phytoextracts aginst F.semitectum

5/28/2018 phytoextracts aginst F.semitectum

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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)



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



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



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


eucalyptus extract


Moringa extract


onion extract


garlic extract


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.

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