This study identified pathogenic microbes associated with the deterioration of sweet orange fruits in Nigeria. Samples were collected from four open markets and tested for microbes. The following microbes were isolated: Aspergillus spp, Staphylococcus spp, Escherichia spp, Rhizopus spp, and Shigella spp. Rhizopus spp and Aspergillus spp were found to be the most active and pathogenic microbes, able to infect both infected and uninfected orange samples. The prevalence of microbes varied between markets, with Oja-Oba market found to have the highest overall microbial load. Proper harvesting and storage techniques are recommended to reduce microbial growth and spo

1. Sci. Agri.
13 (1), 2016: 19-22
© PSCI Publications
Scientia Agriculturae
www.pscipub.com/SA
E-ISSN: 2310-953X / P-ISSN: 2311-0228
DOI: 10.15192/PSCP.SA.2016.13.1.1922
Genotypic pathogenic microbes associated with deterioration
of sweet orange (Citrus sinensis L) fruit in Nigeria
Omolaran B. Bello1*
, Odunayo J. Olawuyi2
, Opeyemi S. Adebisi3
, Alafe H. Azeez4
, Oladimeji DT1
1. Department of Biological Sciences, Fountain University, Osogbo, Osun State, Nigeria
2. Department of Botany, University of Ibadan, Ibadan, Oyo State, Nigeria
3. Department of Crop Protection and Environmental Biology, University of Ibadan, Ibadan, Oyo State, Nigeria
4. Department of Crop, Soil and Pest Management, Federal University of Technology, Akure, Ondo State, Nigeria
*Corresponding author email: obbello2002@yahoo.com
Paper Information A B S T R A C T
Received: 27 October, 2015
Accepted: 4 December, 2015
Published: 20 January, 2016
Citation
Bello OB, Olawuyi OJ, Adebisi OS, Azeez AH,
Oladimeji DT. 2016. Genotypic pathogenic microbes
associated with deterioration of sweet orange (Citrus
sinensis L) fruit in Nigeria, 13 (1), 19-22. Retrieved from
www.pscipub.com (DOI:
10.15192/PSCP.SA.2016.13.1.1922)
This study was carried out in Osogbo township of Osun State, Nigeria to
isolate and determine prevalence and pathogenicity of microorganisms
associated with deterioration of sweet orange fruits. Twenty samples of 20
infected and 20 non-infected sweet oranges (Citrus sinensis L) were
collected from four open markets (Akindeko, Igbonna, Oja-Oba and Sabo
markets) each. The samples were transported immediately to Fountain
University Microbiology Laboratory for pathogenic analysis. The oranges
were rinsed with distilled water and serially diluted in 10 folds. The
highest three dilutions were considered for microbial count analysis. Each
of the orange was cut and the liquid content inoculated on nutrient agar and
potato dextrose agar, incubated at 370
C and 250
C respectively. They were
observed for seven days, and the different colonies isolated using the slide
culture technique. Biochemical analyses of the culture showed that
Apergillus spp, Staphylococcus spp, Escherichia spp, Rhizopus spp and
Shigella spp had the highest load. Pathogens prevalence revealed that
Staphylococcus spp had highest (12.63%) at Sabo, 4.94% at Igbonna and
10.43% at Akindeko. Aspergillus spp with 6.60% and 17.58% loads were
identified at Sabo and Oja-Oba respectively. Rhizopus spp had 21.97% at
Oja-Oba, E. coli, 17.58% at Igbonna and Shigella spp, 8.24% at Akindeko.
Rhizopus spp and Aspergillus spp were the most active microbes with
respective 100% and 90% infections, while the least active microbes were
Staphylococcus spp and Shigella spp. Harvesting fruits at the suitable
periods and stored the harvested orange fruits under controlled conditions
could aid in retarding the microbial growth of post-harvest spoilage of
pathogenic microorganisms.
© 2016 PSCI Publisher All rights reserved.
Key words: Microorganism, pathogenicity, prevalence, infection, Citrus sinensis.
Introduction
Sweet orange (Citrus sinensis L) is one of the most cultivated commercial fruit for household and industrial
consumption globally (FAO, 2004). It is an ever green tree with the height between 0.9m and 10 m (Arora and Kaur, 2013).
Fresh fruits are source of vitamins C and A which form good healthy diet. Besides their delicious taste and flavors, the fruits
contain anti-oxidants that reduced several chronic ailments such as cancer and heart diseases (Madhuri et al., 2014; Olielo,
2014). It is usually taken at breakfast in most of Asian countries and consumed raw for replenishment before and after meal.
Fruit juice, jams, beverages and wines industries utilize sweet orange fruits as raw materials (Olielo, 2014).
Oranges are cultivated in many countries like Spain. China, USA United Arab Emirate, India, Brazil, UK, France,
Germany, Bangladesh, South Africa, Ghana, Benin, Mali and Nigeria (Milind and Dev 2012). Sweet orange is an international
commodity for export to many countries around the world, including United Arab Emirate, India, Brazil, Bangladesh, South
Africa, Ghana, Benin, Mali and Nigeria. With respect drop fruit, either direct contact with animals, their faeces or contact with
surfaces/equipment can cause contamination of fruits. This includes Planting, harvesting, packing, processing and
transporting, handling by food vendors and consumers (Mihajlovic et al 2013). It is recommended that an intake of 400g of
fruit per day would prevent and alleviate micro-nutrient deficiencies in human.

2. Sci. Agri. 13 (1), 2016: 19-22
20
In developing countries including Nigeria, microbial contamination of fruits usually occurs on the fields, due to
contaminated irrigation water, unhygienic fruit handling particularly during harvesting periods (Ali et al., 2011). Food borne
diseases are harmful illness mainly affecting the gastrointestinal tract and are transmitted through consumption of
contaminated food or drink. Fresh fruits borne infections are usually caused by Pseudomonas spp, Vibrio spp., Streptococcus
spp., Staphylococcus spp., Aeromonas spp., Listeria monocytogenes Salmonella spp. and Escherichia coli,. These microbes
contributed immensely the array of diseases causing morbidity and mortality in the developing and developed nations
(Sivapriya et al., 2011; Rahman and Noor 2012; Fadipe et al., 2013).
The occurrence of high microbial counts obtain in orange fruits usually renders them unsuitable for human
consumption, and thus undesirable to the quality mindful markets. Over the years, there has been an increase in the need to
identify and isolate the microorganisms associated with the spoilage as a way of finding a means of controlling it (Akinyele
and Akinkunmi, 2012). In order to determine the magnitude of these problems, this study was therefore conducted with the
objective of identifying pathogenic microbes associated with sweet orange (Citrus sinensis L) decay at four open markets in
Osogbo township of Osun State, Nigeria.
Materials and Methods
Twenty samples of 20 infected and 20 non-infected sweet oranges (Citrus sinensis L) were collected from four open
markets (Akindeko, Igbonna, Oja-Oba and Sabo markets) each. in Osogbo township of Osun State, Nigeria. The samples were
separately packages, labeled and carefully transported immediately to Fountain University Laboratory.
Preparation of culture media
Nutrient agar of 28g powder, make up with 1 litre of deionized water was prepared and allowed to soak for 10
minutes, mixed then sterilized by autoclaving for 15 minutes at 121o
C. It was cooled and poured in the Petri dishes. Also,
potato dextrose agar of 39 g, made up with 1 litre of distilled water was boiled to dissolve medium completely. It was then
sterilized by autoclaving at 121o
C for 15 minutes. The pH was adjusted to 3.5, by adding 10 ml of lactic acid solution, to
facilitate growth of fastidious fungi. The medium was cooled to 55o
C and poured into the Petri dish. The oranges were rinsed
with distilled water and serially diluted in 10 folds. The highest three dilutions were considered for microbial count analysis.
Each of the orange was cut and the liquid content inoculated on nutrient agar and potato dextrose agar, and incubated
respectively at 37o
C and 25o
C and thereafter observed for 24 hours to 7 days. Different colonies obtained were identified using
the slide culture technique. The isolated microbes were characterized the basis of their cultural, morphological and physical
properties. Microscopic examination was carried out after gram staining the bacteria isolates with Lactophenol blue stain. The
isolates were further subjected to biochemical tests viz: catalase, oxidase, coagulase, indole, methyl red-voges proskauer,
carbohydrate fermentation and motility tests.
Table 1. Number of isolates and prevalence of microorganisms obtained from uninfected and infected sweet orange samples collected from
four markets in Osogbo, Osun State, Nigeria
Market Microorganism Number of Isolates of
Uninfected samples
Prevalence
(%)
Number of Isolates of
Infected samples
Prevalence
(%)
Total prevalence
(%)
Sabo Staphylococcus spp 23 12.63 53 15.63 28.62
Apergillus spp 12 6.60 49 14.45 21.05
Oja-Oba Apergillus spp 32 17.58 36 10.61 28.19
Rhizopus spp 40 21.97 38 11.20 33.17
Igbonna Escherichia spp 32 17.58 32 9.43 27.01
Staphylococcus spp 9 4.94 52 15.33 20.27
Akindeko Staphylococcus spp 19 8.24 40 11.79 20.03
Shigella spp 15 10.43 39 11.50 21.93
Total 182 100 339 100
Table 2. Pathogenicity of five isolated microorganisms associated with sweet orange fruits damage collected from four markets in Osogbo,
Osun State, Nigeria
Samples Staphylococcus spp Escherichia spp Shigella spp Apergillus spp Rhizopus spp
1 - + - + +
2 - + - + +
3 - - - + +
4 - - - + +
5 - + - + +
6 - + - + +
7 - - - + +
8 - - - + +
9 - + - + +
10 - - - + +
Keys: - represents not infected, + represents infected

3. Sci. Agri. 13 (1), 2016: 19-22
21
Figure 1. Total percentage prevalence of five pathogenic microorganisms obtained from sweet orange fruit samples collected across four
markets in Osogbo, Osun State, Nigeria
Figure 2. Total percentage prevalence of five isolated microorganisms at four markets in Osogbo, Osun State, Nigeria
Results and Discussion
Five microorganisms isolated from the culture media after biochemical test were Apergillus spp, Staphylococcus
spp, Escherichia spp, Rhizopus spp and Shigella spp (Table 1). Figure 1 shows the relative percentage prevalence of these
pathogens across the four open market sources in which Staphylococcus spp had highest percentage of 28.62 % in Sabo,
followed by Igbonna (20.27%) and Akindeko (20.03%) markets. The fruit samples of Oja-Oba market had no Staphylococcus
spp. However, Oja-Oba market high loads of Apergillus spp (24.18%) and Rhizopus spp (28.19%) followed by Sabo with
Apergillus spp (28.19%). Escherichia spp (27.01%) and Shigella spp (21.93) were identified in Igbonna and Akindeko markets
only. These revealed that each market host at least two pathogenic microbes that affected sweet orange spoilage in the
township.
28
24.18
21.97
17.55
8.24
Staphylococcus spp Aspergillus spp Rhizopus spp Eschericia spp Shigella spp
Percentage prevalence (%)
0
10
20
30
40
Sabo Igbona Oja-oba Akindeko
Percentageofprevalence%

4. Sci. Agri. 13 (1), 2016: 19-22
22
Table 2 shows Rhizopus spp being most active pathogen infected all orange fruit samples including healthy oranges,
followed by Aspergillus spp 90% spoilage. Escherichia spp infected 50% of the samples, while Staphylococcus spp and
Shigella spp. were not isolated. This indicated that Rhizopus spp is most active pathogen that could cause microbial damage to
healthy sweet oranges, if allowed to attack the fruits in any means, and this is followed by Aspergillus spp and Escherichia spp.
Tafinta et al., (2013) also observed similar report for high activities and pathogenicity of Rhizopus spp and Aspergillus spp.
Figure 2 shows total percentage relative population of pathogenic microorganisms recorded at each market where Oja-Oba had
the highest of microbial load (39.55%), followed by Igbonna (22.52%). However, Sabo and Akindeko had relative low isolates
with 19.23% and 18.67% respectively. This indicated that sanitation of both Oja-Oba and Igbonna was very poor compared
with the Sabo and Akindeko markets with appreciable level of sanitation with adequate waste disposal.
Conclusion
Apergillus spp, Staphylococcus spp, Escherichia spp, Rhizopus spp and Shigella spp had the highest load.
Staphylococcus spp had highest isolates in Sabo market and Igbonna markets. Akindeko, Aspergillus spp with 6.60% and
17.58% loads were identified at Sabo and Oja-Oba respectively. Rhizopus spp and Aspergillus spp were the most active
microbes with respective 100% and 90% infections, while the least active microbes were Staphylococcus spp and Shigella spp.
Recommendations
Harvesting orange fruits at the suitable periods and stored the harvested orange fruits under controlled conditions
could aid in retarding the microbial growth of post-harvest spoilage of pathogenic microorganisms. Washing of fruits with
clean water of potable quality could reduce the microbial load, prevent and control the widespread of the pathogens for
consumers’ health safety.
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