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EFFECT OF SELECTED PHYTO EXTRACTS ANDPLANT OILS ON IN VITRO GROWTH OFFusarium
semitectumISOLATED FROM TOMATO ROOTS
SAFIA AHMED
DEPARTMENT OF AGRICULTURE AND AGRIBUSINESSMANAGEMENT
UNIVERSITY OF KARACHI
2013
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EFFECT OF SELECTED PHYTO EXTRACTS ANDPLANT OILS ON IN VITRO GROWTH OFFusarium
semitectumISOLATED FROM TOMATO ROOTS
By
SAFIA AHMED
A PROJECT SUBMITTED IN THE PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
BACHELOR OF STUDIES (B.S)
DEPARTMENT OF AGRICULTURE AND AGRIBUSINESSMANAGEMENT
UNIVERSITY OF KARACHI
2013
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EFFECT OF SELECTED PHYTO EXTRACTS ANDPLANT OILS ON THE INVITRO GROWTH OF
Fusarium semitectumISOLATED FROM TOMATOROOTS
APPROVED
DR. SALEEM SHAHZAD
CHAIRPERSON
DEPARTMENT OF AGRICULTURE AND AGRIBUSINESSMANAGEMENT
UNIVERSITY OF KARACHI
2013
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DEDICATED
TO
Ammi & Baba
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TABLE OF CONTENTS
ABSTRACT.. 1
INTRODUCTION. 2
LITERATURE REVIEW.. 5
MATERIALS & METHOD.. 8
A. ISOLATION OFFusarium semitectum:B. COLLECTION OF PLANT MATERIALS AND CHEMICALS:C. PREPARATION OF THE PHYTOEXTRACTS:D. PREPARATION OF PHYTOEXTRACT AMMENDED MEDIA:E. PREPARATION OF FUNGICIDE DILUTION:F. ANTIFUNGAL ACTIVITY OF PHYTOEXTRACTS AGAINST FUSARIUM
SEMITECTUM:
G. ANTIFUNGAL ACTIVITY OF OILS AGAINST FUSARIUM SEMITECTUM:
RESULT & DISCUSSION.17
REFERENCE. 32
LIST OF TABLES..38
LIST OF FIGURES 39
ACKNOWLEDGMENT 41
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ABSTRACT
The aim of this work was to find an alternative to chemical fungicides currently used in
the control of plant pathogenic fungi Fusarium semitectum causal agent of root rot. The
pathogenic fungus was isolated from infected roots of tomato and identified on the basis
of morphological and cultural characteristics. The in vitro efficacy of different plant
extracts viz. Neem, Eucalyptus, Moringa, Onion, Garlic at 30,50 & 70 % concentration
and plant oils viz. Clove oil, Pepper mint oil & Black pepper oil tested to control
Fusarium semitectum. Fungicide carbendazim was used to compare the results. Results
showed that mycelial growth of the tested organisms was significantly impaired by the
addition of the extracts in the culture medium. Garlic extract completely inhibited (100%)
the test pathogen at all concentrations, followed by neem which inhibited the pathogen by
74% at 30% concentration, whereas onion also showed significant inhibition at higher
concentration. The moringa extract was found least effective against the pathogen. The
oils showed very low inhibition of 51% by pepper mint oil, 15% by clove oil whereas
black pepper oil showed least inhibition by 3% having negligible effect on the growth of
F.semitectum.These results support the potential use of these plant extracts and oil in the
management of diseases caused by tested plant pathogenic fungus.
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INTRODUCTION
From past many years a number of different chemical and synthetic compounds
have been used against phytopathogens. Indiscriminate use of these chemicals has led to
development of fungicide resistance [1-3] and more importantly, environmental
pollution, posing a potential risk to animal and human health. It does possess the
advantages of speed of control in situations of massive pest outbreak against biological
and cultural control practices which work over a longer span of time. However, there are
serious ecological and environmental problems with over reliance on pesticides.
Persistence of pesticides in the food chain [4] and the development of resistance in pests
towards pesticides [5] are the two serious problems encountered [6]. Other than the
environmental and health risks it is evidenced that resistance in pathogens is developed
against the fungicides has rendered certain fungicides ineffective.
In recent years, a large number of synthetic pesticides have been banned in the
western world because of their undesirable attributes such as high and acute toxicity, long
degradation period, accumulation in food chain and an extension of their power to
destroy both useful organism and harmful pests. [7-9]. Many pesticidal compounds are
directly introduced into agricultural land for combating the soil borne disease and pests.
These chemicals upon reaching the soil influence the microbial balance of soil [10, 11,
12]. Due to the aforementioned considerations, necessitate the search for alternative
control measures to reduce the dependence on the synthetic fungicides.
To control the pathogens, plant extracts have been used as antifungal agents [13].
These extracts can be easily prepared by farmers [14] .The presence of antifungal
compound is an important factor for disease control in higher plants [15]. Thesecompounds are biodegradable and toxic to a considerable value for suppressing some
plant diseases [16]. The pre harvest losses due to fungal diseases in world crop protection
may reach up to 12% or even higher in developing countries [17, 18]. Modern
agrochemical research influence the application of plant derived fungicides and it has
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enormous potential against microbial pathogens attack due to presence of secondary
metabolites in plants [19].
The study of biological activity of some compounds found in plants offers an
opportunity to discover new and effective bioactive compounds for pest control [20-
22].Some of these phytochemical compounds are tannins, flavonoids, lignans and
terpenes, which play an important role in the defense mechanisms of fruits and vegetables
and could be considered as potential promoters of the safety of fresh fruits when applied
exogenously, or promote their activity through controlled abiotic stress
mechanisms[23,24].Consequently, the aim of new antifungal strategies is to develop
drugs that combine sustainability, high efficacy, restricted toxicity, safety for humans,
animals, host plants and ecosystems with low production cost. Since fungicides of
biological origin have been demonstrated to be specifically effective on target organisms
and are also biodegradable, biological control has become popular worldwide [25, 26]
Medicinal plants remain a rich source of novel therapeutic agents. Many plant
species are still unevaluated chemically or biologically. Several studies regarding the
action of plant extracts against some phytopathogenic fungi have been performed. The
quality and quantity of the biologically active compounds from the plant extracts
significantly depend on the species, the plant organ and harvest time [27-29]Medicinal
plants represent a rich source of antimicrobial agents [30] Many of the plant materials
used in traditional medicine are readily available in rural areas at relatively cheaper price
[31] Medicinal plants extracts are promising as alternative or complementary control
means because of their anti-microbial activity, nonphytotoxicity, systemicity as well as
biodegradability.[32] Although hundreds of medicinal plants are used medicinally in
different countries as a source of many potent and powerful drugs and the vast majority
of them have not been adequately explored against plant pathogenic fungi. Plants are the
sources of natural pesticides that make excellent leads for new biopesticide development
[33, 34]. Essential oil bearing plants constitute a rich source of bioactive chemicals,
which have been reported to have various antifungal properties [35, 36].
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Considering the vast potentiality of plant as sources for antimicrobial drugs with
reference to antimicrobial agents, a systematic investigation was undertaken to screen the
antifungal activity of medicinal plant species Neem, Eucalyptus, Onion, Moringa, Garlic ,
and the essential oils namely as clove oil, peppermint oil, black pepper oil against the
fungal strains of Fusari um semitectumBerk &Revnel
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LITERATURE REVIEW
Fusarium species is ubiquitous in soil and have been isolated from various soil
types in tropical and temperate regions from desert soil to artic and alpine soils. However,
the majority of Fusarium species were recovered in cultivated soils especially near the
soil surface. Fusarium species occurred widely in cultivated soils and often associated
with plant roots either as parasites or saprophytes [37].
Many economically important crops are infected by pathogenic Fusarium species
causing various types of diseases such as vascular wilt of banana, root rot and stem rot on
vegetables and ornamentals, and fruit decay, wilting and post-harvest diseases. The
disease can cause economic losses as yield will be reduced if proper control methods are
not taken. Fusarium species occur widely in the soil and exist as colonizers of living
plants or plant debris within the soil or adjacent to the soil surface [38] in the soil,
Fusarium species are able to persist as mycelium, chlamydospore and conidia [37].
Fusarium is one of the most important genus of plant pathogenic fungi [39, 40]. It causes
infection in plants, animals and human beings [41-44].
Among the different Fusarium species,F. semitectumwas found to be responsible
for causing diseases like wilts, blights, root rots, and cankers in coffee, pine trees, wheat,
corn, rice, cereals, carnations and grasses [45]. Knight and colleagues [46] reported the
crown-rot disease of bananas caused by F. semitectum. Recently, Hawa et al. [47] found
F. semitectum associated with red fleshed dragon fruit disease (Hylocereus polyrhizus) in
Malaysia. In Pakistan,Fusarium spp., followed byMacrophomina phaseolina(Tassi) has
been found to produce stalk rot disease of maize [48,49]. Fusarium semitectum Berk &Ravnel occur frequently among the fungal micro flora associated with seedling disease.
They are a major cause of seedling death in some countries [50].
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Harvested tomatoes are susceptible to infections caused by Fusariumspecies due
to its succulent epicarp which enable the fungi hyphae to penetrate deep into the fruit
[51]. As a result, the yield of this economically important farm product is affected, hence
lowering the production rate [52]. Fusariumspecies causes fruit rot or decay on tomato
and other vegetable. The disease causes the vegetable fruits unmarketable as consumer
will only choose that are fresh [53] Fusariumspecies has been recovered from decaying
tomato fruits [54]. Tomatoes contaminated with Fusarium species are lethal to human
and animals health if consumed in feeds as some of them produce mycotoxins [53].
Latiffah et al. reported presence of Fusarium semitectum from silt clay loam soils on
plantation of rubber and paddy crops [55] F. semitectum has been known to produce
mycotoxins such as trichothecenes and zearalenone, beauvericin [56] and moniliformin
[57].
Fusarium semitectum was reported to have been recovered from decaying okra,
bitter gourd, loofah, red chilli and cucumber [53] Fusarium semitectum however, was
reported to have caused diseases on banana fruits [58] melons [59] beans [60], sorghum
[61], walnut [62] and storage rot of mushrooms[63] Agbenin et al reported that fresh
neem leaves extract showed antifungal activity against fusarium oxysporum with
increasing concentrations while neem seed kernels showed 100% mycelial inhibition of
the same pathogen [64 ]. During a survey of Sargodha and Faisalabad districts maximum
frequency (%) of the Fusarium semitectum (36.84%) along with a heavy population of
nematode (T. semipenetrans) was recorded. In some orchards nematode population was
above economic threshold level [65] .
Apart from parasitic nature, F. semitectum is also used as biocontrol agent.
Mikunthan and Manjunatha [66] reported the use of F. semitectum as a potential
mycopathogen against thrips and mites in chilli. In India, chilli (Capsicum annuum L.)
suffered with a characteristic leaf curl symptoms due to the attack of mite, (Polyphagous
tarsonemus latus)and thrips (Scirtothrips dorsalis) or both. In such cases F. semitectum
was found to be active against these mites and thrips. In another study, Manjunatha and
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group [67] studied the biocontrol nature of F. semitectumand found that F. semitectum
showed the significant reduction in the attack of tobacco aphidMyzuspersicae.
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MATERIALS & METHODS
H. ISOLATION OFFusarium semitectum:
The plants showing clear symptoms of disease including yellowing, discoloration ofleaves, droopy appearance and stunted growth with stems showing visible browning and
discoloration were collected from tomato fields growing on the outskirts of Karachi.
The plant samples were stored in cool and dry conditions until further procedure. The
roots were then detached from the plants and washed with tap water to remove soil
particles. The roots were then dipped in 1% Sodium hypochlorite. The roots were
immersed in diluted solution for about 4-5 minutes to get of rid and any contaminant. The
roots were then washed with Sterile Distilled Water (SDW) thoroughly to ensure that no
traces of sodium hypochlorite remained. After the roots were washed they were placed on
blotter paper sheets to dry and then cut into 1 cm long pieces.
The root pieces were placed on potato sucrose agar (potato 200gm; agar 20gm; water 1L)
containing antibiotics Penicillin and Streptomycin were added into the medium at the
rate of 100,000 units/1000ml and 0.2g/1000ml respectively.
The plates were incubated at 29 0C for 5 days the growing mycelium was sub-culturedafter 4 days. A second culturing was done to avoid contamination. A third sub-culture
was again done to ensure a complete pure culture. Stock culture of each Fusarium
semitectum strains were maintained on PDA slants in the refrigerators, and sub-cultured
fortnightly. Whenever an experiment was to be carried out the fungus was raised on
sterile poured PDA plates for 4-5 days.
I. COLLECTION OF PLANT MATERIALS AND CHEMICALS:The selected plants to study their antifungal against Fusarium semitectum included
Neem, Eucalyptus, Moringa, and Garlic & Onion. The leaves of neem& eucalyptus
leaves were collected from the campus of University of Karachi, whereas moringa leaves
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were obtained from the plantation around steel mills. Onion and garlic were bought from
a local vegetable seller. The essential oils that were used in the study included Clove oil;
Peppermint oil and Black pepper oil obtained from a local oil extraction shop. All the
collected leaves were stored in dry conditions while the oils were stored in brown glass
bottles and under cold and dark storage conditions so that the effectiveness of active
ingredients in oils does not degrade. The fungicide Carbendazim was obtained from
Department of Agriculture & Agribusiness Management (DAAM), University of
Karachi.
Table 2: List of plants used during the course of study
COMMON NAME SCIENTIFIC
NAME
FAMILY PLANT PART
USED
Neem Azadirachta indica Meliaceae Leaves
Eucalyptus Eucalyptus obliqua Myrtaceae Leaves
Moringa Moringa oleifera Moringaceae Leaves
Garlic Allium sativum Amaryllidaceae Bulb
Onion Allium cepa Amaryllidaceae Bulb
Clove Syzygium
aromaticum
Myrtaceae Seed
Peppermint Mentha piperita Lamiaceae Seed
Black pepper Piper nigrum Piperaceae Seed
J. PREPARATION OF THE PHYTOEXTRACTS:About 200 gm. Leaves of Neem, Moringa, Eucalyptus and bulbs of Onion and garlic
were washed in running tap water to dirt, surface sterilized in 1% bleach for about 5
minutes and again washed with sterile distilled water. After adding 200 ml sterile
distilled water, the leaves were grinded until a fine suspension was prepared. The
suspension was filtered through muslin cloth 3-4 times until a uniform solution was
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obtained; the solution was then filtered through whatman filter paper to obtain the crude
extract. The stock solution was given a water bath at 40-50 oC for 10 minutes to evade
any contamination.
K. PREPARATION OF PHYTOEXTRACT AMMENDED MEDIA:To prepare phytoextract amended media poisoned food technique was used [70]. The
stock solution of the plant was incorporated into potato sucrose broth to obtain 30, 50 and
70% concentrations. The required amount of sugar and agar was added and media
sterilized at 15 psi for 20 minutes. To inhibit bacterial growth, Penicillin and Penicillin
and Streptomycin were added into the medium at the rate of 100,000 units/1000ml and
0.2g/1000ml respectively. The media poured in sterile Petri plates and labeled with their
respective concentrations.
L. PREPARATION OF FUNGICIDE DILUTION:Two gram of carbendazim was dissolved in the 100ml potato dextrose broth (PDB) to get
10,000 ppm dilution. The fungicide solution was passed through a series of dilutions by
adding 20ml of stock solution in the 80 ml of PDB and so on to achieve the required
concentrations of 10, 100 & 100ppm. The required amount of agar was added into the
PDB and media was sterilized at 121o
F (15) psi for 20 minutes. The pesticide amendedmedia was poured in the 90mm Petri plates under sterile conditions and was left to
solidify.
M.ANTIFUNGAL ACTIVITY OF PHYTOEXTRACTS AGAINST FUSARIUMSEMITECTUM:
A 5mm disc inoculums of Fusarium semitectumcut from a 7 day old culture with the
help of cork borer and placed in the center of each Petri plates in the phytoextracts andfungicide amended media. PDA plates with no phytoextracts or fungicide served as
control. The plates were incubated at 29 oC and the colony diameter of Fusarium
semitectum was recorded daily until the control plates were plates were filled by the
fungal growth. The inhibition percentage was calculated by the following formula:
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I = Cdia- T dia x 100
C dia
Where,
I = percentage inhibition of mycelial growth of the pathogen
Cdia= colony diameter of mycelial growth of pathogen in control set
T dia = colony diameter of mycelial growth of pathogen in treatment set
N. ANTIFUNGAL ACTIVITY OF OILS AGAINST FUSARIUM SEMITECTUM:
Eight wells were punched in 1 cm thick poured PDA plate with the help of sterile corkborer under aseptic conditions. The wells were filled with the either of the oils clove ,
black pepper and pepper mint oil with the help of a dropper carefully so that the wells do
not overflow with oil. 5mm inoculums disc from a 7 day old culture of Fusarium
semitectum was placed in the center of each Petri plates in sets of triplicates with
addition of control sets. The plates were placed in the incubator at 29 oC. The colony
diameter of all the plates measured when the growth of Fusarium semitectumfilled the
control sets. The inhibition percentage was calculated by the following formula:
I = C dia- T diax 100
C dia
Where,
I = percentage inhibition of mycelial growth of the pathogen
Cdia= colony diameter of mycelial growth of pathogen in control set
T dia = colony diameter of mycelial growth of pathogen in treatment set
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RESULTS & DISCUSSION
Antifungal activity of various selected phytoextracts and oils against Fusarium
semitectum was studied and the results were compared with the fungicide carbendazim.
F.semitectum showed complete inhibition by garlic extract (fig.5). The
F.semitectum colony did not grow on the garlic amended plates at all but showed a clear
zone formation on 30% & 70% which may be because of starch degradation by the
pathogen (Table 3). The starch degradation by the pathogen was confirmed by placing the
iodine crystals on the inverted plate, the results showed clear hyaline zone. Extracts
showed significant to moderate mycelia inhibition percentage, Neem extract showed a
significant inhibition of 74% at 30% concentration, however the inhibition ofFusarium
semitectum on neem extract decreased with the increasing concentration (fig.1).
Eucalyptus extract showed moderate effectiveness against F.semitectum at 50%
concentration, 66% inhibition observed (fig.2), Onion extract showed significant
inhibition the efficacy increased with increasing concentration and at 70% concentration ,
73% inhibition growth of F.semitectum observed( fig.4). The least effective inhibitory
effect on F.semitectum was shown by moringa extract which decreased with the
increasing concentration. The lowest inhibition was 47% observed when moringa extract
used at 70% .
Although all the readings were recorded on the 5th day after incubation as
F.semitectum control plates were fully grown (fig.19) the treatment sets were not
discarded and were incubated at same temperature. After 10-12 days it was observed that
the colony diameter on the treatment sets eventually increased which implied that in case
of field trials and practical application of the study the extract application should be done
at different intervals as the botanical extracts may eventually degrade over the period of
time.
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Table 1: Effect of phytoextract on growth ofF.semitectum(mm)
Table 2: Mycelial inhibition (%) ofF.semitectumagainst phytoextracts
Table 3: Starch utilization byF.semitectumagainst garlic extract ammended plates
All the oils showed less inhibition percentage as compared to the phytoextracts
with peppermint oil being the most effective giving 51% inhibition, while clove oil
showed 15% inhibition, the black pepper oil showed the minimum inhibition i.e. 3% as
Fusarium semitectum even colonized the wells. The treatment sets of black pepper oil
Concentration
Replicate 1 2 1 2 3 1 2 3
Neem 26.5 25.5 32.5 22.5 27.4 34.5 36.5 34.5
Eucalyptus 34.5 33.5 30 35 26 31.5 32.5 31.5
Moringa 32 28 45.5 43.3 44.5 41.5 46.5 50.2
Onion 26 27 20 27 23.5 26.5 25 21
garlic 0 0 0 0 0 0 0 0
3
30%
31.5
30.1
23.5
25
0
Diameter Of Colony Growth Of Fusarium semitectum Against Phytoextracts
Treatments
50% 70%
Concentr
ation
Neem
Eucalyptus
Moringa
Onion
garlic
Mycelial Inhibition(%) Of Fusarium semitectum Against Phytoextracts
30% 50% 70%
Treatments
71
100
70
66
51
73
100
66
64
74 61
65
47
100
73
Concentration
Replicate 1 2 3 1 2 3 1 2 3
13.5 15 6 14 13 8
Diameter Of Zone Formation Of Fusarium semitectum Against Garlic Extract
70%
no zone formed
30% 50%
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and peppermint oil showed colonization ofAspergillus flavusonly around oil wells which
indicated that the oils were contaminated withA.flavus.
Table 4: Colony growth ofF.semitectumon oil amended plates (mm)
Table 5: Mycelial inhibition (%) ofF.semitectumby plant oils
For the comparative study the fungicide carbendazim was used which is known to
be quite effective for the control of root rot. During in vitro experiment, the fungicide
completely inhibited the growth F.semitectum in all the concentrations, which showed
that the garlic extract and fungicide were equally effective against the pathogen.
1 2 3
74 77 78
40 45 46
blackpepper oil 84 87 90
Peppermint oil
Clove oil
Diameter Of Colony Growth Of Fusarium semitectum Against
Volatile Oils
Peppermint oil
blackpepper oil
Mean Mycelial Inhibition %
Mycelial Inhibition(%) Of Colony Growth Of
Fusarium semitectum Against Volatile Oils
Clove oil
51
15
3
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Table 9: Diameter ofF.semitectumcolony on carbendazim
Diameter Of Colony Growth Of Fusarium semitectum
Against Carbendazim
10 ppm 100 ppm 1000ppmmycelial
inhibition(%)
1 no growth no growth no growth 100
2 no growth no growth no growth 100
3 no growth no growth no growth 100
From the experimental analysis it is shown that all the phtyoextracts showed
antifungal properties against Fusarium semitectum at various levels, with garlic being
highly effective followed by neem and onion, extract whereas, eucalyptus and moringa
extract exhibited moderate to low inhibitory effect on the growth of Fusarium
semitectum. However the oils showed no notable inhibitory effect on the growth of
F.semitectum.
1. Graphical representation of mycelial inhibition (%) of Fusarium semitectumagainst neem extract
10
20
30
40
50
60
70
80
90
100
30% 50% 70%
(%)
Concentration
neem
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2. Graphical representation of mycelial inhibition (%) of Fusarium semitectumagainst eucalyptusextract
3. Graphical representation of mycelial inhibition (%) of Fusarium semitectumagainst Moringaextract
10
20
30
40
50
60
70
80
90
100
30% 50% 70%
(%)
Concentration
EUCALYPTUS
10
20
30
40
50
60
70
80
90
100
30% 50% 70%
(%)
Concentration
Moringa
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4. Graphical representation of mycelial inhibition (%) of Fusarium semitectumagainst onion extract
5. Graphical representation of mycelial inhibition (%) of Fusarium semitectumagainst garlic extract
10
20
30
40
50
60
70
80
90
100
30% 50% 70%
(%)
Concentration
Onion
10
20
30
40
50
60
70
80
90
100
30% 50% 70%
(%)
Concentration
garlic
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6. Graphical representation of mycelial inhibition (%) of Fusarium semitectumagainst oils
Uzma et al. (2008) tested antifungal activity of asafetida (Ferula asafoetida),
black cumin seed (Nigella sativa), neem (Azadirchta indica) and mustard (Brassica
compestris) oils against Fusarium semitectumand other eight phytopathogens ; their
results indicated that except the black cumin oil, all the oils showed considerable
inhibitory effect on the growth of Fusarium semitectum[72], Chandra and Singh [73]
described that plant extract of Calotropis procera, Eucalyptus globulens, Jatropha
multifida, Azadirchta indica, Allium sativum significantly reduced the wilt incidence in
Cicer arietinum. Mycelial growth of various Fusarium species were inhibited by plant
extracts ofAzadirachta indica, Cinnamomum camphora and Ocimum sanctum [74].
The fungitoxic effects of the phyto-extracts indicate the potential of selected plant
species as a source of natural fungicidal material. These extracts exhibit significant
fungicidal properties that support their traditional use as antiseptics. Antifungal activity
was confirmed by all of the selected plant species and the results revealed neem and
0
5
10
15
20
25
30
35
40
45
50
55
60
Black pepper oil pepper mint oil clove oil
(
%)
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garlic as the most effective inhibitor for the mycelia growth of the tested pathogen. The
finding of the present investigation could be an important step towards the possibilities of
using natural plant products as biopesticides in the control of plant diseases caused by
Fusarium semitectum. Further studies are needed to determine the chemical identity of
the bioactive compounds responsible for the antifungal activity.
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Fig 7: Diseased samples of roots from tomato plant
Fig 8: Fusarium semitectum isolated from infected tomato roots
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Fig 9: Extract of Moringa, Eucalyptus, Neem
Fig 10: Preparation of stock solution of carbendazim and amendment in PDA
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Fig 11: Growth of F.semitectum on neem amended PDA plates 30%, 50%, 70%
Figure 12: Growth of F.semitectum on eucalyptus amended PDA plates 50%, 70%
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Fig 13: Growth of F.semitectum on moringa amended PDA plates 50%
Fig 14: Zone formation on garlic amended media due to starch degradation by F.semitectum
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Fig 15: No growth on the carbendazim amended media 10ppm, 100pp, and 1000ppm
Fig 16: Growth of F.semitectum against clove oil
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Fig 17: Growth of F.semitectum against pepper mint oil
Fig 18: Growth of F.semitectum against black pepper oil
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Fig 19: Growth of F.semitectum on control PDA plates
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LIST OF FIGURES
Fig 1: Graphical representation of mycelial inhibition (%) of Fusarium semitectum against
neem extract
Fig 2: Graphical representation of mycelial inhibition (%) of Fusarium semitectum against
eucalyptus extract
Fig 3: Graphical representation of mycelial inhibition (%) of Fusarium semitectum against
Moringa extract
Fig 4: Graphical representation of mycelial inhibition (%) of Fusarium semitectum against
onion extract
Fig 5: Graphical representation of mycelial inhibition (%) of Fusarium semitectum against
garlic extract
Fig 6: Graphical representation of mycelial inhibition (%) of Fusarium semitectum against
oils
Fig 7: Diseased samples of roots from tomato plant
Fig 8: Fusarium semitectum isolated from infected tomato roots
Fig 9: Extract of Moringa, Eucalyptus, Neem
Fig 10: Preparation of stock solution of carbendazim and amendment in PDA
Fig 11: Growth of F.semitectum on neem amended PDA plates 30%, 50%, 70%
Fig 12: Growth of F.semitectum on eucalyptus amended PDA plates 50%, 70%
Fig 13: Growth of F.semitectum on moringa amended PDA plates 50%
Fig 14: Zone formation on garlic amended media due to starch degradation by F.semitectum
Fig 15: No growth on the carbendazim amended media 10ppm, 100pp, 1000ppm
Fig 16: Growth of F.semitectum against clove oil
Fig 17: Growth of F.semitectum against pepper mint oil
Fig 18: Growth of F.semitectum against black pepper oil
Fig 19: Growth of F.semitectum on control PDA plates
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LIST OF TABLES
Table 1: Diameter of colony growth ofF.semitectumagainst phytoextracts (mm)
Table 2: Mycelial inhibition (%) ofF.semitectumagainst phytoextracts
Table 3: Diameter zone formation ofF.semitectumagainst garlic extract
Table 4: Diameter of colony growth ofF.semitectumagainst oil (mm)
Table 5: Mycelial inhibition (%) ofF.semitectum against oils
Table 6: Diameter of colony growth ofF.semitectumagainst carbendazim (mm)
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ACKNOWLEDGEMENT
I would like to express my special appreciation and thanks to my advisor Professor Dr.
Saleem Shahzad, Chairperson Department of Agriculture and Agribusiness Management for
being a mentor and source of inspiration. His valuable guidance, remarks and supportthroughout the course of study will always be remembered in high regards. Thank you for
your immense cooperation in every step of the project.
I would also like to express my gratitude to all the lab assistants with special thanks to Mr.
Waseem and Ms. Bushra for always being available and helpful
It is my deepest regard to acknowledge my parents and my family for their constant support,
faith and encouragement in every step of my life.
Last but not the least I would like to extend my thanks and appreciation to Malahat Munir for
her willingness to help me in any way possible throughout the research and always.