Fungal infestation of onion bulbs during storage in sack and on its antioxidant properties

Contents

Abstract

1.0 Introduction
1.1 Background of the Study
1.2 Aim of the Study
1.3 Objectives of the Study

2.0 Literature Review
2.1 Uses of Allium cepa
2.2 Post-harvest losses in onion
2.3 Microorganisms Associated with Onion Spoilage in Nigeria
2.4 Antioxidant effects of A. cepa
2.5 Antimicrobial activity

3.0 Materials and Methods
3.1 Study Area
3.2 Materials used and Sterilization
3.3 Collection of Samples
3.4 Storage of Onion Bulb in Sack Bag
3.5 Preparation and sterilization of media
3.6 Assessment of Fungal Infestation of the Onion bulb during Storage
3.6.1 Preparation of pure cultures of fungal isolates
3.7 Identification of fungal isolates
3.8 Determination of Frequency of Occurrence of Fungal Isolates
3.9 Preparation of Onion Extract
3.10 Determination of antioxidant Properties of Onion
3.10.1 DPPH Free radical scavenging assay
3.10.2 Measurement of reducing power of the crude extracts of Onion Bulb
3.10.3 Determination of antioxidant activity of crude extracts of the Onion
3.11 Statistical Analysis

4.0 Results
4.1 Fungal Infestation of Onion Bulbs during Storage in Sack
4.2 Frequency and Percentages of Fungal Infestation of Onion Bulbs
4.3 DPPH radical scavenging activity of Fungal Infested Onion Bulbs during Storage in Sack

5.0 Discussions and Conclusion
5.1 Discussions
5.2 Conclusion
5.3 Recommendation

References

ABSTRACT

The aim of this study is to determine fungal infestation of onion bulbs during storage in sack and on its antioxidant properties. The fungal infestation of onion bulbs during storage in sack shows that five (5) fungi each were isolated from the onion bulbs during storage in sack at day zero (0) and day seven (7) respectively while at day fourteen (14), six fungal infestation was isolated from the onion bulbs during storage in sack. A total of sixteen (16) fungi infested the onion bulbs during storage in sack. The fungi belong to four (5) genera and six (6) species of fungi infested the onion bulbs during storage in sack. Aspersillus niger and Aspersillus flavus were the most frequently isolated fungi with 31.25% each followed by Fusarium oxysporum and Aspergillus fumigatus with 12.50% each while Penicillium chrysogenum and Rhizopus stolonifer recorded 6.25% each. The antioxidant analysis of onion extract after storage in sack for 14 days recorded at all concentrations (0.5, 0.25, 0.125, 0.0625 and 0.03125 mg/ml), show that the antioxidant property of the onion extract at day zero (0) and day 7 was higher than that 14. The inhibition of free radical DPPH at day zero (0) and 7 were 29.92 %, 49.21%, 69.29%, 70.07% and 47.63% while at day 14, the percentage (%) of inhibition of free radical DPPH were 29.33 %, 49.02%, 69.01%, 69.77% and 47.21% .

1.0 INTRODUCTION

1.1 Background of the Study

The onion (Allium cepa L .) is an important vegetable crop in Nigeria based on consumption and economic value to farmers. The crop is grown for its bulbs which are used daily in every home for seasoning and flavouring of foods. Onion is a valuable ingredient in the diet due to its content of sugars, vitamins and minerals (Ole et al., 2014). The crop is grown mainly in the north, during the dry season (October to April). The onion farmers in Nigeria almost always store, their onions after harvest for one to five months to ensure a continual supply through seasons when fresh produce is unavailable. Bulb rots are a common cause of onion loss during storage.

Fungi, especially moulds are important pathogens of fruits and a vegetables particularly under tropical and sub-tropical conditions (Adebayo and Diyaolu, 2013). The importance of storage rots includes reduction in the quantity and quality of onion which affects the market value (Dogondaji et al., 2015). Other important consequence often overlooked, is mycotoxin contamination of the affected material (Muhammad et al., 2014).

Biological effects attributed to onions have been commonly ascribed to the volatile sulfur-containing compounds, such as thiosulfinates, mainly responsible for the characteristic taste, aroma and lachrymatory effects (Krest et al., 2012). These compounds are formed from cysteine sulfoxide precursors and the effect of the enzyme alliinase which is released from cell vacuoles when tissues are damaged (Ioku et al., 2011). However, these volatile products are highly unstable and recently, attention has been focused on the effects of phenolic compounds, such as flavonoids, which are more stable (Fossen et al., 2011). Onion is known for being a good natural source of flavonoids mainly represented by the flavonols - quercetin and kaempferol, which are present as their glycosides.

About 15 different fungal species and 5 bacterial species are found responsible for the onion diseases in the storage and transit all over the world. The loss due to these diseases is considerable and may go up to 40% (Dimka and Onuegbu, 2010). In storage various diseases destroy the onions such as Black mould rot (Aspergillus niger), Blue mould rot (Penicillium spp.), Fusarium bulb rot (Fusarium spp.), Basal rot (Fusarium moniliforme), Aspergillus rot (Aspergillus spp.), Dry rot (Macrophomina phasiolina), Soft rot (Erwinia spp.), Smudge (Colletotrichum circinans), Grey neck rot (Botrytis allii), Green mold rot (Penicillium spp.),White rot (Sclerotium cepivorum) and Anthracnose (Colletotrichum chardonianum) (Dimka and Onuegbu, 2010). Among these, black mould rot (A. niger) is more severe in storage. A. niger and A. flavus infect onion at high temperature and high relative humidity. Whereas Penicillium spp. destroys onion at low temperature. Sometimes Penicillium spp. produces mycotoxin, Penitrem A, which has been previously implicated in tremorgenictoxicosis (Adebayo and Diyaolu, 2013). It is reported that the predominant fungal pathogens associated with the storage diseases of onions were Aspergillus sp., Penicillium sp. and Fusarium sp. (Adebayo and Diyaolu, 2013).

1.2 Aim of the Study

The aim of this study is to determine fungal infestation of onion bulbs during storage in sack and on its antioxidant properties.

1.3 Objectives of the Study

The specific objectives of the study are to:

i. Assess fungal infestation of onion bulb during storage in sack
ii. Identify the isolated fungi from onion bulbs stored in sack
iii. Determine the antioxidant properties of the onion bulbs stored in sack

2.0 LITERATURE REVIEW

2.1 Uses of Allium cepa

Allium cepa has been traditionally used for its remedial characteristics in the management of various ailments. The essence of A. cepa proliferated into ancient Greece where it was used as a blood purifier for athletes. During the invasion of Rome, gladiators used to rub down onion juice to firm up the muscles. The Greek and Phoenicians sailors consumed it to prevent scurvy. Moreover, the Greek physician Hippocrates, used to prescribe onion as a wound healer, diuretic and pneumonia fighters. In the 6th century, onion was described as one of the indispensable vegetable or spice and medicine (Kabrah 2010). A. cepa was most regularly used in low developed countries. This could be probably due to the lack of medical facilities and the easy availability of traditional remedies including onion. A. cepa is commonly taken raw or as a decoction for treating infectious diseases. It is also used in a wide variety of preparations for internal and external use to relieve several ailments including digestive problems, skin diseases, metabolic disease, insect bites and others (Silambarasan and Ayyanar 2015).

2.2 Post-harvest losses in onion

Storage is one of the important aspects for post-harvest handling of onion. The storage condition extends the period of availability of fresh onion by arresting the metabolic breakdown and decay. It is achieved by controlling the Relative humidity and Temperature. The storage life of onion is depending on different parameter such as Physiological activity, Biochemical activity and Microbial invasion. Inadequate and improper field curing after harvest, infection by different pathogen, sprouting and also poor storage methods being practiced by the farmers are the main reasons of prevailing losses. In general, the losses due to reduction in weight, sprouting and rotting (decay) were found to be 20-25, 4-5, and 10-12 % respectively (Jaradat et al. 2016). Currently about 35- 40 %of the onion is estimated to be lost as postharvest losses during various post- harvest operations including handling and storage (Akash et al., 2014). Onion suffers from many diseases from pre harvest to post harvest period. The survey conducted at the international level revealed that about 35-40% onion is lost due to damage caused by different diseases (Gupta et al., 2010). A number of microorganisms are responsible for bulb rotting of onion, but among them, fungi are the main causal agent responsible for pre and post-harvest period losses in the onion (Hayta et al. 2014). It makes it prone to the development of various fungal pathogens of different genera and species and in turn leading to the damage by causing rot during the storage. Identification of pathogens which causes diseases in onion is essential for effective inhibition of target pathogens. Various species of Aspergillus pathogens are reported to cause blue mould on onion bulb during storage. The blue moulds are frequently isolated from stored diseased bulbs of local cultivars of onion (Sharma et al. 2014). Aspergillus niger is able to produce mycotoxin which reduces the quality and quantity of food products and feed-stuff which is a potent hepatic- carcinogen in humans and animals (Irkin and Korukluoglu, 2017). The fungus causing black mold is the main member of Aspergillus and is predominantly a plant pathogen responsible for post-harvest deterioration of stored food materials (Kocic-Tanackov et al., 2019).It is responsible for the deterioration of agricultural product during pre and post-harvest stages. It affects the availability of onion to domestic and international trade. The infestation of fungi causes spoilage and ultimately decreases the qualitative attributes and quantity of food (Liguori et al., 2017).Being saprophytic and filamentous in morphology Aspergillus niger resides and perpetuates in soil, forage, organic debris and food products causing black mold disease during post-harvest stage of onion bulbs (Kocic-Tanackov et al., 2019). The most favourable temperature conditions for the growth of the fungus is 28°C-34°C followed by the warm and moist conditions eliciting infection (Liguori et al., 2017). The contamination of pathogens begins at germination stage and remains up till storage period (Ferreres et al., 2010). The pathogen transmission is by infected soil or seed and the infected bulbs shows neck discoloration along with black coloured mycelia and the hiding spores in the outer dry scales (Corzo-Mart et al., 2017).Chemical treatment is found best to inhibit Black mold and other fungal pathogens disease in the onion bulbs (Begum and Yassen, 2015).

2.3 Microorganisms Associated with Onion Spoilage in Nigeria

Onions are associated with micro-organisms which are capable of causing spoilage. This spoilage usually occurs during harvesting, post-harvesting, transportation, marketing and storage. In tropical countries the storage is in ambient temperature (24-32oc) and at a variable relative humidity depending on location and season.

Bulb-rot is a common cause of onion loss or spoilage during storage. They are caused by microorganisms particularly fungi the black mould disease caused by Aspergillus niger is a limiting factor in onion production worldwide. Aspergillus niger has been reported to survive between onion crops as a soil saprophyte in or on bulbs in field or storage and is ubiquitous in nature. Onions are prone to spoilage by fungi during harvesting, handing storing and marketing processes.

Adebayo and Diyaolu (2013) and Gashua et al. (2014) opined that fungi, especially moulds are important pathogens of fruits and vegetables particularly under tropical and sub-tropical condition. Gent and Mohan (2016) estimated that bulb rot account for 10%-15% of storage losses of different varieties during three-month storage period under local condition. Five fungal belonging to different genera are found to be responsible for the spoilage of onion bulbs. These genera include Penicilum spp., Aspergillus flavus, Fusarium spp, Aspergillus niger and Mucor spp. Among the fungi isolated from rotten bulbs, Fusarium species are responsible. Aspergillus spp grow on the surface of onion bulbs but did not cause rotting when inoculated artificially. Generally, spoilage fungi are known to toxigenic or pathogenic under favorable conditions (Adebayo et al., 2012). Al-Hindi et al. (2011) isolated toxigenic fungi from spoiling fruit, pathogenic fungi are cable of causing infections. Aspergillus spp are known to produce several toxic metabolites.

During refrigeration some moulds may also produce mycotoxins. Bulb rottening by storage losses of onions in Nigeria as well as other countries in the world are well documented by Adamick (2014).

Botrytis Neck rot is also a microorganisms capable of causing spoilage in onion his is cause by botrytis alli, a fungus that over winter on plant debris in soil, on infected bulbs, and as sclerotia in soil. Economic effect on the economy of the onion bulbs farmers in the particular and the economy of the country in general. The control of the fungal spoilage of onion bulbs is therefore inevitable. Proper storage conditions, careful harvesting, protection of the bulbs from sunburn, provision of adequate ventilation and regular examination during storage will minimize the entry and proliferation of these organisms in the onion bulbs, thereby reducing the incidence of diseases cause by fungi and also improve the economy by reducing waste resulting from spoilage. Bacteria are of only minor important in the market spoilage of most vegetables e.g onions because of the acid PH value. The soft rot coliform bacteria, Erwinia carotovara and Pseudomonas similar to Pseudomonas marginatia are responsible for most soft rot of onions during transportation or in storage. These micro-organisms develop on onions in the field before harvesting after heavily rains and when leaves are drying. The main source of inoculums is contaminated soil and crop Residues. The bacteria are spread by splashing rain, irrigation water, and insects. Which enter into the bulbs only through wounds such as those cause by transplanting, mechanical injuries or sunscald, it is said that the spoilage of onions occur during storage because Pseudomonas aeroginosa contaminates onion bulbs during harvest by moving through wounds caused by topping, finally causing soft rot. The pathogen can also be seed borne. Botrytis neck rot is caused by a different pathogen from Botrytis leaf spot. This is seen primarily in onion, the spoilage occur more apparently after harvest, while bulbs are in storage. At first the soft neck tissues looks water soaked, and a yellow discoloration moves down into the scales. Bulbs break down into soft mass. A gray mold develops between the onion scales, later producing small to large black bodies (sclerotia) which develop as solid layer around the neck (Carisse et al., 2015).

Spoilage microorganism can also cause spoilage by entering plant tissue during development either through the calyx along the stem, or through various specialized water and gas exchange structures of leafy matter. Blue mold of onion spoilage this is caused by several Penicillium species. These fungi, attack a wide range of vegetables, bulbs, and seeds they are common in the soil growing in infected animal and plant debris. These organisms develop on lesions when bulbs are cut open, one or more of the fleshy scales may be discolored and water soaked. These microorganisms are responsible for poor plant stand in the field and storage delay. The presence of fungi in onions bulbs is also attributed to the environmental conditions, state of handling and processing, state of storage facility and the onions the fungal load of the handlers and the quality of the onion bulbs. These fungi have been known to cause disease of humans and animals.

2.4 Antioxidant effects of A. cepa

Oxidative stress is characterised by over production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) (Abdel-salam et al. 2014). These free radicals, mainly nitric oxide, superoxide anion, hydroxyl radical and hydrogen peroxide, can cause oxidative damages to nucleic acids, proteins, and lipids. Thus, excess production of free radicals under pro-inflammatory conditions may initiate various diseases (Alpsoy et al., 2013). Natural antioxidants are compounds that can delay or inhibit oxidative reactions by scavenging free radicals. The most important of these compounds are phenolic acids, polyphenols, flavonoids, alkaloids and terpenoids (Brewster, 2018). Therefore, suppression of oxidative stress could be achieved by using potential sources of natural antioxidants such as medicinal plants (Celik, 2012). Essential oils derived from these plants, are rich sources of antioxidant components with different biological activities (Colina-Coca et al., 2017). A. cepa contains high levels of phenolic compounds mainly flavonoids, which have antioxidant properties besides other pharmacological effects such as antibiotic, antidiabetic, anti-atherogenic and anticancer activities (Liguori et al. 2017). Flavones, flavanones, flavonols, isoflavones, flavanonols, chalcones, and anthocyanins which are subclasses of flavonoids and flavonols, are the most abundant flavonoids in A. cepa (Liguori et al. 2017). Several studies reported the antioxidant activities of A. cepa and its constituents and introduced the plant as a potential source of natural antioxidants (Razavi et al., 2016). Alterations in oxidant/antioxidant markers including lipid peroxidation (LPO), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and MDA were observed by studies that investigated the effects of A. cepa and its constituents (Liguori et al., 2017).

2.5 Antimicrobial activity

Allium cepa has been described as a potent antimicrobial agent to fight against infectious diseases. Many bacteria, fungi, and viruses were found to be susceptible to different solvents extracts of A. cepa. Sulphur compounds have proven to be the principal active antimicrobial agent present in onion (Rose et al. 2015). Many studies (Liguori et al. 2017; Thomas and Parkin, 2010; Vazquez-Armenta et al., 2014) have reconsidered the effect of organo-sulphur containing compounds on the growth of microorganisms. A. cepa also possesses other antimicrobial phenolic compounds including protocatechuic, p-coumaric, ferulic acids, and catechol. Quercetin and kaempferol have been found as significant contributors to this activity. The effectiveness of kaempferol was greater than quercetin in inhibiting bacterial growth of B. cereus, L. monocytogenes, and P. aeruginosa and was as effective as quercetin in inhibiting the growth of S. aureus and M. luteus (Santas et al. (2010). Other studies also showed that quercetin oxidation products from yellow onion skin such as 2-(3,4-dihydroxyphenyl)-4,6-dihydroxy2-methoxybenzofuran-3-one demonstrated selective activity against Helicobacter pylori strains while 3-(quercetin-8-yl)- 2,3-epoxyflavanone showed antibacterial activity against both multi-drug resistant Staphylococcus aureus and H. pylori strains (Ramos et al. 2016). Benkeblia (2014) observed that essential oil of three types of onion (yellow, green and, red) displayed marked antimicrobial activity against specific pathogens, including Staphylococcus aureus, Salmonella enteritidis, Aspergillus niger, Penicillium cyclopium, and Fusarium oxysporum (Benkeblia 2014). Several researchers such as Begum and Yassen 2015; Hamza 2015; Palaksha et al. 2013 and Zohri et al. (2010) have studied the activity of onion extracts on the Gram-negative bacteria Klebsiella spp. However, contradicting results were obtained from Srinivasan et al. (2011) and Gomaa (2017) whereby there was no inhibition of K. pneumonia with onion extracts. The antibacterial activity of the red variety of A. cepa extract was found to be higher compared to yellow and white varieties (Sharma et al. 2017). Interestingly, Azu et al. (2017) found that A. cepa was effective against P. aeruginosa isolated from patients suffering from urinary tract infections indicating its potential in the management of such condition. In vivo study of Ur Rahman et al. (2017) showed that birds fed with onion at a rate of 2.5 g/kg of feed had a decrease of E. coli population and a significant increase of Lactobacillus spp. Interestingly, a recent study conducted by Lekshmi et al. (2012) showed how nanoparticles synthesized from onion displayed a positive effect in inhibiting Klebsiella spp. Saxena et al. (2010) also reported the synthesis of silver nanoparticles by using onion extract and demonstrated that these nanoparticles, at a concentration of 50 lg/mL, presented a complete antibacterial activity against E. coli and Salmonella typhimurium. Onion extracts are potent against fungal species, and its essential oil inhibits the dermatophyte fungi (Zohri et al. 2010). Aspergillus niger and Fusarium oxysporum were strongly inhibited (minimum fungicidal concentration (MFC) ¼ 75 and 100 mg/mL, respectively) by the ethyl alcohol extract of dehydrated onion (Irkin and Korukluoglu 2017; Irkin and Korukluoglu 2019). Anti-fungal saponins (ceposide A and C) discovered by Lanzotti et al. (2012) were able to inhibit the growth of soil-borne pathogens (R. solani), air-borne pathogens (A. alternata, B. cenerea, Mucor spp and Phomopsis spp) and antagonistic fungi (T. atroviride and T. harzianum). High inhibitory effect against M. furfur (minimum inhibitory concentration (MIC) ¼ 8.062 mg/ml) and C. albicans (MIC ¼4.522 mg/ml) were reported by Shams-Ghahfarokhi et al. (2016). Kocic-Tanackov et al. (2019) stated that essential oil of A. cepa, at a concentration of 7%¸ had complete inhibition on the growth of two yeasts (C. tropicalis and S. cerevisiae) and this was also confirmed by the study of Kivanc and Kunduhoglu (2010). High concentration of the essential oil also weakened the growth of molds (A. tamarii and P. griseofulvum) as well and complete inhibition was observed for E. astelodami. Goren et al. (2012) conducted a clinical experiment to find out if dehydrated A. cepa could be used in the treatment of AIDS. Eight persons (from 28 to 30 years old) who were HIV positive started a dietary regimen comprising of 9–13 g/day of A. cepa extract. After the treatment, all the HIV positive patients experienced a total remission of clinical symptoms associated with AIDS and were able to resume their healthy lifestyle.

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