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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
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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
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Dedication
3?c/eciica£& tAi& to&fYt/ to-
-- SfiatAer^
-- Ols£er&
-- (]6rotAer&
^/(nda/ldear^riead&
3? da£cat& tni& aM>rfi/
sincere//00&.
n
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Acknowledgements
Sfiir&frj o^a/l^a/?vj^atefiil to- yU/aAjpre<zt?/d&s&m<p.
&ince/ie/ tAanA& and ^ratltude^ jjuy to- m///
Intere&&> and/eade/x&Ant^ tArouoduntt? tAi& studw
Q)&e& tAa/iAs/ ar& di& to- &atima> JUU&/HIJI//O^ Aer-
contl/uioti^ <ui^e<sltoyhP.
^Mor& tAanAss ^u>- to- nu// code€Ufue& !ffa&&an/,
fflussien/, yt^mayv and fftavru^Jot^ tAeir- su££ort> and
encmircwemenfr.
Sfcna/u^ rru^y tAanA& ao- to- eiheroy on& a/Ao-
amtrioated l/v tA& tA&sLss.
in
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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
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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
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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
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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
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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
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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.
i \
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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
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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.
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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).
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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
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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
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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
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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
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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.
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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
<•)
Page 22
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
Page 23
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
Page 24
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
Page 25
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
Page 26
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
Page 27
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
Page 28
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
Page 29
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
Page 30
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
Page 31
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
Page 32
+- 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
Page 33
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
Page 34
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
Page 35
Figure 4 (A). Effect of neem callus exctract on S. aureus
Figure 2 (B). Effect of neem leaf exctract on C. albicans
23
Page 36
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
Page 37
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
Page 38
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
Page 39
.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
Page 40
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
Page 41
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
Page 42
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
Page 43
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
Page 44
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
Page 45
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
Page 46
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
Page 47
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
Page 48
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
Page 49
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
Page 50
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
Page 51
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
Page 52
• 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
Page 53
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
Page 54
Reference
Allan P. (1991). Culture of Neem. //;: Plant cell and tissue culture. Stafford A.and Warren G
(eds.) Open University press Milton Keynes, pp. 1-24.
Anderson J. and Ley, S. (1990). Chemistry of insect antifeedants from Azadirachta indica (part
7): preparation of an optically pure hydroxyacetal epoxide related to azadirachtin.
Tetrahedron Letters. 31:3437-3440.
Andrews P., Thyssen J. and Lorke D. (1983). The biology and toxicology of molluscicides,
Bayluscide. Pharmaceutical Therapy. 19:245-295.
Antje W. (1998). Callus cultures of Azadirachta indica and their potential for the production of
azadirachtin. Phytoparasitica. 26(l):47-52.
Augusto J., 'I'omassini B., Xavier D., Ribciro M., Silve G. and Filho M. (2003). Molluscicidal
activity of Physalis angulala L. extracts and fractions on Biomphalaria ienagophila
(d'Orbigny, 1835) under Laboratory Conditions. Mem Inst Oswaldo Cruz, Rio de Janeiro,
98(3):425-428.
lidding 1). (1964). 'Textbook of parasitology, third edition. Appleton - Century - Crafts. New
York./)/;. 1061.
Bhatnar I)., Zeringue II. and McCormick P. (1990). Neem leaf extracts inhibit aflatoxin
biosynthesis in Aspergillus Jlavus and Aspergillus parasiticus. In: Proceedings of a
workshop on neem's potential in pest management programs. Locke, J.C., and Lawson,
R.I 1. (eds.), USDA-ARS, Beltsville,/;/;. 118-127.
Hutterworth .1. and Morgan L. (1968). Isolation of a substance that suppresses feeding in locusts
Chem. Commun. (London): 23-24.
42
Page 55
Bhowinick, B.N. and Vardhan, V. (1981) Antifungal activity of some leaf extracts of medicinal
plants on Curvularia lunula. Indian Phytopalhol. 34:385-386.
Dcvakiiinar C , Saxena S. and Mukerjee K. (1984). New sesamol ethers as pyrethrum synergistsf
Agric. Biol Chem. 49 (3):725-730.
El Kheir Y. and Id Tohami M. (1979). Investigation of molluscicida activity of certain Sudanese
plants used in folk-medicine. American Journal of Tropical Medicine and Hygiene. 82:
237 241. ,
VA Khoby T.. Cialal N. and f'enwick A. (1998). The USAlD/government of Egypt's
schistosomiasis research project (SRP). Parasitology Today. 14: 92- 96.
(•eralil P. and Michael .1. (2002) Experimental design and data analysis for biologists. Cambridge
University press pp 337.
(iliewande M. (1989). Management of foliar diseases of groundnut (Aruchis hypogaea) using
plant extracts. Indian .I. Agric. Sci. 59: 133-134.
(r'oiii inafli S., Alam N., Sriniv asan /\.. Bet/el C. and Singh P. (2000). Structure of the bifunctional
inhibitor of tripsin and a-aim lase from ragi seeds at 2.2 A° resolution. Acta Crystallogr.
56:287-293.
Crant 1. and Schmutterer II. (1987). Effects of aqueous neem seed kernel extracts on ostracod
(Class Crustacea) development and population density in lowland rice. In: Proceedings 3rd
International Neem Conference. II. Schmutterer and-K.R.S. Ascher (eds.), Nairobi, Kenya
Ci'I'Z, Eschborn, Ciermany. pp. 561-591.
Iloben II. and Somasegaran P. (1982). Comparison of the pour, spread, and drop plate methods
for enumeration of Rhizohium spp. in inoculants made from pre-sterilized peat. Appl.
Environ. Microbiol. 44(5): 1246-1247.
43
Page 56
ISTA (1966). International rules for seed testing association. Norway (ISTA). Seed service and
technology, International Seed Testing Association No 4.
Jacobson M. (1990). Review of neem research in the United States. In: Proceedings of a workshop
on neem's potential in pest management programs. Locke .1., and Lawson, R. (eds.),
USDA-ARS, Beltsville, Ml). ARS-86, pp. 4-14.
John C. (2001). The Ultimate Herb Pilgrims, in Neem. Publishing Varanasi Kafhmandu,/)/;>. 177.
Kausik B., Ishita C , Ranajit K. and Uday B. (2002). Biological activities and medicinal
properties^)!'neem (Azmlirachtu iiulica). Current Science. 82 (1 1): 1336-1337.
Krishna A. and Ojha N. (1986). Antifungal evaluation of leaf extracts for the control of some
cucurbitaceous fruit rot diseases. Indian Phytopathol. 39:153.
Larson R. (1990). Commercialization of the neem extract margosan-O in a USDA collaboration.
In: Proceedings of a workshop on neem's potential in pest management programs. Locke, J.
and Lawson, II. (eds.), USDA-ARS, Beltsville, Ml). ARS-86, pp. 23-28.
Lee S., Olsen L, Schweizer P. and Klocke A. (1988). 7-deacetyl-17a-hydroxyazadiradione, anew
limonoid insect growth inhibitor from Azadirachta indica. Phytochemistry. 27: 2773-2775.
Liiulquist K., Adams J.. I kill. P.. and Adams 11. (1990). Laboratory and greenhouse evaluations
of margosan-O against bifenthrin-resistant and-susceptible greenhouse white flies.
Trialeurodcs vaporarionun (llomoptera: Aleyrodidae). In: Proceedings of a workshop on
neem's potential in pest management programs. Locke .1., and Lawson R. (eds.), USDA-
ARS, Beltsville, Ml). ARS-86,/?/;. 91-99.
Locke J. (1995). Effects on viruses and organisms-fungi, In: The neem tree, sources of unique
natural products for integrated pest management, medicine, industry and other purposes. H.
Schmutterer (ed.), VCII. Weinheim. Federal Republic of Germany, pp. 118-127.
44
Page 57
Locke J. (1995). Fungi, in: The neem Tree. Source of unique natural products for integrated pest
management, medicine, industry and other purposes. Schmutterer, II. (ed.), VCH,
Weinheim, Germany,/;/?. 1 18-127.
Locke J. (1990). Activity of neem seed oil against fungal plant pathogens. In: Proceedings of a
workshop on neem's potential in pest management programs. Locke, J., and Lawson, R.
(eds.), USDA-ARS, Beltsville. MD. ARS-86,/>/>. 132-136.
Mathur S. and konsdal O, (2003). Common laboratory seed health testing method for
deleetinfurfgi. L' edn., Published by International seed testing association (ISAT),
Basserdorf. Switerland, />/>A25.
Menu J. (1990). IISDA interest in neem research, hi: Proceedings of a workshop on neem's
potential in pest management programs. Locke, J., and Lawson, R. (eds.), USDA-ARS,
Beltsville, MD.ARS-86./;/;. 1-3.
Morse S., Johnson S., Middle, .1. and Roberts, C. (1986). High-level of tetracycline resistance in
Neisseria gonorrhoeae is a result of acquisition of streptococcal tet M. determinant. Journal
of Antimicrobial Agent and Chemotherapy. 30(5): 664-670.
Murnshige T. and Skoog l; (1962) A revised medium for rapid growth and bioassays with tobacco
tissue cultures. Physiol Plant. 15(3): 473-497.
Muhisiin M., Lisa L., Latima M. and Hind A. (2007). Neem (Azadiraehta Indica A. Juss) callus
induction and its larvaecidal activity against anopheles mosquito. International Journal of
Biotechnology and Biochemistry. 3( 1): 83-92.
Nwachukwu E. and Umechuruba. (2001). Antifungal activities of some leaf extracts on seed-
borne fungi of African yam bean seeds, seed germination and seedling emergence Journal
of Applied Sciences & Environmental Management. 5(1): 29-32.
45
Page 58
Okenio P., Mwatha K., Chhabrab S. and Labryc W. (2001). The killing kinetics of Azadirachta
inc/ica A Juss. (Meliaceae) extract on Staphylococcus aureus, Escerichia coli Pseudomonas
aeruginosa and Candida albicans. African Journal of Science and Technology. 2 (2) : 113-
118. .
Olufolaji B. (1999). Control ol' wet rot disease of Amaranthus sp. caused by Choanehora
cucurbitarum with extracts of Azadirachila indica. .1. Sust. Agric. Knviron. 1(22): 183-190.
Parinielazhagan T. and Francis K. (1999). Antifungal activity of Clerodendrum viscosum against
Curvularia lunala in riccsccds. Journal of Mycology and Plant Pathology. 29 (1): 139-141.
Parvecn (i. and Alain M. (1993). Bioaetivity against plant pathogens, in Neem Research and
Development. Society of Pesticide Science. Randhawa, N.S. and Pannar, B.S. (eds.). New
Delhi, India, pp. 144-153.
Redlcin R.Warlhen J., Uebel L. and Mills J. (1980): The aiitifeedant growth disrupting effects of
a/adirachlin on Spodoptcra Jrugiperda and Oncopellus Fasciatus) Proc. 1st Int. Neem
Conf; Roltaclv hgem,/;/>. 129-136.
SchnuiUcrer 11. (1995). The neem tree, source of unique natural products for integrated pest
management, medicine, industry and other purposes. VCH, Weinheim, Germany.
-Schimittcrcr II. (1988). Potential ofazadirachlin-eonlaining pesticides for integrated pest control
in developing and industrialized countries. 24p.
Sclimuttcrcr II. 1990a. future tasks of neem research in relation to agricultural needs wordwide.
In: Locke, J.C.. and Law son. R.I1. (eds.) Proceedings of a workshop on neem's potential in
pest management programs. USDA-ARS, Beltsville, MD. ARS-86, pp. 15-22.
SchimiUcrcr II. 1990b. Properties and potential of natural pesticides from the neem tree,
Azadirachta indica. Annual Review ot'Lntomology.35: 271-297.
46
Page 59
Schroder P. (1992). NeemA/al / NeemAzal-F in the aquatic environment. Proceedings of the 1st
workshop, Wetzlar, Germany pp.
Schuniutterer ll.and Asche K. (1986). Natural pesticides from the neem tree (Azadrachta indica
A..kiss) and other tropical plants. Proceeding of the third international neem tree conference
. Proceedings of the Third International Neem free Conference (10-15 July 1986), Nairobi.
.Shall N., Khan M .and Azam F. (1992). Seed microflora of cowpea and its control with extract of
Argemone mexicana.Bioved. 3 (2): 176-168.
Shoeb 11. and Hassan A. (1984). The molluscicidal activity of Agavaceoe and Liliaceae extracts
against Lymnaca cuilhnulL .1. Fgypt. Vet. Med. Ass. 44 (3): 33-38.
Sinha P. and Saxena, S.K. (1987) I Tibet of neem leaf powder and extract on the development of
fruit rot caused by Aspergillus nigcr. Neem Newsl. 4:45-47.
Smith M. Pepin M. (1999). Stimulation of bioactive flavonoid production in suspension and
bioreactor-based cell cultures. In:. Plant Biotechnology and In Vitro Biology in the 21st
Century. Allman A, Ziv M, 1/har S., (eds.), Dordrecht: Kluwer Academic Publishers,;?/).
333-336.
Stahl E. (1969). Thin layer chromatography. Springer Berlin, N Y. P. pp. 643.
Verkerk .1. and Wright .1. (1993). Biological activity of neem seed kernel extracts and synthetic
azadirachtin against larvae of I'lulclhi xylostcllu F. Pesticide Science. 37:83-91.
Vidhu S. Narasimhan S., and Nair G. (2006). Bioproduction of azadirachtin-A, nimbin and
salannin in callus and cell suspension cultures of neem (Azac/irachta inclica A. Juss.)
Cueerent Science. 91:22-24.
WHO (1961 ).IVlolluscicidal: Second report expert committee on bilharziasis. Who. Tech.Rep.Ser.
No214.
47
Page 60
od T. (1990). Efficacy of neem extracts and neem derivatives against several agricultural
insect pests. In: Proceedings of a workshop on neem's potential in pest management
programs. Locke, J., and Lawson, R. (eds.). USDA-ARS, Beltsville, MD. ARS-86, pp. 76-
84.
lolt'rey li. and Enright M. (1990). Ampicillin killing curve patterns for ampicillin-susceptible
nonlypeahle haemophilus inlluenzae strains by the agar dilution plate count method.
Antimicrobial Agents and Chemotherapy. 39: 1074-1087.
48