different dilutions of Bacillus subtilis together with F. oxysporum.

46

4.5.2. Application of Bacillus subtilis on one day old F. oxysporum culture Figure 4.14. Shows the inhibitory effect of various dilutions of B. subtilis isolate

on F. oxysporum one day old F. oxysporum culture. Table 4.4. Illustrates the

average diameter of inhibition zones produced by serial dilutions of B. subtilis.

Table 4.4. The average diameter of inhibition zones produced by applying different dilutions of

Bacillus subtilis on one day old F .oxysporum culture.

Bacterial dilution Diameter of

inhibition zone (mm)

Stock culture 6 10-1 3 10-2 0 10-3 0 10-4 0 10-5 0

Figure 4.14. A photograph illustrating the inhibition zones produced by applying

different dilutions of Bacillus subtilis on one day old F .oxysporum culture.

47

4.5.3. Application of Bacillus subtilis on two day’s old F. oxysporum culture Figure 4.15. Shows the inhibitory effect of various dilutions of B. subtilis isolate

on two days old F. oxysporum culture. Table 4.5. Illustrates the average diameter

of inhibition zones produced by serial dilutions of B. subtilis.

Figure 4.15. A photograph illustrating the inhibition zones produced by applying different dilutions

of Bacillus subtilis on two days old F .oxysporum culture.


Bacillus subtilis on two days old F .oxysporum culture.

Bacterial dilution Diameter of

inhibition zone (mm)

Stock culture 6 10-1 6 10-2 4 10-3 0 10-4 0 10-5 0

48

4.5.4. Application of Bacillus subtilis on three days old F .oxysporum culture. Figure 4.16. Shows that the inhibitory effect of various dilutions of one B. subtilis

isolate on three days old F .oxysporum culture. Table 4.6. Illustrates the average

diameter of inhibition zones produced by serial dilutions of B. subtilis on three

days old F .oxysporum culture.

Figure 4.16. A photograph illustrating the inhibition zones produced by applying different dilutions

of Bacillus subtilis on three days old F .oxysporum culture.


Bacillus subtilis on three days old F .oxysporum culture.

Bacterial dillution


(mm) Stock culture 6

10-1 0 10-2 0 10-3 0 10-4 0 10-5 0

49

4.6. Symptoms observed on infected plants There is a marginal yellowing progressing to a general yellowing of the older

leaves, and wilting of one or more runners. In some cases, sudden collapse

occurs without any yellowing of the foliage. On stems near the crown of the plant,

a linear, necrotic lesion may develop, extending up the plant and usually on one

side of the vine Figure 4.17.

Figure 4.17 Necrotic lesion of watermelon stem

One runner on a plant may wilt and collapse, with the rest of the runners

remaining healthy. Gummy, red exudates may ooze from these lesions, but this

may also be caused by gummy stem blight and insect injury. Vascular

discoloration should be evident and is very diagnostic as indicated in Figure 4.18.

50

Figure 4.18 Vascular area discoloration as viewed internally

4.7. Pot trials The results of a pot trial are shown in the figures 4.19, 4.20, 4.21. Here we have

four groups, one of which had no pathogen added (the negative control), one to

which the pathogen was added (the positive control), one with the pathogen and

positive biocontrol Bacillus antagonists from the plate competition studies above

(biologically treated), and the last one had both the pathogen and the antifungal

agent carbendazim (chemically treated).

51

In watermelon, cucumber and muskmelon Figures 4.19, 4.20, and 4.21,

respectively the results showed that the negative control group is growing well

indicating that the growth conditions are normal. The pathogen infected (positive

control) group showed poor growth (as expected). The biologically treatment was

efficient in reducing disease and allowed the plants to grow pretty well. The

chemically treated group showed that the carbendazim ability to reduce disease

is little more efficient than the biological treatment.

Chemically

treated Biologically

treated Negative control

Positive control

Figure 4.19. A photograph of watermelon grown under different treatments

52

Positive control

Negative control

Biologically treated

Chemically treated

Chemically treated

Biologically treated

Negative control

Positive control

Figure 4.20. A photograph of cucumber grown under different treatments

Figure 4.21. A photograph of muskmelon grown under different treatments

53

4.7. Statistical analysis Statistical analysis were conducted using the general SPSS (version 11.0.) Data

were analyzed using standard analysis of variance (ANOVA) with interactions.

The significance was evaluated at P< 0.05 for all tests.

4.7.1. Effect of different treatments on watermelon.

4.7.1.1. Dry weight Table 4.7 illustrates the significance in the mean values of the chemical treated,

biologically treated groups as compared to the positive control group.

Table 4.7. Effect of biological and chemical control on dry weight of watermelon

Group Mean ± SD P value

Chemically treated (n=16)

2.4 ± 0.2 0.01

Biologically treated (n=16)

2.6 ± 0.2 0.01

Positive control (n=16)

1.2 ± 0.2

4.7.1.2. Stem length Table 4.8 illustrates the significance in the mean values of the chemical and

biological treated as compared to the positive control group.

Table 4.8. Effect of biological and chemical control on stem length of watermelon



35.3± 3.0 0.01


25.0 ± 5.0 0.01


14.8 ± 4.0

54

4.7.1.3. Root length Table 4.9 illustrates the significance in the mean values of the biologically treated

group as compared to the positive control group but chemically treated group

was did not significant differences.

Table 4.9. Effect of biological and chemical control on root length of watermelon



10.1 ± 1.0 0.31


11.3 ± 1.5 0.03


8.8 ± 2.4

root length (cm)stem length (cm)dry w eight (g)

Mea

n

40

30

20

10

0

CHEMICAL

BIOLOGIC

POSITIVE

Figure 4.22. Compares the means of dry weight, stem length and root length in the three different

groups (chemically treated, biologically treated and positive control) of watermelon.

55

4.7.2. Effect of different treatments on Cucumber 4.7.2.1. Dry weight Table 4.10 illustrates that the chemically treated and biologically treated groups

are significance as compared to the positive control group.

Table 4.10. Effect of biological and chemical control on dry weight of cucumber

Group Mean ± SD P value Chemically treated

(n=16) 3.0 ± 0.5 0.01


2.6 ± 0.5 0.01


1.2 ± 0.2

4.7.2.2. Stem length

Table 4.11 illustrates the significance in the mean values of the chemically and


Table 4.11. Effect of biological and chemical control on stem length of cucumber



24.6 ± 8.0 0.01


24.5 ± 6.0 0.01


11.0 ± 4.0

4.7.2.3. Root length Table 4.12 illustrates the significance in the mean values of the biologically

treated as compared to the positive control group but chemically treated was did

not significant differences.

56

Table 4.12. Effect of biological and chemical control on root length of cucumber


(n=16) 16 ± 2.0 0.59


17.5 ± 3.0 0.02


15.0 ± 3.0


Mea

n

30

20

10

0

CHEMICAL

BIOLOGIC

POSITIVE


groups (chemically treated, biologically treated and positive control) of cucumber.

4.7.3. Effect of different treatments on Muskmelon 4.7.3.1. Dry weight


biological treated groups as compared to the positive control group.

57

Table 4.13. Effect of biological and chemical control on dry weight of muskmelon


(n=16) 5.8 ± 0.6 0.01


2.9 ± 0.4 0.04


2.4 ± 0.6

4.7.3.2. Stem length

Table 4.14 illustrates that significance in the mean values of the chemically and


Table 4.14. Effect of biological and chemical control on stem length of muskmelon



27.6 ± 3.0 0.01


26 ± 4.5 0.01


18.4 ± 4.8

4.7.3.3. Root length


biological treated groups as compared to the positive control group.

Table 4.15. Effect of biological and chemical control on root length of muskmelon



14.0 ± 2.0 0.02


16.1± 3.6 0.01


11.6 ± 2.4

58


Mea

n

40

30

20

10

0

CHEMICAL

BIOLOGIC

POSITIVE


groups (chemically treated, biologically treated and positive control) of muskmelon.

59

CHAPTER V

DISCUSSION

Fusarium wilt disease is caused by pathogenic forms of the soil-inhabiting fungus

F. oxysporum. F. oxysporum is a major disease problem on many crops.

Currently, control of this pathogen is generally by chemical control (fungicides).

Synthetic fungicides are considered the primary means to control Fusarium

disease. However, several reasons, such as the publics growing concern for the

human health conditions and the environmental pollution associated with

fungicides usage and development of fungicide- resistant strains of Fusarium.

Biological control has emerged as one of the most promising alternatives to

chemicals. During the last twenty years, several biological control agents have

been widely investigated for use on different pathogens.

In this study we investigated the effect of various Bacillus species isolated from

soil on Fusarium oxysporum both on plates and on plants grown in pots. The

efficacy of the Bacillus was also compared to the chemical antifungal agent

carbendazim.

5.1. Characteristics of Fusarium oxysporum F. oxysporum usually produces a pale to dark violet or dark magenta pigment on

the agar if incubated in dark place. PDA is used by some researchers for the

isolation of Fusarium species. We do not recommend this medium for this

purpose, as many saprophytic fungi and bacteria also can grow on the medium

and interfere with the recovery of the Fusarium present. If PDA is used for the

recovery of fungi from plant material, then the concentration of potato and

dextrose should be reduced by 50-75 %, and broad –spectrum antibiotics should

be included in the medium to inhibit bacterial growth (113).

Colonies of F. oxysporum are distinctly pigmented on Komada’s medium, and

usually separable from other Fusarium species on this basis (89). Growth of

60

other Fusarium species may be suppressed by the medium, and this medium

often is not a good choice for the recovery of Fusarium communities that contain

species other than F. oxysporum (15). In our study we used three different media

(PDA, Komadas media, and SDA) for the isolation of Fusarium oxysporum. This

combination should be sufficient for the isolation of Fusarium oxysporum.

The hyphae of F. oxysporum are septate and hyaline. Conidiophores of this

organism are short monophialids. Macroconidia are abundant, slightly sickle-

shaped, thin- walled and delicate, with an attenuated apical cell and a foot-

shaped basal cell, 3-5 septate. Microconidia abundant, in false heads only.

Mostly nonseptate, ellipsoidal to cylindrical, straightly curved. Chlamydospores

formed singly or in pairs and may be profuse in some strains (92). Microscopic examination was also employed in this study for confirming the

identify of Fusarium oxysporum.

5.2. In vitro inhibition of Fusarium oxysporum by soil bacterial isolates Bacillus subtilis was isolated from soil samples. The bacterium was identified

according to standard methods including biochemical tests and microscopic

examination.

In the dual culture experiments, all bacterial isolates tested inhibited the growth

of F. oxysporum. Zones of inhibition were observed between the colonies of

pathogen and bacteria. The inhibition zone could be due to the effect of diffusible

inhibitory substances produced by the bacteria, which suppressed the growth of

F. oxysporum. The presence and size of the zones of inhibition have been used

as an evidence of the production of antifungal compounds by the bacteria (113).

In this study we showed that the increase in bacterial concentration increases the

inhibition zones, and the application of Bacillus subtilis and F. oxysporum

together showed larger inhibition zones.

An interaction zone appeared between the Bacillus and Fusarium on plate, the

hyphae from these fungi stained with cotton blue, were free of cytoplasmic

61

content, possibly by the effects of the inhibitory substance(s) produced by the

Bacillus assayed as the biocontrol agent (104). Further studies are needed in

order to define the nature of the inhibitory substance.

5.3. Application of carbendazim as chemical control agent against Fusarium oxysporum 5.3.1. Carbendazim

Carbendazim reduced disease symptoms by over 50% when used at > 50 µg ml-

1, but had little effect at lower concentrations (28).

5.4. Application of Bacillus subtilis as a biocontrol agent against Fusarium oxysporum

Results from bioassays in dual cultures suggest that production of antifungal

substance(s) by these bacteria may be involved in the inhibition of hyphal growth

of Fusarium. This is supported by the following observations: (a) for all bacterial

isolates selected initially, there was no direct contact between fungal mycelium

and bacterial colonies, so that the inhibition of fungal growth was due to

substances that diffused into the agar medium, (b) the PDA medium used for

dual cultures is rich in nutrients and thus competition for them might be excluded,

and (c) antibiosis is the general mode of antagonism observed for Bacillus spp.

Most Bacillus spp produce antibiotics, many of which have antifungal activity

(105).

5.5. Symptoms observed on infected plants

Infection of the host occurs by penetration of the roots, primarily in the area of

elongation, and is aided by wounds. Disease severity is maximum at soil

temperature of 24 0C and declines dramatically above 30 0C. Low soil moisture

favors the pathogen and accentuates the wilting symptoms. Moreover, High

nitrogen and acidic soils favor disease development.

When applied to seeds B .subtilis provided crop protection mostly due to direct

control of soil borne pathogens through efficient production of various fungitoxic

metabolites (62).

62

Several mechanisms have been suggested for the ability of B. subtilis to inhibit

fungal pathogen. Bacillus subtilis inhibits plant pathogen spore germination,

disrupts germ tube growth and interferes with attachment of the pathogen to the

plant. Colonies of Bacillus subtilis take up space on the roots, leaving less space

for occupation by disease pathogens. Bacillus subtilis consumes exudates, which

deprives disease pathogen of a major food source, thereby inhibiting their ability

to thrive and reproduce. Bacillus subtilis has been shown to combat pathogenic

fungi through the production of iturin which inhibits the pathogens growth (62).

When applied directly to seeds, the bacteria colonize the developing root system,

competing with disease organisms that attack root system (62).

Iturin is a class of lipopeptide antibiotics. Iturins help Bacillus subtilis bacteria out-

compete other microorganisms by either killing them or reducing their growth

rate. Iturins can also have direct fungicidal activity of pathogens.

Lazzaretti et al. (109) reported that B .subtilis inhibited the growth of various fungi

including F .oxysporum in vitro. This may be due to production of an agent

containing the antifungal antibiotics bacillomycins.

The pathogen may cause disease by avoiding or overcoming any of these

defense mechanisms or by vascular occlusion. The cause of wilting by the

pathogen may be due to either vascular occlusion resulting in the failure of water

transport or toxin production. With F. oxysporum f. sp. Melonis however, wilt-

resistant muskmelon plants also harbor the pathogen extensively within the

vascular elements (107).

This phenomenon may be due to degradation of fungal cell wall-degrading

enzymes (CWDEs) by the resistant host as a defense response or alternatively,

the fungus may not be expressing these factors in resistant plants, but the

63

mechanisms governing this resistance are unknown. Likewise, the resistance

mechanisms in watermelon have not been elucidated (108).

5.6. Effect of different treatments in pot trials. The observed biological effects of applying the biological agent (B. subtilis) are

also due to changes in the physiology of the plant. In the first place, the tolerance

towards a biotic and biotic stress factors is improved because the root system of

the plant is strengthened, and hence also the uptake of water and nutrients. In

addition, many results indicate that the application of Bacillus subtilis changes

the phytohormone balance in the plant in such a manner that greater quantities

of reserve substances are incorporated into storage organs. Hence the plant root

length, stem length and dry weight are expected to be improved by application of

biocontrol agents.

The results of this study have shown that the dry weight and stem length of

watermelon, cucumber and muskmelon were significantly different in chemically

and biologically treated groups as compared to the positive control group as

shown in Tables 4.7, 4.8, 4.10, 4,11, 4.13, 4.14.

Also we have observed that the root length was significantly different in only

biologically treated group in watermelon, cucumber and muskmelon but not in the

chemically treated group again in comparison to the positive control group as

shown in Tables 4.9, 4.12, 4.15.

In conclusion, we have been able to isolate bacterial isolate of Bacillus subtilis,

which have shown to have on observable antifungal activity against Fusarium

oxysporum. Therefore we suggest that Bacillus subtilis can be used in the control

of Fusarium oxysporum in agriculture, in particular watermelon, cucumber and

muskmelon.

In addition we suggest further studies, on the different types of antifungal that are

produced by these bacteria.

64

CHAPTER VI

CONCLUSION AND RECOMMENDATIONS

• The application of soil isolating B. subtilis inhibited the hyphal growth of F.

oxysporum and the optimum inhibition was observed when both B. subtilis

and F. oxysporum were inoculated together.

• Results from bioassays in dual cultures suggest that production of

antifungal substance(s) by these bacteria may be involved in the inhibition

of hyphal growth of F. oxysporum.

• Application of B. subtilis to plant seeds was observed to provide crop

protection which could be due to direct control of F. oxysporum by

fungitoxic material.

• Dry weight and stem length of watermelon, cucumber and muskmelon

were significantly different in chemically and biologically treated groups as

compared to the positive control group.

• Root length was significantly different in only biologically treated group in

watermelon, cucumber and muskmelon but not in the chemically treated

group again in comparison to the positive control group.

RECOMMENDATIONS

• Further studies are needed in order to define the nature of inhibitory

substance(s), which are produced by B. subtilis against F. oxysporum.

• It is recommended to use B. subtilis in the control of F. oxysporum in

agriculture, in particular for watermelon, cucumber and muskmelon, and

this will help reduce the use of chemical fungicides, and therefore reduce

its risks on the environment and health.

65

CHAPTER VII

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