Material and Methods

Sampling Site

Sharada: is located at Gwale local government area of Kano statea distance of about 50 Klm away from Kano University of Science andTechnology, Wudil. It is one of the large vegetable markets in Kanostate. There are no fruits and vegetables grown in Kano state thatare not found at Sharada. Despite being one of the largest vegetablegrowing and selling markets at Kano metropolitan, there are no goodstorage facilities at Sharada market. Some marketers store their fruitsand vegetables on the floor of the stores, while others kept theirspacked in baskets. Marketers hardly used chemicals on their fruitsand vegetables. They however washed them either with water ordetergents.

Yankaba: is located at Nassarawa Local Government area with adistance of about 50klm away from Kano University of Science andTechnology, Wudil. It is one of the large vegetable markets in Kanostate. There are no fruits and vegetables grown in Kano state thatare not found at Yankaba. Despite being one of the largest vegetablegrowing and selling markets at Kano metropolitan, there are no goodstorage facilities at Yankaba market. Some marketers store their fruitsand vegetables on the floor of the stores, while others kept theirspacked in baskets. Marketers hardly used chemicals on their fruitsand vegetables. They however washed them either with water ordetergents.

Sample Collection

Lettuce was collected from Sharada and Yankaba markets KanoState. Samples were collected two times in a week, Mondays andThursdays respectively which lasted for three months. The sampleswere collected directly from the market inside polyethene bag(container) to the laboratory in (K U S T Wudil) for analysis.

Isolation Media

Potato dextrose agar was the media used for isolation of postharvestfungi in lettuce sample. Thirty nine grams of PDA wereweighed using weight balance and suspended in 1litre of distilledcool water. The contents were sterilized by autoclaving machine at1210c for 15 minutes. These were allowed to cool for 5 minutes ona laboratory bench until the temperature fell to 450oc.ten millilitersof lactic acid were added to inhibit bacterial growth. The media wasdispensed into sterilized Petri dishes of 9cm diameter.

Procedure for Isolation of Fungi

The isolation was carried out according to the method describedby [14-16] as follows 39g of powdered PDA was weighed usingweighing balance and dispense in one liter of distilled water, stirredthoroughly before been place inside the flask bottles and autoclavedinside the machine. The media was later poured into sterilized petridishes and allowed to solidify. The sample obtained from Kwakwachiirrigation area was sterilized by washing in running tap water andallowed to dry. Portions (2mm) were cut with a sterilized cork borer.Cut pieces were placed on PDA and incubated at 25.7 ±2 c for 3 days.

Colony Count and Subculture

Fungal colonies that grown were observed accounted andrecorded. Each distinct colony was sub cultured into fresh PDAmedia to facilitate possible identification.

Determination of Pathogenicity of Isolated Micro Organism

Pathogenicity tests were conducted to prove Koch postulate.Healthy leaves of lettuce vegetable surface sterilized in 10% (v/v)sodium hypochlorite solution and rinsed in 3 changes of running tapwater and allowed to dry. A ruler was used to mark a (2mm) diametercircle on each sample and sterilized needle was used to streak fungalhyphae on the marked portions. Controls were inoculated withdistilled water. Both were placed on the laboratory bench. Sterilizedforceps were used to remove portions from the diseased areas on the4th day and placed on freshly prepared PDA plates and incubated at25.7 ±2oc for 3 days. Fungal growth that appeared was recorded.

Microscopic Examination

For each examination, a streak of fungal mycelium was placed ona clean glass slide. One drop of cotton blue lacto phenol was addedand the cover slip placed. The slide was mounted on the microscopeand observed at magnification of ×10, ×40 and ×100. Morphologicalcharacteristics of fungi isolated were determined and identified usingmethod described by [17]. Lengths of the hyphae were determinedwith eyepiece graticule by using colonial and morphologicalcharacteristics.

Photography

Photographs of fungal mycelia were taken from mounted slideusing camera Lucida at Biology Department, Kano University ofScience and Technology, Wudil.

Statistical Analysis

Data collected on fungal colonies was analyzed using s Chisquareanalysis. This was achieved using computer program GrapPAD insert Version 1.15 copyright 1990 [18].

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Results

Infection of fungal species was recorded on all the leaf samples. Atotal of 987 colonies were counted and recorded which belong to theC. albican and the three species of Aspergillus. Thus were Candidaalbican 294(%) A. niger {307 (31.1%}), A. flavus {135 (13.8%}), A.fumigatus {252 (25.5%)}. Control plates showed no growth (Table 1).

Table 1: Number of colonies isolated from the two markets on Monday and Thursday.

Variation of the colony counted in Sharada and Yankabamarket

Sharada area recorded the highest colony count with 617 (62.5%).Where a total of 211 (56.1%) and 406 (66.4%) colonies was recordedon Monday and Thursday respectively. The least colony countwas recorded at Yankaba, market area with a total of 370 (37.4%),comprising 165 (43.8%) and 205 (33.5%) on Monday and Thursdayrespectively.

Variation in the colony counted on Monday and Thursday

A total of 376(38.0%) were recorded in the Monday, while 611(61.9%) were recorded during Thursday exposure (Table 2).

Table 2: Variation in the colony count of all the species for the two selected sites on samples collected on Monday and Thursday.

Pathogenecity test

The results of the pathogenicity test confirmed all the four criteriaoutline in Koch postulates for identification of the causative agent ofa particular disease. The pathogen where present in all cases of thedisease. The same pathogens was isolated from the diseased hostand grown in pure culture. When inoculated into a healthy sample of lettuce plant the pathogen from the pure culture causes the samedisease. The same pathogen was reisolated from the new host andshown to be the same as the originally isolated pathogen (Table 3).

Table 3: Pathogenicity Test

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Discussion

A total of 387 colonies were isolated during the study. Fungiisolated include A. niger, A. flavus and C.albican (yeast). The generaA. niger was the most frequently occurring with the percentageoccurrence of 31.1% followed by C. albican with percentageoccurrence of 30.1%, A. fumigatus with percentage occurrence of25.5% the least occurring colony was A. flavus with percentageoccurrence of 13.8%. Pathogenicity test confirmed the pathogensas originally isolated pathogen of lettuce sample from Sharada andYankaba markets. More colonies where recorded at Sharada thenYankaba markets. Also high number of colonies was recorded onMonday exposure than Thursday exposure.

Alao [19] reported that many of the post-harvest diseases offruits and vegetable, grains and legumes are the result of infectionsby pathogens in the field which continue to develop after harvest. Ina related study [20] reported that fungi are the most common causeof spoilage on fruits and vegetables and several fungi like penecilliumspp, cladosporium spp, and altternaria phomopsis are known to causelarge scale storage loss of fruits and vegetables from harvest to storage.

The finding of this research support the report of [13] whostudied fungal deterioration of some vegetables in northern Nigeriaand found that losses in spinach is attributed to the activities of A.niger, A. fumigatus , Mucor, and Rhizopus stolonifer. The findingalso agrees with the work of [21,20] who isolated, A. niger, A. flavus,Rhizopus, and Mucor from samples of vegetables grown at Nassarawalocal government area of Kano state.

The higher count obtained on Monday could be due to heavyactivities with high influx of customers from different locations onthe day for buying and selling. Statistical difference of (P >0.05) wasobtained between the two seasons (Table 2).

It can be concluded that the four fungal species namely Candidaalbican, A. niger, A. fumigatus, and A. flavus, are the common postharvest fungi associated with lettuce on sale at the studied markets.The results obtained in this study indicate that Yankaba, site is themost suitable for marketing of fresh and healthy vegetables. Thisis because in Yankaba area there is total absence of household andindustrial effluents in the area surrounding the market this accountedfor the least colony count. Sharada site is the least suited for marketing of vegetables because, effluents from household and industries in the surrounding area were the source of infection. The effluents mightcontain toxic chemicals that on long time exposure could pose serioushealth hazards to the consumers of these vegetables. Therefore, tosafe guard the consumers from buying produce which may be ofhealth hazard effort should be made ensure that all effluents fromhousehold and industries be adequately treated before discharge intothe surrounding environment. Likewise marketing of crops shouldbe prohibited in any area close to household and industrial effluentsdischarge [23].

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