LUCKY MASEGO MABAKA
201803496
BIO 309
PRACTICAL 2: MYCOLOGICAL
ANALYSIS OF FRUITS AND
VEGETABLES
ISOLATING FUNGI ASSOCIATED WITH FRESH BANANNA AND CABBAGE
COMMONLY SOLD BY STREET VENDORS AROUND IN GABORONE
Fruits and vegetables are food substances that mostly consumed by people all over the world
because of the nutrients they possess (FDA, 2000). This is because fruits and vegetables
supply the body with essential vitamins and minerals that function to keep the body in
healthy condition. However, they are the most food substances that are frequently attacked by
fungal organisms.
Fungal contamination can occur at every step in the production chain of fresh-cut fruit from
the farm until packaging process at the processors. Notably, no treatments during the
production of fresh-cut fruit could ensure the total elimination of microorganisms that might
be present on the surface of fresh-cut fruit. It is well known that fresh-cut fruit create a
favourable environment for proliferation of spoilage organisms which can be a major cause
of spoilage of fresh-cut fruit and shelf-life limiter (Bartz et al, 1995). This is because fungi
are ubiquitous in nature and are present in both indoor and outdoor environments. The fungal
spores remain suspended for longer time in the air, their presence depend on the various
factors like temperature, humidity, sunlight, seasonal variations (Tom and Church, 1975).
Suspensions of organic and inorganic material also effect the distribution of microbes in the
air. Airborne fungi are considered to act as indicator of the level of atmospheric bio-pollution.
Fruits and vegetables normally carry a non-pathogenic epiphytic microflora2 (Patel and Joshi,
2008). The inner tissues of healthy plants and fruits are free of microorganisms, however, the
surfaces of raw vegetables and fruits are contaminated with a variety of microorganisms, this
depends on the microbial population of the environment from which the food was taken, the
condition of the raw product, the method of handling, the time and conditions of storage.
Regardless, many fruits and vegetables present nearly ideal conditions for the survival and
growth of many types of microorganisms (Cole and Kendrick, 1968). This is because their
internal tissues are nutrient rich and many, especially vegetables, have a pH near neutrality.
The structure of fruits and vegetables are comprised mainly of the polysaccharides cellulose,
hemicellulose, and pectin. Starch is the principal storage polymer (FDA,2000). Spoilage
microorganisms exploit the host using extracellular lytic enzymes that degrade these
polymers to release water and the plant’s other intracellular constituents for use as nutrients
for their growth. Fungi in particular produce an abundance of extracellular pectinases and
hemicellulases that are important factors for fungal spoilage (Miedes and Lorences, 2004).
Generally, even during refrigeration some microbes such as moulds and other fungi produce
mycotoxins of various types that are harmful to the consumers (Patel and Joshi, 2008). These
mycotoxins have low molecular weight and toxic secondary metabolites from some species
of fungi. They are dangerous even in minute quantities and present extreme toxicity due to
their ability to withstand heat (Tom and Church, 1975). However, the pathogenic microbes
cause infections or allergies. Cabbage (Brassica oleracea capitata) and other fresh-cut
vegetables are offered as salads in more than 70 per cent of fast food establishments and
family resturants. Banana (Musa paradisiaca) on the other hand is frequently included in
breakfast meals and lunch box meals for kids. These vegetables and fruits are, however,
capable of causing human diseases while still on the plant in fields or orchids, or during
harvesting, transport, processing, distribution and marketing, or in the home (FDA, 2000; and
Patel and Joshi, 2008).
The aim of this study was to evaluate the mycological quality of banana and cabbage
purchased from local market and present a comprehensive report on the fungi isolated.
MATERIALS AND METHODS
In order to prepare and dilute the sample, 25g sample of banana and cabbage portion into a
sterile stomacher bag and exactly nine times of the weight or volume of diluent were added to
give a 1 in 10 (10‾¹) suspension. Using a stomacher, the suspension was homogenized for 2
minutes after which the homogenate was transferred to 90 ml bottle of diluent avoiding
contact between the pippete and the diluent. The contents were mixed thoroughly and
carefully, subsequently 10 ml of the sample homogenate was transferred to another 90 ml
bottle of diluent, these were mixed thoroughly. Using another fresh pipette, 1 ml of the above
homogenate was transferred into a 99ml bottle to achieve 10‾⁴ dilution. The next step was
playing where 0.1 ml of each sample dilution was pipetted into each of the agar plates. After
adding the inoculum, it was spread evenly over the agar surgace with a sterile bent glass rod.
The rod was sterilized by flaming it with ethanol before use. The plates were then incubated
upright in a 25⁰ C incubator for 7 days.
Direct plating
The food particles were immersed in the sodium hypochlorite solution provided in beakers
for 2 min stirring occasionally with a pair of sterile forceps to dislodge air bubbles. The
chlorine solution was drained and the solution was used only once. Subsequently, the food
particle was rinsed once with sterile water. Using a 1 min treatment, with stirring, the water
was poured off. The particles were placed onto agar plates ( PDA and MEA) at the rate of 620 particles per plate using a pair of sterile forceps. Lastly the plates were incubated upright
at 25⁰C for 7 days.
In week 2, a small piece of hyphae and/or spores were placed on a fresh plate as a point
inoculum, preferably near the center of the plate as this will allow the best colony
development and sporulation in most fungi. After this, simple staining was done by taking a
small quantity of culture and mix with a drop of sterile distilled water to prepare a smear. The
smear was allowed to air dry and heat fixed. The smear was then stained with crystal violet
solution for 60 seconds, after which it was rinsed, blot dried and observed under oil
immersion lens. The identification of filamentous fungi was achieved by placing a drop of the
stain on clean slide with the aid of a mounting needle, where a small portion of the aerial
mycelia from the representative fungi cultures was removed and placed in a drop of
lactophenol. The mycelium was well spread on the slide with the needle. A cover slip was
gently placed with little pressure to eliminate air bubbles. The slide was then mounted and
viewed under the oil immersion lens.
RESULTS AND ANALYSIS
The fungal isolates were identified using cultural and morphological features such as colony
growth pattern, conidial morphology, and pigmentation.
Table 1: colonial and microscopic Characteristics of from the Fresh Banana
Isolate
PDA 10‾¹
PDA 10‾²
MEA 10‾²
MEA 10‾⁴
colonial
Colony are
filamentous with
spores
Colony are
white filaments
Cream white
colonies
White mycelia
with black
spores
Microscopic
Brownish hyphae
with no septa
Suspected fungi
Aspergillus
alliaceus
Greenish and
yellow hyphae
with septa
Purple rod like
hyphae
Brownish hyphae
with a long line in
the center
Basipetospora
rubra
Aspergillus
fumigatus
Aspergillus niger
Table 2: colonial and microscopic Characteristics of from the Fresh Cabbage
Isolate
PDA
Colonial
Black Colony
with grey
mycelia
Microscopic
Brownish hyphae
with septa and
thick walled,
unbranched with
rounded ends that
bore conidia
Suspected fungi
Aspergillus
niger
MEA
Yellow colonies
clustered
together. Black
spores appear on
edges of plates
Purple rod-shaped Rhizopus
stolonifer
fungal cells
grouped in
colonies
DISCUSSION
The fungi associated with fresh banana and cabbage commonly sold by street vendors around
Gaborone, Botswana were studied. The filamentous fungi isolated from the banana fruits
were identified as Aspergillus niger, Aspergillus alliaceus, Basipetospora rubra and
Aspergillus fumigatus (Table 1).
Tom and Church (1975) also isolated Aspergillus niger, Aspergillus alliaceus, Basipetospora
rubra and Aspergillus fumigatus from the fresh bananas displayed for sale in five different
market places in St Lucia in windward islands. Christophe and Carlin (1994) also isolated
Aspergillus niger and Aspergillus fumigatus from processed fresh fruits in Ibadan,
Southwestern Italy. When isolating Aspergillius niger, Patel and Joshi (2008) described that
Aspergillus niger colonies growing on MEA had 55-65 mm diameter in 7 days at 25⁰C. they
further explained that the colonies have black conidial areas with white mycelia and reverse
pale yellow which corresponds with our study where we also found out that aspergillus niger
has white mycelia. However our aspergillus niger had already matured by the time the results
were analyzed hence there were black spores present in the plate which is in contrast with
Patel and Joshi’s study.
From table 1, Aspergillus alliaceus was isolated in banana growing on PDA at 10‾¹ however
this is contrast to Christophe and Carlin’s study in that they isolated aspergillus alliaceus
growing on CYA and MEA. Their findings suggest that on MEA, Aspergillus alliaceus grow
up 45-60 mm diameter in 7 days at 25⁰C with white mycelia and the sclerotia white becoming
creamy by age. Tom and Church (1975) concluded in their study “Fungi associated with
crown-rot disease of bananas from St. Lucia in the Windward Islands” that Aspergillus
fumigatus is a species that is characterized by rapid growing dark green low colonies,
uniseriate, with phialides in parallel position to each other. However, we are in concert since
we found cream white colonies in 7 days which corresponds to their study. Basipetospora
rubra was isolated in banana on PDA and greenish and yellow hyphae was observed under
the microscope which matches Cole and Kendrick’s study where they also found white
colonies on PDA, mycelia white and pale yellow septate hyphae.
From table 2, aspergillus spp were isolated from cabbage growing on PDA however it was
quite uncertain as to which the species is exactly. From Van tiechem’s study, it could be
concluded that the fungi isolated in cabbage growing in PDA is Aspergillus niger. The
reasons could be that they resemble the black colony with grey mycelia in aspergillus niger
and their rounded ends that bore conidia. In addition, the isolated fungi in cabbage growing
on MEA was found to be Rhizopus stolonifer .this is supported by Gams and Bissett (1998)
where they state that Rhizopus reproduces asexually by sending up vertical stalk called
sporangiophore which lumps at the tip to produce a sporangium. The cytoplasm in the
sporangium divides repeatedly to release a mass of spores, each with a nucleus. When the
sporangium breaks open, the spores are dispersed in the air, and each can grow to form a new
mycelium on an apt medium.
Presence of these bacteria and fungi on cabbage, most especially Aspergillus spp poses a
serious threat to health of consumers as the organism especially Aspergillus could produce
mycotoxins, which are lethal when consumed which poses a serious threat to health of
consumer and sellers of cabbage at Gaborone. It is therefore necessary and important that
both the farmers and sellers to take necessary and appropriate precautions in preventing
contamination and eating of contaminated vegetables. This will however reduce the risk of
mycotoxins associated with fungi contamination which are deleterious to human health
(FDA, 2000).
The errors present in the experiment might have been because the agar in which the isolates
were streaked was rather soft which made streaking exceedingly difficult. In the next
experiment, this could be improved by using a hard agar.
CONCLUSION
The filamentous fungi isolated from the banana fruits were identified as Aspergillus niger,
Aspergillus alliaceus, Basipetospora rubra and Aspergillus fumigatus. In addition, the fungi
isolated from the cabbage fruits were identified as Aspergillus niger and Rhodopus stolonifer
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