in vitro control of fusarium oxysporum by soil isolates of
TRANSCRIPT
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
4.7.2.1. Dry weight 55
4.7.2.2. Stem length 55
4.7.2.3. Root length 55
4.7.3. Effect of different treatments on Muskmelon 56
4.7.3.1. Dry weight 56
4.7.3.2. Stem length 57
4.7.3.3. Root length 57
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
Table 4.10. Effect of biological and chemical control on dry weight of
cucumber
55
Table 4.11. Effect of biological and chemical control on stem length of
cucumber
55
Table 4.12. Effect of biological and chemical control on root length of
cucumber
56
XIII
Table 4.13. Effect of biological and chemical control on dry weight of
muskmelon
57
Table 4.14. Effect of biological and chemical control on stem length of
muskmelon
57
Table 4.15. Effect of biological and chemical control on root length of
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
Figure 4.23. The Means of dry weight, stem length and root length in
the three different groups ( chemically treated, biologically treated and
positive control ) of cucumber.
54
Figure 4.24. The Means of dry weight, stem length and root length in
the three different groups ( chemically treated, biologically treated and
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)
Diameter of inhibition zone
(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
Diameter of inhibition zone
(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.
Table 4.5. The average diameter of inhibition zones produced by applying different dilutions of
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.
Table 4.6. The average diameter of inhibition zones produced by applying different dilutions of
Bacillus subtilis on three days old F .oxysporum culture.
Bacterial dillution
Diameter of inhibition zone
(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
Group Mean ± SD P value
Chemically treated (n=16)
35.3± 3.0 0.01
Biologically treated (n=16)
25.0 ± 5.0 0.01
Positive control (n=16)
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
Group Mean ± SD P value
Chemically treated (n=16)
10.1 ± 1.0 0.31
Biologically treated (n=16)
11.3 ± 1.5 0.03
Positive control (n=16)
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
Biologically treated (n=16)
2.6 ± 0.5 0.01
Positive control (n=16)
1.2 ± 0.2
4.7.2.2. Stem length
Table 4.11 illustrates the significance in the mean values of the chemically and
biologically treated groups as compared to the positive control group.
Table 4.11. Effect of biological and chemical control on stem length of cucumber
Group Mean ± SD P value
Chemically treated (n=16)
24.6 ± 8.0 0.01
Biologically treated (n=16)
24.5 ± 6.0 0.01
Positive control (n=16)
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
Group Mean ± SD P value Chemically treated
(n=16) 16 ± 2.0 0.59
Biologically treated (n=16)
17.5 ± 3.0 0.02
Positive control (n=16)
15.0 ± 3.0
root length (cm)stem length (cm)dry w eight (g)
Mea
n
30
20
10
0
CHEMICAL
BIOLOGIC
POSITIVE
Figure 4.23. Compares the means of dry weight, stem length and root length in the three different
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
Table 4.13 illustrates the significance in the mean values of the chemically and
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
Group Mean ± SD P value Chemically treated
(n=16) 5.8 ± 0.6 0.01
Biologically treated (n=16)
2.9 ± 0.4 0.04
Positive control (n=16)
2.4 ± 0.6
4.7.3.2. Stem length
Table 4.14 illustrates that significance in the mean values of the chemically and
biologically treated groups as compared to the positive control group.
Table 4.14. Effect of biological and chemical control on stem length of muskmelon
Group Mean ± SD P value
Chemically treated (n=16)
27.6 ± 3.0 0.01
Biologically treated (n=16)
26 ± 4.5 0.01
Positive control (n=16)
18.4 ± 4.8
4.7.3.3. Root length
Table 4.15 illustrates the significance in the mean values of the chemically and
biological treated groups as compared to the positive control group.
Table 4.15. Effect of biological and chemical control on root length of muskmelon
Group Mean ± SD P value
Chemically treated (n=16)
14.0 ± 2.0 0.02
Biologically treated (n=16)
16.1± 3.6 0.01
Positive control (n=16)
11.6 ± 2.4
58
root length (cm)stem length (cm)dry w eight (g)
Mea
n
40
30
20
10
0
CHEMICAL
BIOLOGIC
POSITIVE
Figure 4.24. Compares the means of dry weight, stem length and root length in the three different
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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