Sudan Academy of Science, SAS

Biosciences, Advanced Technology and Environment Council

Bioactivity of Neem {Azadirachta indica)

Callus Extract

Ibtihaj Mukhtar Ahmed

B.Sc. Omdurman Islamic University

Faculty of Science and Basic Medical Science

Department of microbiology

November 2001

Thesis submitted to the Sudan Academy of Science in partial

fulfillment for the requirements for the degree of Master of

Science in Biotechnology

Supervisor:

Dr. Eisa Ibrahim El Gaali

April 2008

Bioactivity of Neem (Azadirachta indica)

Callus Extract

By

Ibtihaj Mukhtar Ahmed Osman

Examiner's Committee:

Name

Dr. Suhair Ahmed Abdelwahab

Dr. Awad Galal Osman

Dr. Eisa Ibrahim El Gaali

Title

External examiner

Internal examiner

Supervisor

Signature

,^y\J

<^V d P —

Date of Examination: 6/ 4 /2008

Dedication

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Acknowledgements

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amtrioated l/v tA& tA&sLss.

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TABLE OF CONTENTS DED1CTION ii ACKNOWLEDGEMENTS iii TABLE OF CONTENTS v LIST OF TABLES vi

LIST.OF FIGURES vii ABSTRACT viii

ARABIC ABSTRACT x

Page

CHAPTER 1

INTRODUCTION 1

CHAPTER 11 3

2. LITERATURE REVIEW 3

2.1. Origin of the lice 3

2.2. Botanical description 3

2.3. Neem chemistry 4

2.4. Secondary metabolites 4

2.5. Extraction of the secondary melabolites 5

2.6. Medicinal usage 5

2.7. Melluscicidal effect 6

2.8. Effect on phytopalhogens 7

2.'). Neem tissue culture 7

CHAPTER 111 8

3. MATERIALS AND METHODS 8

LI. Plant material 9

L2. Disinfection oflhe explains material 9

-'•->• Callus initiation and production 10

3.3.1 Meelia preparalion 10

3.3.2 Culluring of explains material 10

3.3.3 Extract preparation 10

3.4. Thin layer chromatography (TEC) 10

3.4.1 Preparation of'I'LC plates 12

3.4.2 Fractionation oflhe extracts 12

IV

3.5. Antimicrobial Assay to some human pathogens 13

3.5.1 M icroorganisms 13

3.5.2 Growth media 13

3.5.3 Preparation of counting media 14

3.5.4 Antimicrobial activity test 14

3.6. Antifungal assay to some plant pathogens 14

3.6.1 Plant pathogens 15

3.6.2 Fungal growth media 15

3.6.3 Anli fungal activity test 15

3.7 Control of seed-borne fungi 15

3.7.1 Seed samples 15

3.7.2 Effect of the equeous extract on seed fungi 16

3.8. Assay of melluscicidal activity on Biomphalaria 16

3.8.1 Analysis of data 17

CHAFTER IV 18

4. RESULTS AND DISCUSSION 18

4.1. Initiation of compact callus aggregate cultures 18

4.2. TEC screening of the extract 18

4.3. Antimicrobial activity 22

4.4 Antifungal assay to some plant pathogens 27

4.5 Antifungal activities on seed-born fungi 33

4.6 Effect on Bioinphlurki snai1 36

CONCLUSIONS 39

References 42

\

LIST OF TABLES

Page

Components of the MS medium 11

Retention factor (R|) values of the methanol extracts obtained

from neem callus and leaves on thin layer chromatography TLC

plates 21

Suppressive effect of different concentrations ofneem extracts on

the incidence ol seed-bori/fungi on sorghum seeds 34

Mortality rate ol" hiomphalaria snails as affected by treatment

with neem callus (A) and neem leaf (B) extracts 37

VI

Figure List of Figure Paye

1. TLC separation of the compounds extracted from Azaclirachla indica

callus (C) and leaves (L) on silica-gel G 60 plate using the chloroform:

methanol solvent and sprayed with vanillin: sulfuric acid reagent 20

2A. effect of neem callus extract on Candida albicans 23

2B. effect of neem leaves extract on ('andida albicans 23

3A. effect ol'neem callus on Escherichia coli 24

3 B. effect of neem leaves on Escherichia coli 24

4A. effect of neem callus on Slaphylo coccus arureus 26

4 B. effect of neem leaves on Slaphylo coccus arureus 26

5A. effect of neem callus on the radial growth of Drechslera rostrala 28

5B. effect of neem leaves on the radial growth of Drechslera rostrala 28

6A. effect ol" neem callus on the radial growth of Fusariimi oxysparum 29

6B. effect of neem leaves on the radial growth of Fusarium oxysparum 29

7. effect of neem callus on the radial growth of Allerneria alternate 30

711 effect of neem leaves on the radial growth of Allerneria alternate/ 30

8. Antifungal activity of neem callus extract (20 mg/ml) on the growth of

Drechslcra roslraia alter 72 hours of incubation 32

9. f.fleet of high concentration of neem callus extract on the growth of

sorghum seed-born fungi when germinated on PDA plate for 24 hours.. 35

V II

Abstract

This study was conducted in order to explore the possibility of utilizing plant tiusse

culture techniques lor production of secondary metabolites from callus culture of

Azadrachta iin/ica (neeni) and to investigate the bioaclivity of the established callus

extract in comparison with the extract from the intact leaves.

The presence of secondary metabolites in the extracts was detected by thin layer

chromatography (TIC). Both the callus and leaf extracts eluted five fractions of

compounds and it were observed that callus extract had a good resolution.

Various extract concentrations (5. 10. and 20 mgdnl) were determined for the rate and

extent of inhibition kinetics agamsl Staphylococcus aureus. Iischcrichia colh and

I'liinlic/a albicans. Results showed that callus extract of A. inclica wiped out all viable

cells of ('. albicans within lXhours and the subsequent concentrations 5 and 10 mg/ml

retard the growth after 24h. A higher concentration of 20 mg/ml had the same effect on

,S'. aureus after oh and the /.. coli cells were completely inhibited by the extracts after

24h. Similar kinetics were showed by leal'extract but in slight rate as compared to the

callus extract. In general both extracts posses antimicrobial activity with notable

efficient rales.

for assaying of the inhibitor) effect on .some ph) topathogens the effect of different

concentrations of the callus and leaf extracts on the radial growth of Drcchslcra

roslratii, laisarium oxvsporuui and Allcrncria allcrnala were //; vitro assessed. Obvious

inhibitory effect was observed on the mycelia radial growth of the three treated fungi.

The level of inhibition increased with the increase of the extract concentration. I he

maximum inhibitor) el led (<S-I" o) was recorded with Drcchslcra rastrata when

inoculated in media contain 20 mg'inl of callus while the inhibition rate ol" mycelial

growth of the same species reaches o I" <, when inoculated in a medium contain the same

viii

concentration o f the neem leaf extract. The subsequent concentrations o f the cal lus and

leal'extracts gave similar trends o l ' inh ib i t ion on the fungi cultured on extract amended

agar plates.

As for seed-borne fungi obvious inhib i tor) rales observed upon the treatment o l '

sorghum seeds when compared w i th contro l , the high concentrat ion o f cal lus extracts

(21) mg/ml) completely inhibi t the fungal g twoth . wh i le the inh ib i t ion rate was reached

75% when the leal"extract was used.

The study o f the efficiency against fresh water snails BioDiphlaria j^lahraia wh ich is the

vector ol" Schistosoma mansoni showed mol luscic idal act iv i ty against the snails o f / ? .

^lahrala. Different concentrations (5. 10. 15 and 20mg/ml ) o f the callus and leaf

extracts were used and both o f them showed excellent morta l i ty responses at h igh

concentrations alter 2-1 h. I low ever, there was clear dif ference in the ef f ic iency between

Ihe callus extract and the leaf extract and the callus extracts proved to be more effect ive

than that ol' the leaves. Ihe highest and more rapidly mor ta l i ty rate (100%) was

obtained by adding 20 ml o f the callus extract to the rearing med ium. Decreasing the

amount o f the extracl to 15. 10 and 5 m l . reduced the morta l i ty to 97, 92 and 85%)

respectively. Simi lar concentrations o f ihe leaf extract found to be less ef fect ive.

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XI

CHAPTER ONE

INTRODUCTION

The neem tree has been described as Azadirachta inclicci as early as 1830 by De Jussieu

and its taxonomic position as fol lows:

Order: Rulales

Sab order: Rotinae

fami ly : Meliaceae ( mohogang lam i l \ )

SuhlainiU: Melioideae

Iribe: Meieae

(ienus: Azadinichta

Species: iiulica

The neeni tree (Azcnlirnclihi indica A .hiss), has been tised tradit ional ly for centuries in

both agriculture and medicine (Al lan., I u u l ) . Al though neem is one o f the most ancient

and most widely used herbs on earth, intense scientific investigations o f the properties o f

neein are now being undertaken. I hese studies are quickly ver i fy ing the eff ic iency ol its

traditional uses and e\en more uses for neein could be applicable. This illustrates that i f the

traditional wisdom scicnti l icalK accessed, can guide the efforts o f modern science in

discovering remedies for luiman ailments, f r o m the very beginning o f recorded human

history, people ha\e laken advantage of the remarkable neem tree. Even before ancient

herbalists discovered the analgesic qualities o f the w i l l ow tree- from which aspirin is

derived- people used branches, fruit and leaves from the neem tree to cure man) diseases.

Its medicinal qualities are outl ined daled back to \e ry remote times. Up to date, rural

1

throughout the world refer to the neem tree as a village pharmacy because it cures diseases

and disorders ranging from bad teeth and bed bugs to ulcers and malaria (John, 2001).

Parts of the neem tree are commonly used for medicine, shade, building materials, fuel,

lubrication, and most of all as pesticides. The use of this tree as an insecticide that now

draws great interest from industrialized countries as it is considered as an environmentally

safe alternative to synthetic pesticides (Wood. 1990). To date over 195 species of insects

are affected by neem extracts al concentrations ranging from 0.1 to 1,000 ppm, and many

insects that have become resistant to synthetic pesticides could controllable with these

extracts (Undquisl ci al.. 1990: Menu. 1990). Modern scientists are Uncling even more

uses for this remarkable tree. I he seeds bark and leaves contain compounds with proven

antiseptic, antiviral, antipyretic, anti-inllammalory, anti-ulcer and antifungal use.

~>

CHAPTER TWO

LITERATURE REVIEW

2.1. Origin of the tree

The neem tree is believed to have originated in Assam and Burma of South Asia, but other

reports suggest various areas of Pakistan. Sri Lanka, Thailand, Malaysia, and Indonesia.

The tree also grows well in other tropical and subtropical areas around the world (Verkerk

and Wright., I'W3). Neem trees have successfully been established in Australia. Haiti,

West Africa, the Dominican Republic. Lcuador. I'uerlo Rico, the Virgin Islands, and in the

continental United Stales in I lorida. California. Oklahoma, and Arizona (Jacobson, 1990).

2.2 Botanical description

Aziuliruchla iiu/ica. is a member of the family Meliaceae. It is broad-leaved evergreen tree

which can reach heights of/U) meters with a trunk girth of 2.5 meters and can live for over

two centuries. Its deep root system is well adapted to retrieving water and nutrients from

the soil profile, but this deep root system is very sensitive to water logging. The neem tree

thrives in hot. dry climates where shade temperatures often reach 5°C and annual rainfall

ranges from 400 lo 1.200 mm. The tree can withstand many environmental adversities

including drought and infertile, stony. shallow, or acidic soils. The neem produces

ellipsoidal drupes, which are about two centimeters in length, borne on axillary clusters.

These fruits contain kernels that have high concentrations of secondary metabolites

(Schmulterer. IWOa).

.5

2.3. Neem chemistry

The chemicals that have pesticidal activity can most efficiently be extracted from neem

seed kernels. Neem trees begin their reproductive stage at about three to five years of age

but don't become a fully reproductive until they are ten years old. From this time on, the

tree yield an average of about 20.5 kilograms of fruit per year, with maximum production

reaching 50 kilograms per year (Larson, 1990). Of the fruit yield, only about ten percent is

attributed to seed kernels, and desired biologically active compounds comprise only ten

grams per kilogram of kernel weight. This means that an adult neem tree will only

produce about 20 grams of pesticidal compounds in a season (Schmutterer, 1990 b).

2.4 Secondary metabolites

Many biologically active compounds can be extracted from neem, including triterpenoids,

phenolic compounds, carotenoids, steroids, and ketones. The tetranortriterpenoid

azadirachtin has received the most attention as a pesticide because it is relatively abundant

in neem kernels and has shown biological activity on a wide range of insects. Azadirachtin

is actually a mixture of seven isomeric compounds labeled as azadirachtin-A to

azadirachtin-G with azadirachtin-A being present in the highest quantity and azadirachtin-

E regarded as the most effective insect growth regulator (Verkerk and Wright, 1993).

Many other compounds have been isolated and they showed antecedent activity as well as

growth regulating activity on insects. Polar and non-polar extractions yield about 24

compounds other than azadirachtin that have at least some biological activity (Jacobson,

1988). This cocktail of compounds significantly reduces the chances of tolerance or

resistance developing in any of the affected organisms. However, only four of the

4

compounds in neem have been shown to be highly effective in their activity as pesticides:

a/adirachtin. salannin. melianlriol. and nimbin (Jacobson. 1990).

2.5. Extraction ol the secondary metabolites

The secondary metabolites can be extracted by many methods and leaching with water is

the oldest method. On the other hand, more than one non-polar solvent are used to obtain

more varied mixture of chemicals. 1 lexanc, penlane, ethanol, methanol, esters, and

dichloromethane are used in extractions as well as mixtures of these solvents with water.

Once metabolites extracted, several separation techniques, such as IIPI.C fractionation, IR

spectrum analysis, and 1.1 (' NMK and III NMK spectrum are used for analysis and

identification of the isolated compounds (I ,ee cl ul.. 1988).

2.6. Medicinal usajje

Neem fruits, seeds, oil. leases, roots, and bark has long been used in the traditional

medicine. Thus, oxer thousands of \ ears, millions of Asians haxe used neem medicinally.

In addition, in places where the tree has been introduced in recent times, such as tropical

America and Africa, it has also established a reputation as a useful cure for various

ailments, lodax. the best-established ami most widely recognized uses are based on its

merits as a general antiseptic. Neem has proxed effective against certain fungi that infect

the human bodx such as .l\/>cr^illus Ihivus which causes increasing problems that difficult

to he controlled bx sxnthelic fungicides (Hhaliuir cl <//.. 1990). Many preparations ofneem

extracts are rcporlcdlx efllcacious against a variety of skin diseases, septic sores, and

infected burns. I he leaxes. applied in the form of poultices or decoctions, are also

recommended for boils, ulcers, and eczema. I he oil is used for skin diseases such as

scrofula, indolent ulcers, and ringworm. ( Tires for many more ailments have been claimed

5

but have not been independently confirmed by trials under controlled conditions.

Nonetheless, there are intriguing indications that neem might in future be used much more

widely. These promising applications include antimicrobial, anti-inflammatory,

hypertensive, and anti-ulcer treatments (Locke. 1990). Vector and disease control is

another potential important use of neem. Neem insecticides are potent insect growth

regulators against mosquito larvae: neem oil and other derivatives can be effective personal

repellents against biting adult mosquitoes; and certain neem fractions have anti-malarial

action.

2.7. Melluscieulal ellecl

Snails are the small creatures that attract great attention through their association with snail

borne diseases such as schistosomiasis which associated with Biomphalarea spp.

Eradication of the snails is the hope of eliminating the most parasitic diseases of man,

animals, birds and llshes. I he optimal chemical molluscicide is not yet been developed

and must meet certain restrictions (Belding. 1964). Therefore, the use of natural products

of plant origin emerged as a substitute for snail control. Molluscicides of plant origin are

cheap, safe, and easily available and Held applicable with simple techniques (Shoeb and

Hassan. 1984: Schroder. 1992). Since the products of neem tree (A. inclica) was used as

multipurpose plant in agriculture and for pest control (Grant and Schmutterer, 1987),

extracts from different neem parts could also be tested to assess if they possess a

molluscicide activity. The mode of action of neem extracts is not understood very well. It

is quite possible that the different chemicals or different ratios of chemicals found in neem

tree have varied effects on insects (Anderson and Ley, 1990). The precise effects of the

various neem-tree extracts on a given species are often difficult to pinpoint. Neem's

complexity of ingredients and its mixed modes of action vastly complicate clarification.

Moreover, the studies to date are hard to compare because they have used differing test

insects, dosages, and formulations, further, the materials used in various tests have often

been handled and stored differently, taken from differing parts of the tree, or produced

under different environmental conditions. Although neem's effects on pestiferous insects

are by far the best known, the tree's various products can influence other pest organisms as

well. In the long run. these mav pro\ e important \ a hie (l)e\ akumar el <//.. I 984).

2.S. Ki'iect on phytoputhogens

Subsequent to the isolation of a/adirachlin from neem seed kernels (Butlerworth and

Morgan. I9OK): extensive work has been done on the chemistry and pesticidal properties of

compounds from the neem tree (Schmutlcrer. 1995). Information relating to the antifungal

activities of compounds from neem is limited (Locke. 1995; I'raveen and A lam, 1993).

Neem leaves have been shown to possess antifungal activity cither by direct soil

amendment or as their extracts, active against a number of phytopathogens (Locke, 1995).

Thcv reduced radial growth and spore germination of Curvularia hiiiata (Bhowmick and

Vardhan, 1981). successlullv controlled fruit rots of cucurbitaccous plants caused by

I'lisariiim ci/nisc/i and l-'tisariuni \einilcclimi (Krishna and Ojha., 1986). and significantly

reduced fruit rot of tomatoes caused bv . I spcrgi/lus /hiviis and Aspergillus nigar (Sinha and

Saxena. 1987). Aqueous neem leaf extracts controlled I'ucciiiici circichic/is and

Mycospluicrcllu hcrkclcyi the foliar diseases of groundnut (Ghewande. 1989).

2.9. Neein tissue culture

The term tissue culture generally refers to artificial cultivation of plant tissue (Smith and

Pepin, 1999). for production of commercial amounts of secondary metabolites, for

7

different utilization, constant availability with standardized quality is one of the important

requirements and therefore in vitro cultures production could more be feasible. In the case

of complex chemical compounds such as azadirachtin, the content varies considerably due

to environmental and genetic factors. Therefore, in order to obtain constant amounts of

standardized quality of secondary metabolites, it will be appropriate to employ tissue

culture techniques for its production. Various growth factors might have effect in neem

tissue cultures processes and might also affect the productivity of the secondary

metabolites.it is well known that, establishment of continuous in vitro cultures facilitate

production of secondary metabolites even in quantities that allow economically feasible

production (Antje, llW8).

While neem tree products have some shortcomings as a conventional alternative, they fit in

well as a tool to be used in integrated pest management systems. As more and more

.synthetic chemicals are being pulled from the market, neem is an environmentally benign

alternative. It has significant effect on pests without harming beneficial organisms.

Toxicology studies have indicated it to be quite safe to mammals also (Schmutterer,

1990b). Researchers, however, still have much work ahead of them to characterize the

responses of sensitive insects in the field.

Objectives

1- Testing of biolechnological options as useful efficient method for generation of

effective products (neem callus extract versus leaf extract).

2- Building awareness anil facilitating the use of neem extracts as inexpensive, simple, and

natural useful products in Sudan.

3-Promoting environment friendly technology approach in integrated pest mangement.

8

CHAPTER THREE

MATERIALS AND METHODS

3.1. Plant material

The experimental plant material {Azacliruchni indica A. .kiss) were obtained from

the nursery of National I oresl Department, brought to the green house of the

Commission for Biotechnology and Genetic Fngineering and raised therein. Fresh

young healthy leaves from greenhouse raised neem plants were used as explants

lor callus induction ami as fresh material for extraction. I hcse plants were tised as

source of explains through out this experiment.

3.2. Disinfection of the e\plants material

For disinfection of plant material destined to in-vilro culture, assas s were done varying the

times of exposition and the concentrations of the agent used, l ea l explains were First

prewashcd and cleaned carefull\ under gentle stream of tap water for 15 minutes to remove

all the surface dirt, lollowetl b\ washing for 10 minutes in sterile distilled water containing

15 mg ascorbic acid. 100 mg citric acid and 1 g of activated charcoal. Further, the explants

were washed in three successions of sterile distilled water, and the clean explants were

transferred to a laminar chamber for surface sterilization under optimized condition.

Disinfestations was carried out b\ successive rinsing the explants in 70% ethanol (v/v) for

60 seconds followed b\ rinsing in 10".> ( lorox (5.25% sodium hypochlorite) containing

two drops of I ween .20 ( 0.05% \<\) lor 15 minutes. I mall), explains were rinsed live

times with sterile distilled water. Alter surface sterilization plant materials were placed in

<•)

sterilized Petri dishes and excised with line scissors to small pieces (~ 1.0 cm). Further, the

materials were transferred to basal nutrient medium.

3.3. Callus initiation and production

3.3.1. Media preparation

Callus cultures were initialed and maintained in 0.8% agar solidified MS medium

(Murashigeand Skoog 1962). The components of the medium were shown in Table I. As

described previously by Mulasim el <//.. (2007) callus induction from .1. iiulicci leaf explains

was best achieved on MS medium supplemented with 10 g/l sucrose. 100 mg /I myo-instol

and 0.4 mg l\ lhiamine-11CI in presence of 1.0 mg /I of IBA. The pi I of the medium was

adjusted to 5.8, and then distributed in 25x150 mm culture tubes covered with plastic

Bellco kaplus before auloclaving al I 2 l"( for 15 minutes under pressure of 15 Pis. fhe

media were left to cool in the culture room until use.

3.3.2. Culturiii" of explants material

I Inder strict aseptic conditions in laminar flow cabinet, the leaves were incised into small

pieces using a scalpel and the incised pieces were then partialis dipped into the MS

medium contained into 15 ml culture tube. Cultures were maintained al 25"C ± 2 with a

daily photoperiod of 16 hours light of 1000 lux using fluorescent lamps. Callus cultures

were transferred after 28 da\s of incubation to fresh media.

3.3.3. Extract preparation

Callus cultures were collected after 6 weeks of growth in the incubator. The collected calli

and fresh leaves obtained from the same explants source were then freeze dried separately

for three days till they com cried to tine powdered forms. Both samples. 5 g. of each were

10

Table 1. Components of the MS medium

Component

KNO,

NIljNO.,

Ca('l2.2ll20

MgS04.7ll20

KH2P04

MnSO.,. 41 U )

ZnS04.7ll20

11,130;

Kl

CuS04.5ll20

Na2Mo04.21l20

CoCI2.6IU)

IcS()4.7112(J

Na2i;i)TA.2ll20

Thiamine. 1 K'l

P>rido\inc 11( 1

Nicotinic acid

Myo-inositiol

Glycine

MS (mg/l)

1900

1650

440

370

170

22.3

8.6

6.2

0.83

0.025

0.25

0.025

27.8

37.3

0.1

0.5

0.5

100

2.0

11

then weighed separately and suspended into 250 ml ITIcnmeyer llasks containing 50 ml of

methanol and incubated at room temperature onto rotary shaker for 48 hours. The solvent

was then removed by filtration with Watman filter paper and the nitrate left to evaporate till

dryness. The obtained extracts were then disolved in methanol to form the require

concentrations.

3.4. Thin layer chromatography (TLC)

3.4. 1. Preparation of TLC plates

The thin layer chromatography was carried out according to method of(Slahl, 1969). Glass

IMates (20 x 20 x 0.250) cleaned with acetone so as to remove all grease and fingerprints.

Plates were then placed in plate leveler and the thickness of the coating was adjusted to

0.25 mm. To make the slurry. 30 g of silica gel ((J 60) were dissolved in 60 ml of distilled

water by shaking thoroughly in 250 ml lTlenme_\cr Masks till it make uniform consistency

and free from air bubbles. I he si HIT) was spread using Desga spreader of 0.25 mm thick

layer and drawn smoolhl) and quickl) on the surfaces of the plates. I he coated plates were

first air dried at room temperature for 30 minutes and further placed vertically in an oven

to be activated at I I 0 "(' for one hour.

3.4. 2. Frnctionntion of the extracts

A light line was drawn b\ pencil 1.0 cm parallel to the bottom of the plate and samples

were spoiled along the line using capillar) lubes and allowed to evaporate. The mobile

solvent composed of chloroform and methanol in a ratio of 1:3 (v/v) was poured into the

developing chamber, covered and allowed to equilibrate under fume hood for 30 minutes,

fhe plate loaded with the samples was then carefully placed into the developing chamber.

When the front of the mobile phase approaches the top. the plate was removed out and left

12

lo dry al room temperature. The chromalogram was firsl observed under normal light and

UV (365 mil) before being sprayed with vanillin (3 g) dissolved in a sulfuric acid solution

(KM) ml). Next, the plate was left to dry till the separation appeared. When the compounds

appeared on the plate, the distance between the spots and the end of the mobile phase were

measured, and the retardation factor (l\f) values were calculated and recorded.

3.5. Antimicrobial assay to some human pathogens

3.5.1. Microorganisms

The Gram-positive bactaria Sia/>/i\ lococcus aureus , (ATCC 25923) and Gram - negative

bacteria Escherichia coli (A'I ('(' 25922) in addition to, the diplooid fungus yeast, Candida

albicans (ATCC 7596) were obtained from the national laboratory of the ministry of health

(Khartoum, Sudan). I he stock samples were originally obtained from American Type

Culture Collection (ATCC). Roekville. Maryland, USA.

3.5.2. Growth media

Nutrient agar. MacConke_\ agar and malt extract agar were prepared for culturing of .S'.

aureus, E coli and ( '. albicans respeeli\el\ according to the manufactures instructions.

3.5.3. Preparation of counting media

Agar media were prepared for their corresponding microorganism.Immediately after

auloclaving. the media were al lowed to cool at room temperature to about 45 C. The

freshl\' prepared and cooled media was poured into glass, llal-bottomed Petri dishes (IU cm

in diameter) placed on a le\el. horizontal surface to give a uniform depth of approximately

4 mm. The agar media was allowed lo cool and solidify at room temperature and the

bottom of the plates were divided into <S equal sectors via marker pen and incubated at 35UC

for 18-20 hours before use to ensure sterility.

13

3.5.4. Antimicrobial activity test

The minimum inhibitor) concentration and lethal dilution were determined by the drop

plate techniques as described by( I loben and Somasegaran 1962). In this method, cultures

at the end of the exponential growth phase were serially diluted in sterile distilled water to

give approximate!) a range of I (/' cell/ml. Pxlracts were then added to each of the diluted

cultures at final concentrations of 5. 10. and 20 mg/ml. Initial controls were set up by

plating of untreated dilutions for eaeh microorganism. Lethal rate was measured at once

and then at one hour incubation interval for the first 12 hours, then at 6 hours interval for

the next 12 hours. 'I he numbers of \ iable cells were counted using the drop plate method

in which one drop ( 3 pi) from the different treatment was delivered to each sector with

calibrated sterilized Pasteur pipette. Plates were incubated in an inverted situation at 37"C

for at least two davs. I he average number of viable organisms per ml of the stock

suspension was determined by means of the surface viable counting serial technique.

3.6. Antifungal assay to some plant pathogens

3.6.1. Plant pathogens

Three plant fungal pathogens were used in the stud). l:usariitni oxyspurum (wilt disease in

tomato), Divchslcra rosinim (causative laclor wilt disease in tomato) and Altcrncria

ulhTiialci (earl) blight in tomato). All species were obtained from the central planlalholog)

laboratory of plant protection directrate (Ministry of Agriculture, Khartoum. Sudan).

14

3.6.2. Medium for fungal grow(h

Potato dextrose agar (PDA) medium was used for growth and maintenance of the three

fungal species. The medium composed of 0.4% potato starch, 2.0% dextrose, 1.5% agar

and the pi I was adjusted to 5.S. I en ml of the medium was then taken into 15ml

autclavable screw caped Falcon tubes and autclaved at 12I"C for 15 minutes under pressure

of 15 Pis.

3.6.3. Anti fungal activity test

Methanolic extracts of callus and leaf, were suspended into falcon tubes contained the

prepared PDA to form the appropriate required concentrations (5.10 and 20 mg/mlj and

then plated into Petri-dishes.Plates without extracts were used as controls. Suspensions of

the different fungal species (5 ul) were sepcratl) inoculated onto the center of each plate

and the plates were incubated at ?i5"C for 72 houre. The visible growth at the different

concentrations of the two extracts was daily measured as the increment in diameter of the

formed colony and compared to the control. The experiment was prepared in three replica

and mean were calculated.

3.7. Control of seed-born fungi

3.7.1. Seed samples

As sorghum (.sorghum hico/or) is one of the most commonly cultivated and consumed

crop in Sudan, it was selected for earning out the test of controling seed-borne fungi.

Infested slock of sorghum seeds were obtained from the seed pathologv laboratory

(Department of Uolanv. f'acullv of Science. University of Khartoum). Selection of the

infested seed lot was based on the insedence of fungal growth as revealed by standard

n]ollortesl(lSTAl%6).

15

3.7.2. Effect of the cqucous extract on seed fungi

Solutions of neem callus and leaf extracts were freshly prepared at concentration of 20

ing/ml (v/v) and distributed inlo 15 ml screw-caped falcon tubes. Sorghum seeds were

soaked inlo the prepared extracts for 15 minutes and then transferred to sterile Petri-dishes

plated with filler papers to be dried for 30 min at room temperature. The effect of neem

callus and leaf extracts on the seed borne fungi was carried out as described by Mathur and

Konsdal (2003). Sets of three Pelri-dishes containing PDA medium were prepared and

each Pelri-dish was marked to lour equal sectors at the bottom side of the plate, fifteen

seeds from each treatment were carefully placed onto the sectored plate, at a rate five

seeds/sector, to form 3 replicates. A set of live untreated seeds were placed onto the forth

sector to be used as the control and plates were then incubated for overnight at room

lemperaUilre. The fungus incidence was recorded as posalive or negative of seed bearing

fungus growth per replica.

3.8. Assay of mclliiscicidal activity on Biomphalariu

The Biomplialaria snails were collected from irrigation canals at l.l-laki I lashim area

North Khartoum. Selection of the sampling site was based on preliminary observation of

relative abundance of the snails and ensures that there was no treatment with pesticides in

the area. Snails were captured using long metallic scoop with 4 mm mesh. Plastic buckets

filled with lap water were used to keep out the collected snails at room temperature

(3()°C±2) and the snails fed with fresh lettuce (Luciucu .saliva). Three sets of five Petri-

dishes were laden with moisi llller papers and placed on the laboratory bench. Using a

metal forceps, snails were transferred from the plastic bucket into each Petri-dish. Two

separate sets of Pelri-dishes were prepared and labeled a. b. c. d and e. from the originalh

In

prepared callus and leaf extracts of neem (100 mg/l). different volumes were add to the sets

of prepared Petri-dishes. To the first set. 5 ml of the callus extract were added into dish (a),

10 ml'in (b), 15 nil in (c) and 20 ml in (d). In Petri-dish (e), 20 ml of distilled water was

added as control. Different concentrations of the leaf extracts were added to the second set

of Petri-dishes in the same manner. The effects of the four extract concentrations were

monitored by counting the number of dead snails at one-hour intervals for the first 12

hours, then at 6 hours interval for the next 12 hours. Identification of dead snails was made

based on the snails' immobility and the odor of the soft parts. The experiment was designed

as complete randonii/ed block design with three replicates.

3.8. Analysis of data

The data were subjected to statistical analysis of variance. The one-way analysis of

variance (ANOVA) was used to find out the variation resulting from experimental

treatments. Treatments were compared with standard error (Gerald and Michael 2002).

17

CHAPTER FOUR

RESULTS AND DISCUSSION

4.1. Initiation of compact callus aggregate cultures

During the first weeks after transferring the leaf explants to MS medium callus aggregates

started to be formed in the culture media and gradual increase in size was observed during

the three weeks later to form a compact callus. The established callus culture was

composed of green or light green, spherical, smooth surfaced callus aggregates. The

medium was totally clear. No dispersed cells or cell debris was observed in the growth

containers. The calhis grew slowly, doubling the fresh weight within 8 days and continuing

to increase in size. Cavities were formed in the centre of four to five weeks large

aggregates, therefore callus with diameters above 3 cm were incised into small pieces along

the naturally occurring ruptures and transferred to growth containers where new cultures

could be obtained. The growth of the callus cultures was relatively slow. The maximum

biomass obtained was 5.8 g (fresh weight) that meant a nearly six-fold increase.

4.2. TLC screening of the extracts

On screening of the leaves and callus methanolic extracts with thin layer chromatography

(TLC) clear spots were visualized on the developed plate under UV illumination. The two

types of extracts showed similar pattern of spots. Upon spraying the plates with

vinallin/lLSO.i there was development of five major spots with different colors and Re­

values. The colors of the spots range from gray and dark blue to orange and brown. The

range of colors was indicalive of presence of different compounds with different polarities

(Figure 1). Both, callus extract and the leaves extract, have more or less the same TLC

18

pattern and Rf values (Table 2) and there was no major difference based on the separation

of the fractions on TLC plates and number of the compounds eluted. On the other hand

there was little variation in the degree of faintness and deepness of the developed colors.

The callus extract displayed faint spots rather than the leaves extract. From the TLC

screening it is clear that distribution of the secondary metabolites was the same in the two

types of extracts. If we concider the degree of color as a measure for the concentration of

the secondary metabolites we can assume that the higher concentrations of the compounds

were exhibited by the callus extract. Elution with TLC indicates the presence of secondary

metabolites in the callus extract

19

+- 3 -*

<*- 5 •+

^^m L C

Figure 1. TLC separation of the compounds extracted from A indica callus (C) and leaves

(L) on silica-gel G 60 plate using the chlorform: methanol solvent and sprayed vanillin:

sulfuric acid reagent.

20

Table 2. Retention factor (Rf) values of the methanol extracts obtained from

neem callus and leaves on thin layer chromatography TLC plates

Neem extract

Callus

x Leaves

Rf values

1

0.127

0.127

2

0.3

0.3

3

0.55

0.55

4

0.73

0.73

5

0.86

0.86

21

4.3. Antimicrobial activity

, The means of viable cells counting obtained at each concentration of the two types of

i

i extracts (callus and leaf) at different intervals are presented graphically in Figures 2 - 4 . As

! presented in Figure 2 (A & B) C. albican was consistently eliminated by the three

, concentrations of both callus and leaf extracts and the population gradually reduced as the

time of incubation protracted and there was no growth observed after 24h of incubation.

The higher concentration of 20 mg/ml of the callus extract completely wiped out all viable

C. albicans within 18h however much more time was observed with the same concentration

of the leaf extract. Subsequent concentrations of the extracts (10 mg/ml and 5 mg/ml)

showed similar trends of effect completely killed all viable cells of C. albicans in 24h.

In case of E. coli, although the counts decreased from the initial inoculums it did not show

prompt respond even to high concentrations (20 mg/ml) and similar trends were observed

with all concentrations of the callus extracts (Figure 3 A). The different concentrations of

the callus extracts slightly reduced the population of the initial inoculums at the first 6 h,

yet drastically reduction in cell count appear after 12h and the decrease in cell count

continued complete elimination observed after 24h.

Similar results were obtained when leaves extract was used (Figure 3B). All concentrations

initially reduce the population slightly in the first 6h and substantial reduction in viable

cells was observed at the concentration 20 mg/ml especially after 12h of incubation. In

general treatment with both callus and leaf extract inhibit the growth of the cell population

as compared with the controls.

22

Figure 4 (A). Effect of neem callus exctract on S. aureus

Figure 2 (B). Effect of neem leaf exctract on C. albicans

23

2.5

.1 t

i..1

z < t 1 2 3 4 5 6 12

Time(h)

i s

—•—Oinginl

—•— 5 ma ml

—*—10 ing ml

—•—10 ing ml

24

Figure 4 (A). Effect of neem callus extract on E. coli.

2.5

1 S 2

-•1 •A

S 1.5 at 9

* 1 u 1 1 | 0.5

z

l^-t=4-

0 1 2

""fcr-~»_ • i —1—1 1

3 4 5

Time (h)

S 12 18

• 0 me ml

—•— 5 me ml

* 10 ins ml

—•— 20 mc ml

24

Figure (B). Effect of neem leaf extract on E. coli.

24

S. aureus showed very high response to the extracts at all concentrations (Figure 4). The

response of the organism was very drastic and seems that the population was noticeably

reduced immediately after the first 3h especially with the concentrations 20 and 10 mg/ml

of the callus to reach complete elimination with in 4h. However complete elimination was

.observed after 8h at concentrations 20 mg/ml and 10 mg/ml of leaf extract.

The mechanisms by which microorganisms generally survive the action of antimicrobial

agents are poorly understood and remain debatable (Woolfrey et al., 1990). This is true for

the case of E. coli also, because the resistance attributed to Psendomonas could be capsule

related, but that of E. coli could probably be due to genetic factors or due to cell membrane

permeability. A higher dosage of A. indica has the initial effect to decline in growth in the

first 6 h. This is followed by a paradoxical growth effect and a final continued slow

population decrease. S. aureus and C. albicans which are gram positive are wiped out in

less than 24h, unlike the gram negative rods, E. coli is interesting which are not completely

remove. Perhaps the mode of action of A. indica extracts is indeed strongly cell wall

related.

Other studies have shown that some antibiotics affect organisms in different ways than the

cell wall (Morse et al., 1986). Our results agree with proposal of Okemo et. al., (2001) that

neem compounds could be safely used as chemotherapeutic agents if used within

predetermined concentrations. If the minimum inhibitory concentration had been

determined by the disk diffusion methods it would be difficult to assess actual time for

antimicrobial activity for the specific concentration and therefore the drop plate method is

most suitable for the study of the kinetics of plant extracts.

25

2.5

1 , 1

e 2 1-5 — 1

z 0

1

•

^5=^ » 1 2 3 4 5 I

Time(h) S 12 IS

—*—Omgral

—•— 5 mg ml

• lOmaml

—•— 20ma ml

24

Figure 4 (A). Effect of neem callus exctract on S. aureus.

2.5

1. V*

i : z

1 1 2 3 4 5 6 "

Time(h)

—•*—O ins ml

—•— 5 mg ml

—A— lOma ml

—•— 20 ma ml

ft HB ft S 12 24

Figure 3 (B). Effect of neem leafe extract on S. aureus.

26

.4. Antifungal assay to some plant pathogens

figures 1 and 2 illustrated the inhibiting effects of neem callus and leaf extracts on the

adial growth of D. rostrata, F. oxysporum and A. alternata when grown in PDA medium

hat contain neem callus and leaf extracts. Obvious inhibitory effect was observed on the

hycelia radial growth of the three treated fungi as compared to the controls (Figure 8). The

ohibition rates on the radial growth increased with increase of the extract concentrations.

*feem callus extract showed maximum inhibitory effect (84%) with D. rostrata (Figure

\A). High concentrations (20 mg/ml) of the neem callus extracts also showed 63.6% and

>3.3% inhibition rates with A. alternata and F. oxysporum respectively (Figure 6A and

?igure 7A). Similar trends in the rate of inhibition were also observed with the subsequent

joncentrations of the callus extracts on treatment of the three fungi used in the test.

Although the leaf extracts at different concentrations were less in the efficiency than callus

extract they also showed good inhibition rates. The radial growth of D. rostrata was

ohibited by 61% after three days of incubations in the medium contain 20 mg/ ml of the

eaves extract (Figure 5B). Less response was observed with A. alternata and F. oxysporum

Pigure 5B and Figure7B).

27

i i

On mo nil

• ?insjmml

O 10 me ml

O 20 nit! ml

Time (days)

Figure 5 (A). Inhibitory effect of neem callus extract on the growth of D. rostrata.

£ - -

6 •

" 4

I 3 w 2 •

1 -

• 0 me ml

• 5 m; ml

• in nit; ml

D 20 me ml

Time (davs)

Figure 5 (B). Inhibitory effect of neem leaf extract on the growth ofD. rostrata.

28

BOing ml

• 5 ing ml

Old lug ml

OlOmgml

Time (davs)

Figure 6 (A). Inhibitory effect of neem callus extract on the growth of F. oxysporum.

• 0 mg ml

• 5 mg ml

OlOmgml

O20mgml

Time (days)

Figure 6 (B). Inhibitory effect of neem leaf extract on the growth of F. oxysporum.

29

H 6

9 <

fc 3

2 •

1 •

Time(<lavs)

• 0 . 0 me 1

• 5 in g ml

D10 ins ml

Q20ii igml

Fiigure 7 (A). Inhibitory effect of neem callus extract on the growth of A. alternata.

9

S s

I

8 r

n

5

4

3

2

1

Ld

m, DO 0 ins ml

• 5mg ml

• 10 ms ml

O 20 mg ml

1 2

Time (<lavs)

Figure 7 (B). Inhibitory effect of neem leaf extract on the growth of A. alternata.

30

ri general these results showed that the two types of neem extracts (callus and leaves)

oppressed the radial growth of the fungal colony comparing to controls (Figure 5 -7).

pfeny investigations, performed to confirm the fungicidal effects, have revealed that neem

Xtracts have antifungal properties. Our results agree with those obtained by Olufolaji

1999) on wet rot disease of Amaranthus sp. and Choanephora cucurbitarum using neem

oot bark and fruit extracts. Similarly, Nwachukwu and Umechurubal (2001) observed

nhibition of mycelial development of Colletotrichnm gleosporiodes by extracts of neem

eaves and fruit. Gourinath et. al., (2000) stated that the inhibitory activities of plant

(Xtracts vary with the plant part used and the nature of the fungus. This explains the

lifference in the efficiency to the fungus exhibited by the neem callus and fresh leaf

extracts.

4.5. Antifungal activities on seed-borne fungi

Hie effects of the various concentrations of the two extracts on the incidence of seed-borne

Sfiingi as mycelial growth on sorghum seeds are presented in Table 6. Both the callus and

leaf extracts significantly inhibited mycelial emergence and growth of the fungi when

Icompared with the untreated control seeds (Fgure 9). The level of inhibition of mycelial

growth of the fungi varied with the type of extracts and extracts concentration. It is

obvious that callus extract more effective than the leaves extract and the degree of

inhibition increases with the increase in the concentration. Fungal growth was observed at

low concentration (5 mg/ml), while the concentration of (20 mg/ml) was more effective in

elimination of fungal growth.

31

B^^. x.

|T 1

•?>-•

c

Figure 8. Antifungal activity of neem callus extract (20 mg/ml) on the growth of D. rostrata after 3 days of incubation.

(T: medium contain callus extract, C: control)

32

dthough the neem tree has been known to be useful in soil enrichment and for insect, pest

id to some extent disease control, it's potential for the control of soil-borne diseases, our

tults show clearly the potential of neem products for control of plant diseases caused by

echslera rostrata, Alterneria alternata and Fiisarium oxysporum. Leaf spot in sorghum,

mato wilt and early blight caused by these fungi are of the most serious plant diseases.

bey often appear severely and cause considerable yield loss. Most plant varieties in use

»day are moderately or extremely susceptible to fungal diseases, thus chemical controls are

Kessary tolceep high yields. However, the chemical control of the disease has several i

awbacks and is environmentally hazardous. The use of natural products for the control of

tngal diseases in plants is considered interesting alternative to synthetic fungicides due to

eir lower negative impacts on the environment. The antifungal properties reported in this

udy are of the hopes to find such a natural alternative. In recent years much attention has

sen given to nonchemical systems for seed treatment to protect them against seed-borne

ithogens. Plant extracts have played significant role in the inhibition of seed-borne

ithogens and in the improvement of seed quality and field emergence of plant seeds.

imilar results were obtained by Shah et al, (1992), when they found that the seed extract

{Argemone mexiccma was effective in eliminating most of the seed-born fungi of cowpea.

arimelazhagan and Francis, (1999) were also reported that leaf extracts of Delonix regia,

ongamia glabra and Acacia nilotica significantly inhibited spore germination, mycelial

rowth and spore production o[' Alterneria helianthi and Fiisarium solani from sunflower

jeds. In this study there is good evidence that callus and leaf extracts of neem

Azadirachta inclica), have a promising results for seed borne fungi control.

33

Table 3. Suppressive effect of different concentrations of neem extracts on

the incidence of seed-borne fungi of sorghum seeds.

Type of Extract

Callus

Leaves

control

4-+++

++++

Treatments

5

+++

+++

10

+

++

20

-

+

+ + + + = 100% growth of fungus

+ t- + = 75% growth of fungus

+ + = 50% growth of fungus

+ =25% growth of fungus

- No growth of fungus

34

Figure 9. Effect of high concentration of neem callus extract

on the growth of sorghum seed-borne fungi germinated on

PDA plate for 24.

(C is controls and T is treated seeds).

35

It is therefore necessary to search for control measures that are cheap, ecologically sound

and environmentally safe to eliminate or reduce the incidence of these economic important

pathogens, so as to increase seed germination and yield of sorghum.

4.6. Effect on Biomphlaria snails

The objective of this study was to determine the lethal dose and identify the effects of the

different doses of callus and leaf extracts of neem A. indica on the physiology of

Biomphalaria glabrata subjected to treatment for 24 h. The effects of the calls extracts and

leaves extracts on Biomphalaria are presented in Tables 5 A and B respectively. When 20

ml of the callus extract was applied, all the snails were died within 24 h. Decreasing the

amount of the extract to 15, 10 and 5 ml, reduced the mortality to 97, 92 and 85%,

respectively (Table 5 A). For the leaf extract (Table 5B), 58% mortality was recorded when

the snail were treated with 20 ml extract, while 53, 43 and 37% mortality was recorded

with 15, 10 and 5 ml extracts, respectively, after 24h. Significant elevation in the rate of

mortality observed with the elongation of the period of exposure and the increase of the

extract concentration. The result also showed clear difference in the efficiency between the

callus extract and the leaves extract, and the extracts obtained from the callus proved to be

more effective than the leaf one. This result can be attributed to the biocide effects of neem

leave natural products (Vidhu et al., 2006).

Similar results were obtained by Augusto et. al..(2003) when they evaluate the molluscicide

activity of Physalis angulata on B. tenagophila under laboratory conditions. In Sudan El

Kheir and El Tohami (1979), found that root extract of Jatropha citrcas exhibit efficient

molluscicidal activity against B. truncatus.

36

Table 4. Mortality rate Biomphaiaria snails as effected by treatment with neem Callus (A) and neem leaf (B) extracts.

A

Cone, in ml

0

5

10

15

20

Time in 1 0

1

2

3

2

2 0

2

2

3

2

3 0

3

.2

3

4

4 0

4

4

3

4

5 0

5

3

4

3

6 0

4

2

3

4

7 0

5

3

3

3

8 0

5

4

4

4

9 0

3

5

4

4

lours 10 0

3

5

3

3

11 0

2

3

3

3

12 0

4

4

5

5

18 0

4

6

8

9

24 0

5

10

10

10

Total

0

50

55

59

60

%

0

83

92

97

100

Mean

0.0

3.6

3.9

4.2

4.3

SE(±]

0.0

0.24

.56

0.57

0.64

B Cone.

in ml

0

5

10

15 20

1 0

| 0_ 0 1 2

2 0

1

1 2 2

3 0

0

1 2

3

4 0

2 2

3 2

5 0

2

3 2

3

6 0

1

2 3 2

7 0

3

3 3

3

ime in hours 8 0

2

3 2

3

9 0

1

2 1 2

10 0

1

2 2 2

11 0

2

1

3 2

12 0

1

2 3 3

18 0

3

2

3 3

24 0

3

2 3

3

Total

0

22

26 32

35

1 %

0

37

43

53 58

| Mean

0.0

1.6

1.9 2.3

2.5

| SE[±)

0.0

0.30

0.21 0.30 0.20

37

Molluscicidal activities were also observed with hexane extracts of the leaves, fruit, stem,

root, and seeds of Pithecelobium maltiflorum when tested on B. truncatus.

Schistosomiasis is the second most important parasitic disease after malaria in terms of

overall morbidity burden. For the control of the disease,

Multifaceted approaches are desirable (El Khoby et al. 1998), including control of the

intermediate host snails. At present niclosamide (Bayluscide, Bayer, and Leverkusen,

Germany) is the only molluscicide applied on a large scale (WHO 1993). Since this

synthetic mdlluscicide is also toxic to fish (Andrews et al. 1983) and frequently not

affordable in poor, schistosomiasis-endemic areas, alternative possibilities for snail control

is needed to be evaluated. Plants with molluscicidal activity may be exploited to contribute

to schistosomiasis control, particularly if they are already grown locally for other purposes.

A. indica is widespread in Sudan and it can easily be grown on almost most types of soil

and therefore prevents erosion. Most parts of the plant and their extracts are used in

traditional medicine, thus, exploitation of its molluscicidal properties appears to be

possible. Here we report the possibility of controlling Biomphalaria glabrata (intermediate

host of S. mansoni which causes intestinal schistosomiasis). However further investigations

on the tested extracts to explore if there is possible toxic activity man and his animal

products consider to be one of the broad-spectrum pesticide (Schmutterer, 1988). It is well

known that neem natural products contain many active ingredients and the Azadirchtin is

the most famous one (Kausik et al., 2002). Thus, the increase knowledge on the ecological

aspects of the interactions between A. indica and Biomphalaria spp. is essential for the

adoption of efficient measures for the control of Biomphalaria populations in endemic

areas of schistosomiasis.

38

CONCLUSIONS

• This study emphasizes the importance of tissue culture and the use of plant cells for

the production of biochemicals, an issue that represents a new area of

biotechnological exploration. The techniques could be envisioned for industrial

processes if strong emphasis exerted on continuous culture production from single

cell systems. The study examines the availability of some products that similar to

those synthesized by higher plants.

• Whenever a chemical is used to control the insect or microbial population, not only

is the environment polluted but also other desirable fauna is affected by the

introduction of the toxicant in the ecosystem. Simultaneously, the target species is

provoked to develop resistance against a wide range of pesticides as well. The high

cost of chemical pesticides and the environmental hazards as a result of pesticide

usage should encouraged scientists to seek safe and low cost pesticide groups. For

this reasons, antimicrobial agents from natural sources such as the neem tree (A.

indica A. Juss) will be of great interest.

• In this study, methanolic extract from callus and leaf of A.indica showed high

antimicrobial activity against .S'. aureus and E. coli were C. albicans. Although the

exact active components of the extract that showed this effect were not identified,

but antimicrobial activity indicated the presence of at least some of these

compounds. Therefore, modern drugs can be developed after extensive investigation

of its bioactivity, mechanism of action, pharmacotherapeutics, and toxicity and after

proper standardization and clinical trials.

39

• In Sudan very little work has been done on the biological activity of compounds

extracted from natural origin to be used in best control specially phytopathogens,

hence extensive investigation is needed to exploit the antifungal utility to combat

plant diseases. As the global scenario is now changing towards the use of nontoxic

plant products having traditional medicinal use, we tried to develop modern

treatments for the control of various plant fungal diseases and seed borne- fungi.

Applications of antifungal compounds extracted from callus A. indica should

emphasized modern pesticide development programs should isolated from natural

sources.

• In fact, time has come to make good use of centuries-old knowledge on neem

through modern approaches of drug development. Quite significant amount of

research has already been carried out during the past few decades in exploring the

chemistry of different parts of A. indica (neem). Several therapeutically and

industrially useful preparations and compounds have also been marketed, which

generates enough encouragement among the scientists in exploring more

information about this medicinal plant.

• Since Biomphalariu spp. causes a serious threat to human health by transmitting

the infectious disease bilharzias, it is imperative that control must be improved

through the application extracts from a versatile medicinal plant such as the neem.

This study evaluated the physiological effects caused by molluscicides of the neem

extracts in Biumphalaria spp. the main intermediate host of S. mansoni. It was

found that the plant could be considered as one of the most promising

40

molluscicides. Results can utilize to motivate further investigations to find

alternative control of B. glabrata that might minimize the environmental hazards of

chemicals on the ecosystem.

•The study explains that in-vitro production of secondary metabolites could be

possible through plant cell culture. Strategies for improving secondary products in

suspension cultures could be accomplished for establishment of successful cell

culture lines capable of producing high yields of secondary compounds in cell

suspension. Large scale accumulation of secondary products in plant cell cultures

depends on the composition of the culture medium, and on environmental

conditions. Bioreactors are the best solution to offer the optimal conditions for

large-scale plant production for commercial manufacture.

Vector control may be accomplished by manipulation of application of environment

safe products to reduce human contact with infective vectors. The work also

focuses in the control of human schistosomiasis and other harmful diseases through

modern biotechnology in match with environment to developed an alternative

discipline based primarily in locally available and of less adverse environmental

impacts. Strategies such as the suppression of vector populations through the

provision of safe water supplies, proper sanitation, waste management facilities and

sewage manipulation that treated with a natural pesticides should be discussed with

the appropriate examples which abundantly available. An extensive research and

development work should be undertaken on neem and its products for their better

economic and therapeutic utilization.

41

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