ISOLATION AND CHARACTERIZATION OF FUNGI ASSOCIATED

WITH SPOILED TOMATOES

INTRODUCTION
Vegetables constitute commercially and nutritionally important indispensable
food commodity. Vegetable naturally play a vital role in human nutrition by
supplying the necessary growth factors such as vitamins and essential minerals
in human daily diet and that can help to keep a good and normal health.
Vegetables are widely distributed in nature. One of the limiting factors that
influence the fruit economic value is the relatively short shelf-life period caused
by pathogen’s attack (Droby, 2006: Zhu, 2006).
Tomato (Lycopersiconesculentum) pronounced Toh-MAH-to is the most
important vegetable worldwide. Tomato is an annual plant which belongs to
the Solanaceaefamily which includes other well-known species such as Potato,
Tobacco, Pepper and Egg plants (aubergine) and can reach a height of over two
meters. It’s a berry fruit, tomato is grown mainly in soil (Frazier and Westhoff,
2004).Tomato is essential mainly for its dietary needs and can be consumed in
diverse ways. It can be cooked as vegetable, as an ingredient in many dishes
and sauces; in the making of stew, fruit juices and can be eaten raw in salads
(Masefield et al., 2002).
Tomato in West Africa is grown in gardens and irrigation schemes. In
Nigeria, most tomatoes are grown in the northern parts of the country (Erinle,
2007) and there is no record of any systematic or organized traditional storage
method for vegetables and fruits. They are usually sold immediately after
harvesting. They are packed in baskets, cardboard boxes, or wooden crates
ready for transportation to the markets. Tomato is the most perishable
vegetable during handling, transportation and storage. This is because tomato
contains large amount of water which makes them susceptible to spoilage by
the action of microorganisms such as fungi, bacteria and protozoan’s (Ross,
1975). Estimates have shown that about one third of the produce is lost before
reaching the consumer (Erinle, 2007), this loss has been attributed to a number
of factors which include physical (mechanical breakage, bruises), physiological
and also damages caused by pathological agents (Thompson and Kelly, 2000),
market value of the tomato are mainly reduced by this factors.
Watt and Merrill, (2000) defined spoiled food as that which have been
damaged or injured so as to render it undesirable for human consumption.
Various activities may result in food spoilage: insect activities, causing physical
injuries for example, bruising and enzyme activity by microorganism causes
change in colour, taste, smell, texture and quality of the food.The spoilage of
food by microorganisms should not be viewed as a sinister plot on the part of
the microbes deliberately to destroy foods but as a normal function of these
organisms in the total ecology of all living organisms (Watt and Merrill, 2000).
It has also been estimated by Watt and Merrill (2000) that 20% of all fruits and
vegetables harvested for human consumption are lost through microbial
spoilage causing one or more of 250 market diseases. Spoilage of fresh tomato
usually occurs during storage and transit and also while waiting to be
processed.

AIM AND OBJECTIVES

This research work is aimed at identifying the various fungi organisms
associated with the spoilage of fresh tomatoes in Nigeria with the view of
mapping out strategies to curtail these fungal attacks and also to determine the
effects of the isolated fungi on the spoiled tomatoes.

Objectives
To isolate fungi organism from spoilt tomatoes in Nigeria.
To characterize and identify the fungi in spoilt tomatoes.
To determine the prevalence and pathogenicity of fungal organisms.
To create public health awareness about the preponderance of fungal organisms.

LIMITATION OF STUDY
Researches have been conducted to ascertain the fact that various
microorganisms are responsible for the spoilage of tomatoes. Organisms include
Bacteria, for example, Pseudomonas and Xanthomonasspeciesas stated by Watt
and Merrill (2000), Fungi, for example, Alternaria, Colletotrichum,
Fusariumand Penicillium species as stated by Barksdale (2001) and other
Meioidogyne groups of nematode.
MATERIALS AND METHODS

Samples Collection
Tomato samples were purchased from the market in. They were
transported to the biochemistry laboratory in sterile polythene bags for
fungal isolation. The samples were left for one week for spoilage to occur.
The spoilt tomatoes were used for the study.

Materials Sterilization
All the glass wares were properly washed, dried and sterilized in the
oven at 160℃ for one hour. The entire working surfaces were also
disinfected with ethanol to reduce contaminants.

Samples Processing
One gram of each of the spoilt tomatoes was carefully cut with the aid of
a sterile scalpel and enriched in sterile sabouraud dextrose broth for twenty four
hours. Ten fold serial dilutions of the samples were thereafter carried out.

Isolation of Fungi
The pour plate method was used. One milliliter of the serially-diluted
sample (103) was dispensed into a conical flask containing sterile sabouraud
dextrose agar (SDA) and two percent chloramphenicol to inhibit bacterial
growth. The contents were properly mixed and dispensed aseptically into sterile
petri-dishes. Incubation was carried out in an inverted position at 280C for five
days. The colonies that developed were counted and subcultured repeatedly on
sabouraud dextrose agar plates to obtain pure cultures. They were later stored
on SDA slants for characterization and identification.

Characterization and Identification of the Isolates

The pure cultures of the moulds were identified on the basis of their
colony growth pattern, conidial morphology and pigmentation using the slide
culture technique and microscopic examination. The yeasts were characterized
and identified using the Gram stain, Chlamydospores formation, Germ tube,
sugar assimilation and motility tests. The identity of each fungus was confirmed
with the aid of a mycological atlas.

Pathogenicity Test of the Isolates

The procedures of Chukwuka et al, Baiyewu et al and Onuorah et al were
used. Fifteen healthy tomatoes were properly washed with tap water, rinsed
with distilled water and surface-disinfected with ethanol. Sterile cork borers
were used to bore holes in each of the tomato fruits. Each of the isolated fungi
was thereafter inoculated into the fruits after which the cores of the fruits were
replaced. Sterile petroleum jelly was used to seal the holes of the fruits to
prevent contamination. Fifteen tomato fruits wounded with the cork borers but
were not inoculated with the fungi served as controls.
The inoculated tomato fruits and the controls were placed in sterile
polythene bags (one fruit per bag). Each of the fruits was moistened with wet
balls of absorbent cotton wool to create a humid condition. The fruits were
thereafter incubated at 28℃ for five days and observed for spoilage. The fungi
were re-isolated from the fruits and compared with the original isolates. The
decay rate of each fungus in the healthy fruits was also determined by
measuring its rot diameter after two weeks of its inoculation into the healthy
tomato fruit.

REFERENCES

[1]. Abdel-Mallek, A. Y., Hemida, S.K. and Bagy, M.M. K.(1995). Studies on
fungi associated with tomato fruits and effectiveness of some commercial
fungicides against three pathogens. Mycopathologia 130: 109-116.
[2]. Aran, N. Alperden,I. and Topal,O. (1987). Mould contamination problem in
tomato paste and risk analysis system in the critical control place. Journal of
food Industry 2(3): 43-47.
[3]. Asan, A. and Ekmeki, S. (2002). Contribution to the colonial and
morphological characteristics of some Aspergillus species isolated from soil.
Journal of Faculty of Science 25:121-139
[4]. Beuchart, L.R. (1995). Pathogenic Microorganisms Associated with fresh
produce. Journal of Food Production 50(2): 204-216.
[5]. Bashir, O. B., Habib, U., Odunayo, O.J., Opeyemi, A.S.,Alafe, A.H. and
Owode, T.M.(2006). Microorganisms causing post harvest tomato fruit decay in
Nigeria. Journal of Entomology and Zoology Studies 4(1): 374-377.
[6]. Bihn, E.A. and Gravani, R.B. (2006). Role of good Agricultural practices in
fruit and Vegetable safety. In: Microbiology of fresh Produce (Edited by K.R.
Mathews). ASM press, Washington D.C. 328pp.
[7]. Eni, A.O., Ibokunoluwa, O., Oranusi, U.(2010). Microbial quality of fruits
and vegetables. African Journal of Food Science 4(5): 291-296.
[8]. Gosh, A. (2009). Identification of Microorganism responsible for spoilage
of tomato (Lycopersicum esculentum) fruit. Journal of Phytology 1(6):414-416.
[9]. Ibrahim, A.D.,K., Musa, K., Sani, A., Aliero,A.A., Yusuf, B.S. (2011).
Microorganisms associated with the production of volatile compounds in spoilt
tomatoes. Research in Biotechnology.2: 82-89
[10]. Kimura, S. and Sinha, N. (2008). Tomato (Solanum lycopersicum ): A
model fruit Bearing crop. CSH Protoc. Doi: 10.1101/pdb.emo 105
[11]. Mbajiuka, S.C. and Enya, E. (2004). Isolation of Microrganisms
associated with deterioration of Tomato (Lycopersicum esculentum) and
Pawpaw( Carica papaya) fruits. International journal of Current Microbiology
and Applied Sciences 3:501-512
[12]. Onuorah, S. and Orji, M.U. (2015). Fungi Associated with the Spoilage of
Post-harvest Tomato Fruits Sold in Major Markets in Awka, Nigeria. Universal
Journal of Microbiology Research 3(2): 11-16
[13]. Pitchaon M., Maitree S. and Rungnaphar P.(2007). Assessment of
phenolic content and free radical-scavenging capacity of some Thai indigenous
plants. Food Chemistry 100:1409-1418.
[14]. Rashad, R.A., Ahmed, R.A. and Saleh, A.M. (2011). Isolation and
identification od some fruit spoilage fungi:screening of plant cell wall degrading
enzymes. African Journal of Microbiology 5(4):443-448.
[15]. Rodriquez P.,Soares C.,Kozakiewicz, Z.,Paterson R.R.M., Lima, N. and
Venancio A. (2017). Identification and Characterization of Aspergillus flavus
and toxins. Communicating Current Research and Educational topics and
trends in Applied Microbiology 527- 534.
[16]. Sravanthi, J., Gangadhar, S. R. (2015). Phytochemical and antioxidant
composition in Lycopersicum esculentum. Journal of Medicinal Plants Studies
3(4): 107-110
[17]. Ugwu, C. O., Chukwuezi, F.O. and Ozougwu V. C.O.(2014). Microbial
Agents of tomato spoilage in Onitsha metropolis. Advances in Biological
Research 8(2):87-93.
[18]. Wogu, M.D, Ofuase, O.(2014). Microorganisms responsible for the
spoilage of tomato fruits, Lycopersicum esculentum, sold in markets in Benin
City, southern Nigeria. Scholars Academic Journal of Bioscience 2(7): 459-466
[19]. Wokoma E.C.W.(2008). Preliminary report on disease of tomatoes in
Choba, Rivers State. J. Appl. Sci.Environ. 12(3):178-121.
[20]. Yaltao, T. (1985). Physiology of mature tomatoes and harvest.
Proceedings of the last Congress Raising and Evaluating of Tomatoes
Technology,(CRETT’85). British Journal of Botany 4(3):23-33
[21]. Zain, M.E. (2011). Impact of mycotoxins on humans and animals. Journal
of Saudi Chemical Society 15: 129-144.