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. Adebisi

3, Alafe H. Azeez

4, Oladimeji DT

1

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 370C and 250C 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.

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 121oC. 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 121oC 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 55oC 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 37oC and 25

oC 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

Sci. Agri. 13 (1), 2016: 19-22

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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

Per

cen

tage

of

pre

vale

nce

%

Sci. Agri. 13 (1), 2016: 19-22

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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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