Aspergillus niger had the highest occurrence in pineapple, water melon, oranges, pawpaw, tomatoes with a frequency of 38%.The most common fungi that causes apple rot is penicillium expansum and Monillinia fructigena.
Fusarium aveanaceum followed with the occurrence of 31% in fruits such as pineapple, watermelon, oranges, and tomato.
While, penincillium digitatum and rhizopus stolonifer had the least frequency of 4% each in tomato and orange.(Lewis R.A CRC Dictionary of agricultural sciences Boca Raton, FL CRC press 2002)
Mean while in other journals Aspergillus contains about 38.8% frequency, penicillium 17.2%,fusarium 26.9, cladosporium 7.6% and Rhizopus 9.5% of fungal isolates shown.(Zain M.E 2011 impact of mycotoxins on human and animals journal of Saudi chemical society 15:129-144)
2.3 Researchers Conclusions
These pathogenic fungi species associated with fruits spoilage in this study are economical and public health significance. A.niger causes black mold in certain fruits and vegetables. Some strains of A.niger have been reported to produce potent mycotoxins called ochratoxins that can be harmful to human beings and animals. Care should be taken during handling of these fruits, technology based modern preservative methods such as pasteurization, vacuum packing, radiation, pulsed electric field electroporation, high pressure food preservation, and bio preservation are suggested to enhance the keeping quality of fruits.(Lewis RA CRC dictionary of Agricultural Sciences. Boca Raton, FL CRC Press 2002).
Market conditions that favours contamination can be worsened by poor hygiene of vendors, using microbial unsafe container poor handling practice and poor environmental conditions such as sanitarily unsafe marketing environment. The consequences of the problems could be increased loss of fruit due to microbial spoilage and the existence of some human pathogens. Out of the fungi isolated in this study most of the fungal organisms isolated in the study play a vital role in the deteration of food and feed systems and some of them are also able to produce toxic compounds for humans and animals. The mycotoxins produced by these fungi can cause serious health hazards including cancerogenic pathogens. Fungi pathogens are causing losses of marketable quality and hugiene of fruits resulting in major economic problem in Nigeria and the world as a whole(Gultie A, sahile Subramanian C.Assesment of fruits management in Gondar town market of Northwestern).
3.0 CHAPTER THREE
This Chapter is about the practical aspect of the project, I collected all the samples from Utako market starting from 7 February 2018, On Wednesday I collected blemished samples. A total of 15 fruits which comprises of (3 spoilt Apples, 3 spoilt tomato, 3 spoilt pineapple, 3 spoilt water melon and 3 spoilt oranges) for culturing.
After collecting the samples, they were quickly taken to the laboratory. I sterilized them using jik (Hypo), isopropanol, and Dettol. I cultured from them using the following materials;
3.1 MATERIALS AND METHODS
Vaseline /petroleum jelly
3.2 MATERIAL STERILIZATION
Firstly, I started by wearing my lab coat, then sterilizing the entire laboratory to evade any contamination. The fruits were sterilized using the antiseptic mentioned above, then I poured my distilled water into the conical flask of measurement of 500mg and heated in an autoclave to avoid contamination, after the water is boiled for 15 to 25minutes it is then brought out to settle at room temperature for about 5 to 10 minutes.
3.3 AGAR PREPARATION
The agar agar is measured at 14g and the antibiotics mentioned above each of 500mg and small amount of nutrient agar to hasten the rate of the reaction are being added to the boiled distilled water and closed tightly with a foil paper and paper tape to secure it and prevent it from pouring away and contaminants. The boiled water is now kept back in the autoclave to boil for another 15 to 25 minutes to mix well and solidify. The mixture is now brought out of the autoclave to cool at room temperature for 10 to 15 minutes and then poured into the petridishes and allow it to solidify then scalpel was used to caught out a small portion of the spoilt parts and placed on the Agar and closed very quickly and taped with a labeled paper tape (small portion was left not completely covered for ventilation) to avoid any contamination.
3.4 SUBCULTURING TECHNIQUES
After 5 days that’s on the 12th February we subculture into nutrient agar and this was also prepared the same way as mentioned above. And this meant to be grown for maximum of 10 to 14 days. But I did the subculture after 10 days due to the rapid growth of the fungi.
3.5 PATHOGENECITY TEST
Then I went to the market and collected fresh samples for the pathogenicity test. The samples were being inoculated (punctured) and the affected area of the agar being placed on it and sealed with petroleum jelly to prevent contamination.
After the inoculation the tomatoes, apples and oranges are kept in the incubator while the pineapple and watermelon were placed on a disinfected area in the lab at room temperature.
After 7 days there was a massive, beautiful contamination on the fresh fruits which was subcultrured also to see if we have the same disease causing agent. Then we checked under microscope which we got many Aspergillus, and many more and we took the samples for FTIR at shetsco in kwalli Abuja.
The results we got from the microscope were now compared with the ones online and textbooks to be sure.
As a control for the pathogenicity test, the selected fruits were surface punctured with the hollow metal rod but was not inoculated with fungi isolates and sealed with petroleum jelly.