Abstract

Fruits spoilage causes threat to human health and responsible for great economic
loss worldwide. This study was conducted to isolate and identify fungi responsible
for spoilage of fruit commonly sold in University of Maiduguri commercial Area.
A total of 9 spoiled fruits, 3 samples from each different fruit Orange (Citrus
sinensis), papaya (Carica papaya), and melon (Cucumismelo), were collected
aseptically in sterile polyethene bag and delivered to Biological Science Research
Laboratory. Fungi were isolated and identifiedby using culturing and microscopic
methods. Seventy seven fungal isolates belonging to five different genera,
Aspergillus spp., Penicillium spp., Alternaria spp., Rhizopus spp., and Yeastwere
isolated from spoiled fruits. Aspergillus spp. had the highest occurrence in all the
fruits tested with a frequency of 40.26% followed by Penicillium spp. (24.67%),
Alternaria spp. (20.78%), Rhizopus spp. (9.1%), and Yeast (5.19%). The results of
this study suggest that fruits are spoiled with variety of fungi which causes great
economic loss of country. In addition, isolation of pathogenic fungi in this study
highlights the risk to human and animal health associated with spoiled fruits.
Therefore improved preservation methods are required to prolong the shelf life of
fruits and minimize the health hazard. * Corresponding Author: Dr. Sham Lal
[email protected]

Article published on January 15, 2020 , Sapna2, Pardeep Kumar1, Javed Ahmed
Ujjan2 , 244 Qureshi et al.Int. J. Biosci.

2020
Introduction

Fruits have got an important role commercially as well as nutritionally. For

balanced diet and keepinggood health, fruits intake is necessary since they
aresource of vitamins and essential minerals. Althoughfruits are easily available
worldwide, the limitingfactor that affects their economic value is short shelf-life
due to bacterial and fungal attack.

Due to containing high amount of sugar, low pH, andideal water activity fruits are
prone to bacterial andfungal spoilage (Droby, 2006; Singh and Sharma,2007). On
fruits, fungi overgrow bacteria, since theyprefer to grow at low pH. Some fungi
can startspoilage of fruits from the fields, while others canonly contaminate and
proliferate after harvestingwhen the plant natural defense system is
reduced(Tournas and Katsoudas, 2005). It has been reportedpreviously that about
20% of the harvested fruits isspoiled even in developed countries (Droby,
2006;Zhu, 2006). In developing countries, a post-harvest loss of fruit is more
severe due to less storagefacilities.

Globally various researchers have reported post-harvest fungalspoilage. For

example, Akhter et al.,(2009) isolated Colletotrichum species fromstrawberry;
Sharma et al., (2013) isolated 11 fungalspecies from local fruits; El-Gali, (2016)
isolated 12fungal species of 8 genera from various fruits;Mailafiaet al., (2017)
isolated 7 fungal isolates fromvariety of fruits; Biyik (2018) isolated 11
differentfungal species from strawberries.

Fungal spoilage is responsible for organolepticchanges including change in texture

and flavors offruit. In addition, some moldshave been reported toproduce
hazardous metabolic products such aspatulin, byssotoxin A, malformins,
naphthopyronesand related mycotoxins (Frisvad and Samson, 1991;
Pitt and Hocking, 1997; Beuchat and Pitt, 2001;Tournas and Stack, 2001), which
poses threat tohuman and animal health. The use of syntheticfungicides mayreduce
the fungal spoilage; however,use of same fungicides for long time could lead to245
Qureshi et al.resistance among fungi against fungicides (Spotts andCervantes,
1986). Moreover, high residue ofpesticides in fruits limits the use of pesticides.

There is limited published data on fungi which causethe post-harvest diseases in

local fruits. Therefore,present study was conducted to study fungalpathogens
responsible for post harvesting spoilage ofsome fruits available locally.

1.1 Statement of the problem

Fungi are known to destroy fruits, thereby reducing the quantity for consumption
and the profits obtained from sales of fruits. There is need to identify this fungi
especially those that are pathogenic to humans so as to reduce the risk of
contamination and infection arising from handling and consumption of fruits.

1.2 justification of the study

Fungi have been identified with the major cause of spoilage in fruits due to their
high water content, proper isolation in Fruit will greatly reduce the spoilage of
those perishable fruit and as such producers and consumers will be able to protect
their Fruit and also identify spoiled Fruit that have been attacked by fungi.

There is little or no work has been done on the isolation and identification of fungi
on spoiled Fruits in the University of Maiduguri Commercial Area.Therefore, the
major Fungi responsible for spoiled of fruit will be majorly identified and checked.

1.3 Scope of the study

With the increase in the number of the spoiled fruits in the university of Maiduguri
Commercial Area, this Further, the study isolate and identify Fungi associated with
the spoiled of fruits and some health factors of those fruit while consumed.

1.4 significant of the study

The result of this research will be significance to scientific communities by

providing evidence based information on the important of isolating spoil fruits.
Also, it will provide guide to to identify Fungi or type of Fungi responsible for
spoiled fruits.

1.5 Aims and Objective of the study

This research work is aimed to Isolate the various fungi associated with the
spoilage of fruits in University of Maiduguri Commercial Areaand to determine the
effects of the isolated fungi on the spoiled fruits.

Objectives

To isolate of fungi from spoilt fruits in University of Maiduguri Commercial Area.

To characterize and identify of the fungi

3.0 Material and methods

3.1 MATERIALS

3.1.1 Equipment

Petri dishes, Inoculate needle, burner, conical flasks, beakers, test tubes, filter
papers, culture plate with suitable nutrition media, retort stand, centrifuge,
incubator, microscope.

3.1.2 Reagent

Distilled water, Potato Dextrose Agar (PDA), 1% sodium hypochlorite, antibiotics

(streptomycin).

3.2 Sample collection

A total of 9 fruits samples showing rot symptoms comprising 3 samples of three

different types of fruits such orange (Citrus sinensis), papaya (Carica papaya),
Water melon (Citrullus lanatus) will be collected aseptically in sterile polyethene
bag from University of Maiduguri Commercial Area. All samples will then deliver
to Biological Research Laboratory for fungal analysis.

3.3 Sterilization of Apparatus

Sterilization apparatus involved in this experiment will be decontaminated

appropriately. Materials such as agar will be aseptically sterilized in an autoclave
at 1210C for 15 minutes, wire loop will also sterilized in flame until red hot and
allow to cool before using and1% Sodium hypochlorite will be used to wipe the
top work place to prevent contamination (Uker and Chiejina, 2012).

3.4 Preparation of culture media

Preparation of culture media Potato Dextrose Agar (P.D.A) to be used for culturing
fungi will be prepared according to the manufacturers’ instruction and autoclave at
121°C for 15 minutes.

3.5 Isolation of fungal species

The surface sterilization of sampled fruits will be achieved with 1% Sodium

hypochlorite (for 3min) followed by five times wash in sterile distilled water. The
fruit samples will then cut into small (approximately 3mm) pieces and inoculated
onto sterilized potato dextrose agar (PDA) plates in three replicates amended with
antibiotics (streptomycin). The plates will then be kept in incubator at 37°C at
room temperature for five days. After incubation, plates will be observed for
fungal growth. The pure cultures will be prepared for each discrete colony by
taking small portion of fungal hyphae with sterilized mycological needle and stab
inoculation on fresh PDA plate. Incase of yeast like colonies, the pure culture will
be prepared by inoculating culture on fresh PDA plate by streaking. Plates will be
incubated as mentioned earlier (Anwer et al., 2017).

3.6 Identification of isolated fungi

The molds will be identified by observing cultural characteristics (colony growth

pattern, colony color, presence of aerial mycelium, wrinkles and furrows).

The wet mount technique by using lactophenol cotton blue will be performed as
suggested by Oyeleke and Manga, (2008). Concisely, lactophenol cotton blue (one
drop) will be placed on the clean glass slide, where a small portion of mycelia of
fungal culture will be placed with the help of mycological needle. A cover-slip will
be placed over the specimen by applying a little pressure.
The slide will be observed on 10x and 40x objective lenses of microscope for mold
side identifications in accordance with Samson and Varga, (2007), Adebayo-Tayo
et al., (2012), Tafinta et al., (2013) and Samuel et al.,(2015).

Bacteria like colonies were identified as

Table 1.Prevalence of fungal isolates on the spoiled fruits.

No

Fungi

No. of isolates

yeast by performing simple staining method as

suggested by Anwer et al.,

(2017).

Statistical analysis

All experiments were performed in triplicates.

Prevalence of each fungal isolate was calculated in

percentage.

Results and discussion

Fungi responsible for spoilage of fruits after

harvesting were studied. Seventy seven fungal isolates

belonging to five differentgenera were isolated from

various fruits collected from University of Maiduguri Commercial Area

Occurrence (%)

Fruit sample

1.

2.

3.

4.

5.

Aspergillus spp.
Penicillium spp.

Alternaria spp.

Rhizopus spp.

31 it

19

16

Yeast

07

04

The fungi identified in this study were Aspergillus

spp., Penicillium spp., Alternaria spp., Rhizopus spp.,

and Yeast (Fig. 1.). The fungal isolates associated with

spoiledfruitsand their frequency of occurrence in the

various fruits analyzed is shown in Table1. Control

PDA plates did not show growth.

Aspergillus spp. was found to be more frequent

prevalent (40.26%) and was isolated from each fruit

sample tested followed by Penicillium spp. (24.67%)

in strawberry, orange and papaya; Alternaria spp.

(20.78%) in papaya, melon, apple and guava,

Rhizopus spp. (9.1%) in strawberry, papaya and

melon, and Yeast(5.19%) in strawberry, melon and

apple (Table 1). Similar results have been obtained by

other researchers, for example, Aspergillusniger has

been reported to be most frequent fungal isolate in

tomato (Ibrahim et al., 2011; Mailafia et al.,

246 Qureshi et al.

40.26

24.67

20.78

9.1

5.19

+
+

+
+

2017).Kalyoncu, (2005) found Alternariaalternata in

spoiled fruit. Ghosh, (2009) and Akinmusire,(2011)

have reported the presence of Rhizopus spp. in fruit.

The study conducted by Ayoola, (2007); Chukwukaet

al., (2010); Wogu and Ofuase,(2014); Mbajiuka and

Enya, (2014) and Mailafia et al., (2017) has revealed

the presence of Aspegillus spp., Penicillum spp., and

Yeast in spoiled fruits.

In various countries fungi are among most commonly

occurring organisms on fruits and their prevalence

have been recorded by various researchers (Booth,

1976; Amadi and Oso, 1996; Bukar, 2009; Akhteret

al., 2009; Oyetunji et al., 2012; Sharma et al., 2013;

El-Gali, (2016); Mailafia et al., (2017).

Generally, there could be different factors responsible

for contamination of agricultural products such as

Strawberry

Orange

Papaya

Melon

Apple

GuavaInt. J. Biosci.

2020

pre-harvesting invasion in field, improper handling of

fruits during harvesting, poor sanitation, careless

packaging and delivery of fruits to the market.

Therefore, efforts should be made to minimize

wounding of fruits. This would prolong the shelf life

of fruits and make them available for more time at

least in season. In addition, care must be taken for

field sanitation to reduce the load of contaminant

microbes carried from the field to post harvest stage.

In this study, strawberries were found to be spoiled

with all the fungi isolated.

This could be because of their soft peel which could

make them prone to physical damage and easy

penetration by fungi. Other fruits showed growth of

some fungi and absence of other fungi. This could be

due to difference in sugar content, pH and water

content of fruits analyzed in this study.

Fig. 1. Microscopic features of isolated fungi, A-C)Aspergillus spp., D)Penicillium

spp., E-F)Alternaria spp.,

G-H) Rhizopus spp. and I) Yeast.

Since fruits spoiled with fungi can infect other fruits,

it is very important to discard that fruit immediately

to avoid the loss of other fruits. In addition, washing

of fruits could reduce the microbial load however,

after washing if fruits are not consumed they should

be properly dried otherwise presence of moisture on

fruits could encourage the growth of fungi. Some of

247 Qureshi et al.

the fungal genera isolated in this study such as

Aspergillus, Penicillium and Alternariaare

pathogenic and produce mycotoxins. These fungi are

able to grow on fruits at room temperature and pose a

risk to human and animal health. Fungal spoilage of

fruits is minimizing hygiene of fruits and marketable

quality, causing major economic loss in Pakistan,Int. J. Biosci.

2020

therefore, strategies need to be devised for

minimizing spoilage of fruits.

Conclusion

Fruits have high economic and nutritional value.

Their spoilage is not only hazardous to health but an

economic loss of country.Proper handling of fruits

from pre-harvest to post-harvest is required to avoid

spoilage of fruits with fungi.

The use of modern preservation methods such as

radiation, vacuum packing, pasteurization, high-

pressure food preservation and bio preservation

isrecommendedto prevent
andimproveshelf life of fruits.

fungal spoilage

Acknowledgements

“The first authorwants to acknowledgeShah Abdul

Latif University, Khairpur, Sindh (Pakistan), for

providing laboratory facilities used for this research.”

Conflict of interest

The authors declare that they have no conflict of

interest.”

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