MICROORGANISMS ASSOCIATED WITH FRESH

AND DRY PLANTAIN LEAVES

BY:

Henunumwen Mary LAWRENCE (MISS)

LSC0609869

DEPARTMENT OF SCIENCE LABORATORY

TECHNOLOGY (MICROBIOLOGY OPTION),
FACULTY OF LIFE SCIENCES,
UNIVERSITY OF BENIN,
BENIN CITY, NIGERIA.

JANUARY, 2013

1
MICROORGANISMS ASSOCIATED WITH FRESH
AND DRY PLANTAIN LEAVES

BY:

Henunumwen Mary LAWRENCE (MISS)

LSC0609869

A PROJECT REPORT SUBMITTED TO THE

DEPARTMENT OF SCIENCE LABORATORY
TECHNOLOGY (MICROBIOLOGY OPTION),
FACULTY OF LIFE SCIENCES, UNIVERSITY OF
BENIN, BENIN CITY
IN PARTIAL FULFILLMENT OF THE
REQUIREMENT FOR THE AWARD OF DEGREE
OF B.Sc. (HONS) IN SCIENCE LABORATORY
TECHNOLOGY

JANUARY, 2013

2
 CERTIFICATION

This is to certify that this project work was carried by Henunumwen Mary

LAWRENCE (Miss) in the department of Science Laboratory Technology

(Microbiology option), Faculty of Life Sciences, University of Benin, under

my supervision.

___________________ ___________________
Dr. (Mrs). F.E. Oviasogie Mr. E.O. Oshomoh
Project Supervisor Project Coordinator

___________ ______________
Date Date

__________________
Dr. A.B.O. Ogedegbe
(Head of Department)

_________________
Date

3
 DEDICATION

I dedicate this project work to God Almighty and to my parents Mr. and Mrs.

Taiwo Lawrence Orumwense. Thank you for your advice and support.

4
 ACKNOWLEDGEMENTS

I give thanks to God almighty for His grace upon my life and for seeing
me throughout the period of my stay in school.
My profound gratitude goes to my supervisor Dr. (Mrs.) Oviasogie for
her motherly concern, constructive and supportive ideas which have aided this
seminar work effectively. Also to my lecturer Dr. Akpaja I really appreciate
your efforts and corrections. God bless you sir.
My sincere gratitude goes to my wonderful parents Mr. and Mrs. Taiwo
Lawrence for their love, prayers, financial assistance and advice.
I also give thanks to all the lecturers in the Department of Science
Laboratory Technology for their support.
My sincere appreciate goes to my beloved husband Engr. Tony
Braimah who has always been there for me.
My profound gratitude goes to my siblings Osaretin Lawrence, Eseosa
Lawrence and Ewaen Lawrence. I love you all.
I am indeed grateful to my friend Mrs. Henrrietta Eigbadon and to the
rest of my friends too numerous to mention you have all been wonderful.

5
 TABLE OF CONTENTS

Title - - - - - - - - - - i
Certification - - - - - - - - - iii
Dedication - -- - - - - - - - iv
Acknowledgment - - - - - - - - v
Table of contents - - - - - - - - vi
List of tables - - - - - - - - - viii
Abstract - - - - - - - - - ix
CHAPTER ONE
Introduction - - - - - - - - 1

Literature review - - - - - - - - 2

Morphology and reproduction of plantain - - - - 2

Ecology of plantain - - - - - - - 4

Plantain propagation - - - - - - - 5

Nutritional value of plantain - - - - - - 5

Plantains leaves and uses - - - - - - - 6

Pest and diseases - - - - - - - - 7

Banana weevil - - - - - - - - 7

Nematodes - - - - - - - - - 8

Fusarium wilt - - - - - - - - 9

Chemical constituent of plantain - - - - - 10

Proven scientific evidence - - - - - - 10

6
Toxicities - - - - - - - - - 11

Aims and objectives - - - - - - - 12

CHAPTER TWO

Materials and methods - - - - - - 13

Collection of samples - - - - - - 13

Sterilization of materials - - - - - - 13

Microbiological analysis - - - - - - 14

Morphological characteristics - - - - - 15

Biochemical test - - - - - - - 16

CHAPTER THREE

Results - - - - - - - - - 20

CHAPTER FOUR

Discussion and conclusion - - - - - - 28

Conclusion - - - - - - - - - 30

References - - - - - - - - - 31

Appendix A - - - - - - - - - 37

Appendix B - - - - - - - - - 38

7
 LIST OF TABLES

TABLE TITLE PAGE

1: Number of drooped leaves from plantain tree 22

2: Total viable bacterial counts from fresh and

leaves of plantain tree - - - - 23

3: Fungal counts from fresh and leaves of

plantain tree - - - - - 24

4: Cultural, morphological and biochemical

characteristics of the bacterial isolates 25

5: Cultural and morphological characteristics

of the fungal Isolates - - - - - 26

6: Distribution and occurrence of the microbial isolates 27

8
 ABSTRACT

The number of drooped leaves from different plantain tree and the

microorganisms associated with the drooped leaves of plantain tree were

studies for the period of three week. The isolation of bacteria and fungi

colonizing the drooped leaves of plantain were determined using pour plate

method. The total heterotrophic bacteria counts ranged from 2.4×10 3 -7.4×103

cfu/g while the total heterotrophic fungal counts ranged from 4.9×10 2 -

4.4×103 cfu/g. Gram staining reaction and biochemical tests were carried out

to identify the types of bacteria present. The microorganisms isolated were

Micrococcus luteus, Bacillus subtilis, Pseudomonas aeruginosa , Klebsiella

oxytoca, Enterobacter aerogenes, Staphylococcus aureus, Erwinia sp.,

Fusarium sp., Mucor sp., Penicilium sp. and Aspergillus The result showed

that several types of bacteria and fungi were present on the dry leaves of

plantain. Many of these bacteria and fungi species are known organic matter

decomposers, nitrogen fixers and plant pathogens.

9
 CHAPTER ONE

INTRODUCTION

Plantain (Musa spp. AAB genus) constitute the fourth most important

global food commodity (after rice, wheat and maize) grown in more than 100

countries over a harvested of approximately 10 million hectares, with an

annual production of 88 million tones (Frison and Sharrocks, 1999). The all

year around fruiting habit of banana and plantain puts the crop in a superior

position in bridging the hunger gap, between crop harvests. It therefore

contributes the significantly to food and income security of people engaged in

its production and trade, particularly in developing countries. In Africa they

provide more than 25% of the Carbohydrate requirements for over 70 million

people (IITA, 1998) Eastern and Southern Africa produce over 20 million

tones of banana which accounts for 25.58% of total world output (Karamura

et al., 1999).

A healthy diet consists of eating a variety of foods from 5 food groups

but in correct proportions these include; food containing starch, fruit and

vegetables, milk and diary food, foods containing protein and that contains

fats and sugars.

10
 Banana fall in the vegetable group as well as the food group which

mostly contain starch. Sweet desert bananas are generally eaten raw (fruit),

while cooking bananas and plantains are boiled, streamed, fried or roasted.

LITERATURE REVIEW

Plantain originated from South East Asia, a region considered as the

primary centre of diversification of the crop and where the earliest

domestication has occurred (Simmond, 1962). Musa acuminate is said to have

originated from Malaysia, while the hard musa balbsiana originated from

Indocline. The low land areas of West Africa contains the world’s largest

range of genetic diversity in plantains (Musa AAb) (Oritz and Virlisteke,

1994). Conversely in East Africa, banana are highly evolved into an important

zone of secondary genetic diversity for the East African highland bananas

(Musa AAA) (Smale, 2006).

MORPHOLOGY AND REPRODUCTION OF PLANTAIN

Plantain are large perennial herbs with an underground stem called a

corm, which is the true stem of the banana plant. The corm produces aerial

shots which arises from the lateral buds which develops into eyes and later

suckers. The continuous vegetative growth of sucker perpetrates the corm’s

life and hence the perennial status plantains.

11
The aerial shoots is called a pseudostem and grows to height of 2 to 8cm

depending on the variety and the conditions. The pseudostem consists of large

overlapping leaf bases which are tight rolled round each other forming a

cylindrical structure almost 48cm in the diameter. The roots are structure

initiated from the corm and they range 50 to 100cm in length ; occasionally

sub-horizontal roots reach 3cm (Blomme and Oritz, 2000). The corm consists

of the apical meristem from which the leaves and ultimately the flowers are

initiated. On average, each plant produces 35 to 50 leaves in its growth cycle.

When the plantain plants has formed an average of 40 leaves (within 8 to 18

months), the terminal bud of the corm develops directly into the

inflorescence which is carried up on a long smooth unbranched stem through

the centre of the leaf cluster. The inflorescence is a compound strike of

female and male flowers arranged in groups. Each group consists of two rows

of flowers, one above the other, closely appressed to each other, and the

whole collection is covered by a large subtending bract. The bracts and their

axillary groups of flowers are arranged spirally round the axis and the bract

become closely overlapping each other forming a tough conical inflorescence

at the tip. The lower bracts of the axis enclose female flowers; the middle few

bracts enclose neuter flowers (absent in some cultivars) whilst at the tip of the

inflorescence male flowers occur (Purseglove, 1972). In a few cases, (M.

12
schizocarpa, M. accuminata spp. Banksili and M. accuminata spp. Errans)

hermaphrodite flowers are produced (Sharrock et al., 2001). The female

inflorescences develop into fingers that constitute the bunch. Plantain bunches

possess 4 to 12 hands (clusters), each with at least 10 fingers. In wild bananas

both male and female flowers produce abundant nectar and pollen where as n

cultivated bananas, many clones lack pollen. Plantain and banana pollen is

tiny and sticky, being coated with waxes and proteins held in place by

sculpture elements. The quantity of pollen is an important factor to enhance

the germination potential of pollen grains (Dumpe and Oritz 1996). The

female flowers have ovaries that develop first by parthenocarp (without

fertilization) to form pulp which is the edible part of the crop. However, wild

bananas exhibit cross pollination and ultimately fertilization to form seeds

instead of pulp (non-partinocarpic)

ECOLOGY OF PLANTAIN

Plantains are mostly cultivated between 30oN and South of equator

demanding a mean monthly temperature of 27oC for optimal growth. They do

strive in both low and high altitudes for instance plantains grow well in

lowlands while the East African highland plantains (matooke) survive at

altitudes between 1000 and 1800 distributed rainfall of an average of 2000 to

2500mm through out the year and short dry seasons. Although plantains can

13
be grown on a wide range of soils deep well drained retensive loamy soils,

with high humus content are the best (Zake et. al., 2000). Plantains require

considerably amounts of mineral nutrients to maintain yield Nitrogen, potash

and Phosphorus are the major nutrients required in bulk quantities and can be

either be supplied by fertile soils or by commercial fertilizers.

PLANTAIN PROPAGATION

Plantain do not produce seeds and therefore are clonally propagated

using a number of methods. The methods include; tissue culture derived

plantlets, suckers and split corms sometimes called bits. For good

establishments sources and selection of suckers are very important. A new

and most promising planting material consists of In-vitro plants which are

small suckers produced from meristem culture (Iswennen, 1990). Planting

materials can also be collected from an existing old field, and or a

multiplication plot planted only for the production of suckers. Seed

propagation is only possible in wild bananas which produce vast seeds from

open pollinations.

NUTRITIONAL VALUE OF PLANTAIN

Plantain is a carbohydrate source. Its utilizable protein content as

percentage of Caloric ingestion is higher than sago and Cassava, but is much

lower than other staples such as yam, maize, rice, potato and wheat. On per

14
gram consumed basis, plantain’s essential amino acid concentrations are very

low, even lower than cassava. The low fat content of plantain, coupled with

its high starch content makes it possible for geriatric patients. It may also be a

possible food alternatively for people suffering from gastric ulcer, Coeliac

disease and in the relief of colitis. Plantain contains very little beta-carotene.

The vitamin C content of plantain is very similar to those of sweet potato,

cassava and potato, but the concentration may vary with the crop. Maturity at

harvest, soil and farming conditions.

PLANTAINS LEAVES AND USES

The entire above-ground portion of the plantain is not a true woody

trunk, as in other trees but a “ false trunk” or “ false stem” that consist of

leaves and their fused petiole bases, referred to as a pseudostem. The

pseudostem supports a canopy consisting of 6-20 (or more) leaves. Plantain

leaves are used as plates in several tropical regions of the world. Plantain

leaves can exceed two meter in length. They are similar to banana leaves but

are larger and stronger. In latin America, plantain leaves are lightly smoked

over an open fire, which makes them more flexible and improves storage

properties flavor and aroma. In Venezuela they are fairly widely available in

grocery stores or open air market and are used as wrappers in hallacas. It is

15
also widely used as packaging materials for packaging food and any food

flowers (though this is now replaced widely by plastic).

PEST AND DISEASES

Plantain and banana are susceptible to a wide range of pests and diseases.

Some pests and diseases are highly aggressive or very contagious and easily

spread, and once established they are persistent and practically impossible to

eradicate. In general, the severity and occurrence of pest outbreaks and plant

damage depends on several mitigating factors.

- Environmental conditions

- Specific banana variety.

- Specific disease or pest.

Major pests 7 diseases affecting bananas including the following:

BANANA WEEVIL

The banana weevil cosmolitis sordidus german (Coleoptera:

curculionidac) is a major insect in all the banana and plantain growing

areas of the world (Waterhouse and Norris 1987). It is associated with

yield losses or up to 50% even to 100% in severe infestations which may

lead to total crop failure (Mitchel, 1980, Sengooba, 1986). The larvae bore

tunnels in the corm from where they inflict serious damages to the banana.

Plants affected by weevils are characterized by: splitting of leaf sheaths,

16
tunneled corms and pseudosterm snapping serious weevil attacks may lead

to massive toppling of bananas. Other detrimental effects of weevil

infestations include: premature plant death, stunted growth, delayed fruit

maturation, production of small bunches, reduced number of suckers,

reduced sucker vigour and development of water suckers (Abera et al.,

2000). Weevils are more destructive in low land areas than highlands, and

cease to be a problem beyond 1500m above sea level due to the reduced

temperature.

NEMATODES

These are microscopic worms exhibiting a parasitic effect on a number

of plants including bananas. Parasitic nematodes can be or three broad

categories, the root or aerial parasites, the ectoparasites or endoparasites

and either migratory throughout the life cycle or sedimentary for at least

part of female development. All the important species that feed on bananas

are root parasites. Pratylenchus goodeyi (Cobb) Sher and Atten,

Radopholus similes (Cobb) Thorne and Helicotylenchus milticinctus

(Cobb) golden, are migratory endoparatsites. Both meloidogyne species

and rotylenchulus are sedentary endoparasites. These differences have

implications for both the type of damage caused and appropriate

biotechnology for their control (Caswel et al., 1990; Starr and Page, 2000).

17
 Nematodes attack roots, causing lesions thereby reducing water and

nutrient uptake to the upper parts of the plant and also paving way to

pathogenic microorganisms. Nematode infestation is characterized by

poorly developed root system, production of small poorly formed bunches

and plant toppling (complete up rooting) of the plant including the corms).

The most damaging species on bananas including. Radopholus similis

(cobb) throne (Kashaija et al., 1994), Pratylechus coffeae, P. goodeyi

(Namaganda et al., 2000) and Helicotylechus multicinctus.

Fusarium wilt

Fusarium wilt (panama disease), caused by a soil borne fungus

Fusarium oxysporum schlecht. F. sp cubense (E.F. Smith) snyd and Hans is a

typical vascular disease causing disruption of translocation and systemic

foliage symptoms in bananas, which eventually lead to callapse of the crown

and pseudostem (Jeger et al., 1995). Symptoms generally commence as

premature yellowing of the oldest leaves which develops as a band along the

margin and spreads towards the midrib. The leaves hand between the

pseudostem and the middle of the lamina while still green. All the leaves

eventually collapse where the petiole joins the pseudostem and die (Rawal,

1996). Fusarium f. sp. cubense is also capable of infecting and surviving on a

wide range of alternative hosts, including coffee and maize, both of which

18
may be intercropped with bananas. As a result, disease diagnosis and

development of control strategies, involving the use of plant varietals

resistance is hampered by difficulties encountered in identification of

alternative hosts and relevant pathogenic forms present. Fusarium wilt affects

only exotic banana cultivars Gros Michel (Musa AAA), Kisubi (Musa AB,

Sukali Ndizi (Musa AAB) rather than the indigenous East African highland

bananas (Musa AAA-EA).

CHEMICAL CONSTITUENT OF PLANTAIN

Tannis, eugenol, tyramine. High tannin content in the plant and unripe

fruits has antibiotics activity. Serotonin, levarternol, and dopamine are

available in the ripe fruit and peel other chemical constituents are alkaloids,

steroidal lactones and iron.

PROVEN SCIENTIFIC EVIDENCE

The leaves have been studies as treatment for bronchitis and cold.

Studies in rats demonstrate, effectiveness for stone lysis. Plantain juice was

used as an antidote for snake bite. In animal studies, the extract of Musa

paradisiaca green fruits reduced hyperglycemia in normal and diabetic mice

and protected the gastric mucosa from asprin induced erosion stimulations,

gastric and colonic mucosa and had direct vasodilation effect and non specific

relaxing and inhibiting effect on aortic and portal smooth muscles. There was

19
evidence invivo antimicrobial activity of musa paradisiacal root extract. The

root extracts show invitro antimicrobial activity the flowers are astringens.

Banana fruit is mild laxative. It aids in combating diarrhea and dysentery.

Plantain is used for the treatment of inflammation, rheumatism, gripe,

disease, antihypersion. Unripe banana and plantain fruits are astringent and

use to treat diarrhea. The leaves are used for cough and bronchitis. The root

can arrest hemoptolysis and posses strongly astringent anthelmintic

properties. Plantains juice is used as an antidote for snake bite. Other used are

for asthma, burns diabetes, dysentery excessive menstrual flow, fever,

gangrene, gout, headache, hemorrhage, insomnia, intestinal parasite sores,

syphilis, tuberculosis, ulcers, warts. In some traditional medicine, the head

protecting the leaves of the bid was used against heavy menteutic bleeding

(hemorrhagic). Other therapeutic uses were against diarrhea, dysentery,

migraine, hypertension, asthma and jaundice.

TOXICITIES

Stem juice used as arrow poison in Africa. No toxicities and

contraindications are reported in human yet. Musa paradisiaca is a non- toxic

plant. In an animal study, Musa paradisiaca pseudortalk extract was used in

rats to evaluate the toxicities but the product showed non-toxic when rats

orally took a doze of 2g/kg.

20
Aim and objectives

Identification of microorganisms associated with plantain leaves during

storage under different conditions

21
 CHAPTER TWO

MATERIALS AND METHODS

Collection of samples

Plantain leaves used in this study were collected 20 plantain tree and

these tree were identified alphabetically and number of drooped leaves from

each tree were recorded. This was carried out for the period of 3 weeks.

Sterilization of materials

Glasswares such as petridishes, test tubes, glass rods, pipette,

measuring cylinders, beakers and conical flasks to be used for the work were

washed using water and detergent. They were rinsed severally with distilled

water and sterilized after wrapping with aluminum foil paper with autoclave

and oven. They were autoclaved at 1210C for 15 minutes while glass wares

were sterilized using the oven at 160 0C four 1 hour. The table was disinfected

by cleaning with cotton wool and ethanol to disinfect the environment.

Culture Media Used: The media used in this study include Nutrient agar and

potato dextrose agar

Nutrient Agar

Nutrient agar was used for the cultivation of non-fastidious bacteria and

for heterotrophic bacteria plate counts. This medium was prepared

commercially available dehydrated powder, available from suppliers of

22
culture media. Twenty eight grams (28 g) of nutrient powder was dissolved in

1 litre of distilled water in conical flask which was stirred and covered tightly

with cotton and aluminium foil paper. It was autoclaved at 121 0C for 15

minutes after which it was cooled to about 45-50 0C before pouring into petri

dishes (approximately 20 ml).

Potato dextrose agar

Potato dextrose agar was used for the cultivation of fungi and for

heterotrophic fungi plate counts. This medium was prepared commercially

available dehydrated powder, available from suppliers of culture media.

Thirty nine grams (39 g) of potato dextrose agar powder was dissolved in 1

litre of distilled water in conical flask which was stirred and covered tightly

with cotton and aluminium foil paper. It was autoclaved at 121 0C for 15

minutes after which it was cooled to about 45-50 0C before pouring into Petri

dishes (approximately 20 ml).

Microbiological analysis

Ten grammes (10 g) of the sample was measured and ground in a

sterile laboratory mortar and pestle. The ground sample was transferred to a

sterile beaker and 90 ml of sterilized distilled water was added. One milliliter

(1 ml) of the prepared sample was diluted in 9 ml of sterile distilled water

(diluents) and mixed thoroughly by shaking. 1ml o f the resultant mixture was

23
aseptically transformed to 9 mls of sterile water in a test tube. This process

was carried out under sterile aseptic conditions. The dilution was continued

serially until the third dilution was attained (i.e. 10-3). An aliquot of (0.1 ml)

of the 10-3 dilution was inoculated into a sterile Potato Dextrose Agar (PDA)

and Nutrient Agar (NA) plates respectively using pour plate method. The

nutrient agar plates were incubated at 37 0C for 24 hours while the potato

dextrose agar plates were incubated at 28 0C for 72 hours. The number of

colonies formed in each culture plate was counted and expressed as the

colony forming unit (CPU) per gramme of the sample.

Morphological characteristics

Gram staining

The gram staining technique was done on the basis of the component of

the cell wall of the organism. Organisms which retained the primary stain

(crystal violet) are known as gram positive organisms while those which do

not retain the primary stain when decolourized by alcohol are gram negative.

The non retention of the stain is due to the thin nature of the cell wall, thin

peptidoglycan layer and the cell composition; absence of teoichic acid. The

gram stainin reagent include crystal violet (primary stain), Lugol iodine

(mordant), % alcohol (decolourizer), safranin (secondary stain)

24
PROCEDURE: A thin smear made on a clean, grease-free glass slide with an

inoculating wire loop. The smear was heat fixed by passing it over the flame.

The smear was flooded with the primary stain (Crystal violet) and allowed to

react for one minute which the slide was rinsed with distilled water. Gram's

iodine was added (mordant) for 1 minute. The smear was gently rinsed with

distilled water, Alcohol (70% ethanol) was applied to serve on decolorizer for

30 seconds, it was then rinsed with distilled water Finally, it was counter-

stained with Safranin for 1 minute and rinsed again with distilled water, then

allowed to dry. The smear was examined under the microscope using x 100

oil immersion objective lens.

BIOCHEMICAL TEST

Catalase test

Catalase test demonstrate the presence of Catalase enzyme by aerobic

microorganism. Catalase is an enzyme that catalyses the release of oxygen

from hydrogen peroxide. A drop of 3% hydrogen peroxide solution was

added to a slide. The organism to be tested for Catalase production is brought

in contact the solution. The production of gas bubbles indicate positive

reaction, i. e Catalase enzyme is produced.

25
Oxidase test

This was carried out by placing a clean filter paper on the working bench; the

isolates were then placed onto the filter paper using a sterile inoculating wire

loop. After which a few quantity of oxidase reagent was added, a purple

coloration was observed within 10-15 minutes which indicates that the

organism is oxidase positive.

Coagulase test

This test was carried out to determine the presence of enzyme; coagulase. The

test is used to distinguish coagulase positive staphylococcus aureus from

coagulase negative Staphylococcus species. A colony of bacteria was

emulsified with a sterile saline solution on clean grease free slide. A drop of

plasma was added with the emulsion and mixed. Positive coagulase

organisms show clumping, while negative coagulase organism showed no

clumping.

Urease test

This test was aimed at identifying Enterobacteriae that produce urease

enzyme, which hydrolyze urea to give ammonia and carbon dioxide. The test

organism was heavily inoculated onto urea base agar in a bijou bottle using a

sterile wire loop and incubated at C for 24 hours. A change of colour

from yellow to pink in the medium showed positive test.

26
Indole test

This test was carried out for indole production by test organism which

is important in identifying enterobacteria. A sterile wire loop was used to

inoculate a colony of test organism into 2ml of peptone water containing

tryptophan. The tube was stoppered and incubated at 37 0C for 24hours.

Kovac’s reagent was added to the medium. Observation of purple colouration

on the surface layer within 10minutes showed a positive result.

Citrate test

This test is based on the ability of an organism to use citrate as its

source of carbon. It was used to identify the Enterobacteriae. Simon’s citrate

agar medium was prepared in a slant bijou bottle and test bacterial isolates

were inoculated onto the slant medium and incubated at for 48hours after

which it was examined for colour formation. A bright blue colour in the

medium showed a positive citrate test.

Sugar fermentation test

The ability of the isolates to utilize certain sugar as a source of energy was

tested. If the organism does not ferment a particular sugar, acid will be

produced and gas may or may not be produced. The production of acid is

indicated by colour change of the medium from red to yellow and the

production of gas is indicated by a void produced in Durham tube. Inability to

27
ferment sugar is indicated by no colour change of the medium. The

fermentation medium was prepared by 0.1g of peptone, 0.1g of sodium

chloride and 0.1 g of fermentable sugar (glucose) in 10ml of distilled water.

An amount of 9 ml of the medium was pipette into a test tube containing

Durham’s tubes in replicates(based on the isolate under test).5ml of phenol

red indicator was immediately discharged into the test tubes. The test tubes

containing medium were sterilized in an autoclave at 121 0C for 15 minutes.

After sterilization, each isolate were inoculated into glucose medium. The test

tubes were incubated at 37 0C for 24- 48 hours for bacterial isolates. A change

of colour from red to yellow in the medium showed positive result.

28
 CHAPTER THREE

RESULTS

Table 1 shows the number of drooped leaves from each plantain tree.

There was variation in the number of drooped leaves from each tree during

the period of analysis.

Table 2 shows the total heterotrophic bacterial counts from the leaves

of plantain tree. The bacterial counts from fresh leaves of plantain tree was

2.4×103 cfu/g while the bacterial counts from dried leaves of plantain ranged

from 4.5×103 -7.4×103 cfu/g

Table 3 shows the total heterotrophic fungal counts from the leaves of

plantain tree. The fungal counts from fresh leaves of plantain tree was

4.9×102 while the fungal counts from dried leaves of plantain ranged from

2.0×103 -4.4×103 cfu/g

Table 4 shows the cultural, morphological and biochemical

characteristics of the bacterial isolates. The bacterial isolates include

Micrococcus luteus, Bacillus subtilis, Pseudomonas aeruginosa, Klebsiella

oxytoca, Enterobacter aerogenes, Staphylococcus aureus and Erwinia sp.

Table 5 shows the cultural and morphological characteristics of the fungal

isolates. The fungi isolated were Fusarium sp., Mucor sp., Penicilium sp. and

Aspergillus niger.

29
 Table 6 shows the distribution and occurrence of the microbial isolates

from the leaves of plantain tree.

30
Table 1: Number of drooped leaves from plantain tree

No. of tress Wk 1 Wk 2 Wk 3
A 4 2 3
B 4 3 3
C 3 3 1
D 2 3 2
E 3 2 2
F 2 2 2
G 3 2 3
H 4 2 2
I 3 4 2
J 3 1 2
K 3 3 1
L 2 2 1
M 3 2 2
N 2 2 3
O 3 2 2
P 3 2 3
Q 2 3 1
R 3 3 2
S 2 1 2
T 2 2 3
U 2 2 2
V 4 2 1

31
Table 2: Total viable bacterial counts from fresh and dry leaves of
plantain tree
Period of analysis Fresh leaves Dry leaves

Week one 2.4×103 4.5×103

Week two ND 7.4×103

Week three ND 5.9×103

KEY:
ND= Not determined

32
Table 3: Fungal counts from fresh and dry leaves of plantain tree

Period of analysis Fresh leaves Dry leaves

Week one 4.9×102 2.0×103

Week two ND 2.9×103

Week three ND 4.4×103

KEY:
ND= Not determined

33
Table 4: Cultural, morphological and biochemical characteristics of the
bacterial isolates

1 2 3 4 5 6 7
Cultural
Elevation Convex Flat Low Convex Low Convex Convex
convex convex
Margin Entire Entire Entire Entire Entire Entire Entire
Colour Yellow Cream Green Cream Cream Yellow Dirty
white
Shape Circular circular Circular Circular Circular Circular Circular
Morphological
Gram staining + + - - - + -
Cell type Cocci Rod Rod Rod Rod Cocci Rod
Cell Single Chains Single Single Single Cluster Single
arrangement
Spore staining - + - - - - -
Biochemical
Catalase + + + + + + +
Oxidase - - + - - - -
Coagulase - - - - - + -
Citrate + + + + + + +
Urease + + - + - + +
Indole - - - - - - -
Glucose + + + + + + +
1= Micrococcus luteus
2=Bacillus subtilis
3= Pseudomonas aeruginosa
4= Klebsiella oxytoca
5= Enterobacter aerogenes
6 = Staphylococcus aureus
7= Erwinia sp.

34
Table 5: Cultural and morphological characteristics of the fungal isolates

Cultural Microscopy Possible isolates

Black fluffy Septate hyphae conidia occur Aspergillus sp.
colonies with on large radiating heads.
reverse side Conidiophores arises from a
yellow segment of mycelium called a
foot cell
Green flat Septate hyphae conidiophores Penicillium sp.
colonies with with smooth stripe
white periphery
White flat Non-septate hyphae, Mucor sp.
colonies with sporangiophores symbolically
reverse side branced with long and short
colourless criminate branches

Cottony white Micro-conidia, ovoid to Fusarium sp.

colonies with ellipsoidal in shape are borne
reverse side on simple phialides. Macro
colourless conidia
are borne on phialides on
branched conidiospores.
Septate
fusiform, slightly curved and
pointed at both ends

35
Table 6: Distribution and occurrence of the microbial isolates

Bacterial isolates Fresh leaves Dry leaves

Bacillus subtilis + +

Micrococcus luteus - +

Klebsiella oxytoca - +

Staphylococcus aureus - +

Pseudomonas aeruginosa + +

Erwinia sp - +

Fungal isolates

Fusarium sp. + +

Mucor sp. - +

Aspergillus sp. + +

Penicillium sp. + +

KEY:
+= Present
-= Absent

36
 CHAPTER FOUR
DISCUSSION AND CONCLUSION
There was variation in the number of drooped leaves of the plantain

tree. This variation may be possibly to disease caused by plant pathogens,

environmental and genetic factor. Plantain leaves contain lignin and cellulose

which many bacteria and fungi like Basidiomycetes are capable of degrading

with the aid of enzymes. Fungal mycelium excretes extensive enzymes

complexes which can directly attack and degrade these components in

plantain leaves. They therefore use the dried plantain leaves as nutrients for

their growth. The bacterial counts from fresh leaves of plantain tree was

2.4×103 cfu/g while the bacterial counts from dried leaves of plantain ranged

from 4.5×103 -7.4×103 cfu/g. The fungal counts from fresh leaves of plantain

tree was 4.9×102 while the fungal counts from dried leaves of plantain ranged

from 2.0×103 -4.4×103 cfu/g. The microbial loads of the dry leaves of the

plantain tree were comparable higher than the microbial loads of the fresh

leaves. The microorganisms isolated were Micrococcus luteus, Bacillus

subtilis, Pseudomonas aeruginosa, Klebsiella oxytoca, Enterobacter

aerogenes, Staphylococcus aureus, Erwinia sp., Fusarium sp., Mucor sp.,

Penicilium sp. and Aspergillus. The microorganisms isolated from plant

leaves is in agreement with the report of previous studies by Amuneke et al.

(2011) who similar microorganism from dry plantain leaves used for the

37
cultivation of Pleurotus ostreatus. The microorganisms isolated from fresh

and dried leaves from plantain tree may be possible airborne contaminant,

microflora of the leaves and pathogens of plantain tree.

Pseudomonas and Erwinia species are plant pathogens which caused

bacterial wilt disease of plantain. The major characteristics of the disease are

yellowing and complete wilting of the plant starting with the most peripheral

leaves (Tushemereirwe et al., 2003). After wilting the leaves tend to droop

and the plant eventually stops growing and dies. Secretion of bacterial ooze

can be seen on leaves. The most common symptoms are wilting and

premature ripening of the bunch in addition to male. The presence of

Staphylococcus aureus and Microococcus sp. could be due to human handling

during collection of the plantain leaves and the environment. Bacillus species

are spore producing organisms which is ubiquitous in the environment. The

presence of Bacillus species may be possibly due to airborne contamination.

Aspergillus sp. is ubiquitous in the environment and could be a possible air

borne contaminants of the plantain leaves.

Fusarium species are plant pathogens capable of infecting and

surviving on a wide range of plants. Fusarium wilt , caused by a soil borne

fungus Fusarium oxysporum is a typical vascular disease causing disruption

of translocation and systemic foliage symptoms in plantain, which eventually

38
lead to collapse of the crown and pseudostem (Jeger, et.al., 1995). Symptoms

generally commence as premature yellowing of the oldest leaves which

develops as a band along the margin and spreads towards the midrib. The

leaves hang between the pseudostem and the middle of the lamina while still

green. The leaves wilt, the petiole buckles and spreads towards the midrib. All

the leaves eventually collapse where the petiole joins the pseudostem and die

(Rawal, 1996).

Microbial succession from composting to harvesting has shown that the

nature and amount of nitrogen present in the dry plantain leave used as

substrate for mushroom influences the degree of cellulose degradation.

According to Chang (1980) the growth of Pleurotus species is favoured on a

dry plantain leave of low nitrogen content and the nutrient content of

substrates affects the formation of fruit bodies

CONCLUSION

The results of this present study showed that there was variation in the

number of drooped leaves of plantain tree over time. There was also high

microbial contaminant in the dry leaves of plantain tree than the fresh leaves.

The presence of these microbial isolates in the plantain leaves showed that

can survive on dry plantain leaves and utilized it as substrate for growth.

39
 REFERENCES

Abera, A. M., Gold, C. S., Kyamaanywa, S. and Karamura, E. B. (2000).

Banana weevil, Cosmopolites sordidus (Germar), ovipositional

preferences, timing of attack and larval survivorship in a mixed cultivar

trial in Uganda. In: pp. 371-403. Craenen, K., Ortiz, R., Karamura, E.

B., and Vuylsteke (Eds). Proceedings of the International Conference

on Banana and Plantain for Africa. International Society for

Horticultural Science (ISHS), Leuven, Belgium.

Anyankorah, C.H. (2002). Cultivation of Edible Mushroom. Mushroom

Science 30:399-401.

Amuneke E. H., Dike K. S., and Ogbulie J. N. (2011). Cultivation of

Pleurotus ostreatus: An edible mushroom from agro base waste

products. Journal of Microbiology Biotechnology Research 1 (3):1-14

A. O. A. C. (1980). Official methods of Analysis. 15th edn. Association of

Official Analytical Chemists, Washington, D. C. 84pp.

Argent, G.C.G. (1976). The wild bananas of Papua New Guinea. Notes from

the Royal Botany Garden Edinburgh 35: 77-114.

Bahl, N. (1985). Hand book on mushrooms. Oxford Publishing Co. PVT.

New Delhi. 80pp.

40
Blomme, G. and Ortiz, R. (2000). Preliminary assessment of root systems

morphology in Musa. In. pp. 259-266. Craenen, K., Ortiz, R.,

Karamura, E. B. and Vuylsteke (Eds). Proceedings of the International

Conference on Banana and Plantain for African. International Society

for Horticultural Science (ISHS), Leuven. Belgium.

Carreel, F., Faure, S., Gonzalez, de Leon, D., Lagoda, P.J.L., Perrier, X.

Bakry, F., Tezenas du Montcel, H., Lanaud, C. and Horry, J.P. (1994).

Evaluation of the genetic diversity in diploid bananas (Musa spp).

Genetics Selection, Evolution 26(.1): 125-136.

Chang, S.T. (1980). Cultivation of edible mushroom. Journal Mushroom as

Human Food 19: 43-47.

Elliott, C. E (1991). Reproduction in Fungi. Journal of industrial Food

Processing 15(2): 47-52.

Frison, E. and Sharrock, S. (1999). The economic, social and nutritional

importance of banana in the world. In: pp. 113-123. Picq, C., Foure, E.,

Frison, E. A (Eds). Banana and food Security International

Symposium. Douala, Cameroon,

IITA (1998). Plantain and Banana Improvement Program. Annual Report for

1997 International Institute of Tropical Agricultural, Onne, Nigeria.

27pp.

41
INIBAP (2003). Conservation through utilization of bananas and plantains in

the Great Lakes region of East African. Final Report INIBAP,

Montpellier, France. 67pp.

Jeger, M. J., Eden- Green, S., Thresh, J. M., Johnson, A., Waller, J.M and

Brown, A. E. (1995). Banana diseases. In: pp. 317-381. Gowen, S. R

(ed). Banana and Plantains, Chapman and Hall, London.

Jeger, M. J., Eden-Green, S., Thresh, J. M., Johanson, A., Waller, J. M. and

Brown, A. E. (1995). Banana diseases. In: pp. 317-381. Gowen, S.R.

(ed.) Bananas and Plantains. Chapman and Hall, London.

Karamura, E, Frison, E., Karamura, D. A. and Sharrock, S. (1999). Banana

Production systems in Eastern and Southern African. In. pp. 10-14.

Security Picq, C., Foure, E., Frison, E. A (Eds). Banana and Food.

International Symposium, Douala, Cameroon.

Kashaija, I. N., Speijer, P. R., Gold, C. S. and Gowen, S. R. (1994).

Occurrence, distribution and abundance of plant parasitic nematodes on

bananas in Uganda. African Crop Science Journal 2: 99-104.

Mitchel, G. A (1980). Banana Entomology vin the Windward Islands: Final

report, 1974-1978. London, Centre for Overseas Pest Research Pp. 216.

42
Namaganda, J. M., Karamura, E., Namanya P., Gowen, S. R. and Bridge, J.

(2000). Studies on host range of the banana lesion nematode,

Pratylenchus Goodeyi. In. pp 419-425. Craenen, K., Ortiz, R. and

Karamura, E. B. (Eds). Processing of the International Conference on

Banana and Plantain for African. international Society for

Horticultural Science (ISHS), Leuven. Belgium.

Ogundana, S. K and Fagede, O. H (1982). Nutritive value of some Nigerian

edible mushrooms. Foods Chemistry 8:263-268

Ortiz, R. and Vuylsteke, D. R (1994). Future strategy of Musa improvement:

Banana and plantain breeding: Priorities and strategies. INIBAP.

Montpellier, France. 42pp.

Purseglove, J.W. (1972). Tropical crops. Longmans, London. 42pp.

Rasmussen, C. (1996). Mushroom cultivation. Mushroom Science 1:8-15.

Rawal, R. D (1996). Fungial disease of banana: Current scenario in India. In.

pp. 214-226. Signh H. P. and Chadha, K. L (Eds) Banana,

Improvement, Production and Utilization. AIPUB. NHB. Gurgaon,

India.

43
Rawal, R.D. (1996). Fungal disease of banana: Current scenario in India. In:

pp. 211-232. Singh, H. P. and Chadha, K. L. (Eds.) Banana,

Improvement, Production and Utilization. AIPUB, NHB, Gurgaon,

India

Sengooba, T. (1986). Survey of banana pest problem complex in Rakal and

Masaka districts: preliminary trip report. Uganda Ministry of

Agriculture, Kawanda Research Station. 10pp.

Sharrock, S. L., Horry, J.P. and Frison, E. A (2001). The state of the use of

Musa diversity. In: pp. 223-244. Cooper, H.D., Spillane, C. and

Hodgkin. T (Eds.) Broadening the Genetic Base of Crop Production.

CABI, Uk.

Simmonds, N. W. and Weatherup, S. T. C (1960). Numerical taxonomy of the

wild bananas (Musa). New Phytology 115: 567-571.

Smale, M. (2006). Assessing the impact of crop genetic improvement in sub-

saharan Africa: Research context and highlights. In: pp. 207-251.

Melinda, S., Edmeades. and De Groote, (Eds). Promising crop

biotechnologies for smallholder farmers in East Africa. Adventist

University of Africa Printing Press, Nairobi

Stover, R. H. (2000). The intensive production of horn-type plantains (Musa

AAB) with coffe in Columbia. Fruits 38: 765-770.

44
Swennen, R (1990). Herbs: plantain leaf herb - cut. International Institute for

Tropical Agriculture, Food Agriculture 45: 333-336.

Swennen, R. (1990) Plantain cultivation under West African conditions. A

Reference Manual. 11TA, Ibadan, Nigeria.

Thoithi1, G., Kennedy, A. and Abiy, Y. (2000). Biological Activities and

Chemical Constituents of Plants used for Herbal Medicine in Kenya.

Department of Pharmaceutical Chemistry, University of Nairobi,

Kenya. 89pp.

Tushemereirwe, W. K., Kangire, A., Smith, J., Ssekiwoko, F., Nakyanzi, M.,

Kataama, D., Musiitwa, C., and Karyaija, R. (2003). An outbreak of

bacterial wilt on banana in Uganda. Infomusa. 12(2):6-8.

Waterhouse, F. and Norris, K.R. (1987). Biological control-Pacific prospects.

Inkata Press Ltd. Melbourne. 454pp.

Zake, Y.K., Bwaniki, D.P. and Nkwiine, C. (2000). Soil management

requirement for banana production on the heavy soils around Lake

Victoria in Uganda. In: pp. 285-292. Craenen, K., Ortiz, R., Karamura,

E.B. and Vuylsteke. Proceedings of the International conference on

Banana and Plantain for Africa. International Society for Horitcultural

Science (ISHS), Leuven, Belgium.

45
 APPENDIX A

CULTURE MEDIA

Nutrient agar

Beef extract 3.0g

Agar No.2 12.0g

Peptone 5.0g

Sodium chloride 8.0g

Distilled water 1000ml

46
 APPENDIX B

GRAM STAINING AND BIOCHEMICAL REAGENTS

STAIN AND REAGENT

GRAM STAIN

A. Gram crystal violet

Solution A

Crystal violet 2.0g

Ethanol (95ml) 20.0ml

Solution B

Ammonium oxalate 0.8g

Distilled water 80.0ml

Gram iodine

Iodine 1.0g

Potassium iodide 2.0g

Water 300.0ml

3.0 g of medium was dissolved was in 300.0ml of distilled water.

Gram’s safranin

Safranin 0.25g

Ethanol 10.0ml

Distilled water 100ml

47
Biochemical reagents

Indole medium

Peptone 20.0g

Sodium chloride 5.0g

Distilled water 1000ml

pH 7.4

25.0 g of indole medium was dissolved in 1000ml of distilled water and

autoclaved for 15 minutes at 121 0c and dispensed aseptically into sterile test

tubes.

Kovac’s reagent

Amul-alcohol 15.0ml

p-dimethyl-aminobenaldehyde 0.5ml

Concentrated HCl 50ml

Small quantity of kovac’s reagent were prepared by dissolving the aldehyde

into alcohol and adding the acid slowly and then kept inside the refrigerator.

48