ISOLATION AND MORPHOLOGICALLY IDENTIFICATION OF ASPERGILLUS FLAVUS INCIDENCES FROM MAIZE SEEDS IN ABUJA, NIGERIA

Food safety and security well implemented could help in making more crops available for consumption. Maize seed is a crop wel l known to be attacked by fungi such as Aspergillus flavus and reduce its nutrients. This study intends to isolate and morphologically identify the Aspergillus flavus from maize seeds from Abuja, Nigeria. The experimental design was complete randomized design involving untreated yellow (Y) and white (W) maize seeds from7 locations in Abuja, Nigeria. Pure culture of fungal isolate was prepared using Potato Dextrose Agar (PDA) and Sabouraud Dextrose Agar (SDA). Isolates obtained were characterized and identified on the basis of their colonial and morphological characteristics which include macroscopic and microscopic examinations. All maize seeds from the Abaji had no fungi incidences in both SDA and PDA, while all maize seeds from the experimental field show fungi incidences in both SDA and PDA. On SDA, the maize color yellow and white had F (2, 6) static values of 7.083 and 0.212 at p=0.129 and 0.941 respectively. For the PDA, white maize seeds and yellow maize seeds from all the locations had F (2, 6) static values of 0.377 and 0.521 at p=0.850 and 0.773. Aspergillus flavus was isolated in this study. The maize seeds from Kuje district and the experimental field 2 show a high incidence records. All growth of the Aspergillus in the two media used were not significant at p>0.05.


INTRODUCTION
Fungi are known to cause deterioration and loss of nutrients in maize after insect (Debnath et al., 2012). The fungi genera Aspergillus, Bipolaris, Curvularia, Fusarium, and Penicillium which are well known fungi that attack seeds have been linked with maize seeds (Hussain et al., 2013). Aspergillus flavus is the most common member of the Aspergillus species in West African and the United States soils (Gachara et al., 2018). Systemically, fungal attack on maize replete its viability, nutrient quality and quantity, seedling blight, failure in germination, subdued seedling and unappreciable crop performance (Enyiukwu and Ononuju, 2016). Aspergillus flavus is a saprophytic pathogen that thrives largely on many organic nutrient sources with sugars (Amaike and Keller, 2011). It is a fungus with wide economic impact which cut across been a pathogen of animals and insects, plants, cause of storage rots in large number of crops, production of highly regulated mycotoxin, aflatoxin B1 (Klich, 2007). Its aflatoxin contaminants had been reported in some agricultural products (Perrone et al., 2014). A. flavus a well-known and cosmopolitan fungus could survive some series of environmental conditions (Abbas et al., 2009). They have the tendency to survive temperatures within 12 o C to 48 °C , an optimal growth temperature of 28 °C to 37 °C and a high humidity above 80% (Hell and Mutegi, 2011; Yu, 2012). As a storage mold on plants products, Maize seeds have been reported to be infected by A. flavus in the field prior to their harvest and in storage (Klueken et al., 2009). Maize (Zea mays L.) a cereal crop belongs to the Poaceae family and it is rich in vitamins A, C and E, carbohydrate, protein, essential minerals, fibre and calories (Salako et al., 2019). Millions of maize about 8.63 million Metric Tons (MT) i s produced annually in Nigeria (Sule et al., 2014). It is a staple for over 1.2 million individuals in Africa and Americas (IITA, 2009;USDA, 2016). Maize is used as raw material for some industrial production, feed, fodder, and vegetable. It has been reported that poor storage condition, storage period, temperature, humidity levels and suitable climate could lead to infection caused by various storage fungi, such as Aspergillus species (Ezekiel et al. 2008) This study intended to isolate and morphologically identify the Aspergillus flavus from maize seeds from Abuja, Nigeria.

Study area
The maize seeds used in this study was obtained from seven (7) different locations across Abuja, Nigeria, Table 1 and Figure 1 illustrate their geographical location.

Seed collections
Two different color of maize seeds were obtained from six different markets across Abuja and experimental field, University of Abuja (UNIABUJA). Distribution of the location where the maize seeds were obtained is as illustrated in Fig. 1.

Preparation and sterilization of media Sabouraud Dextrose Agar (SDA)
SDA was used in this study and prepared according to the manufacturer's instructions thus, 65g of SDA is dissolved in 1000 ml of sterile water and then sterilized (autoclaved) at 121 °C and pressure of 15pa for 15 minutes

Potato Dextrose Agar
PDA was also used in this study and prepared according to the manufacturer's instructions thus; 39g of PDA powder was added to 1 liter of distilled water and boil while mixing to dissolve completely. The sterilization was done at 121ºC for 15 minutes using the autoclave. The sterilized prepared media was dispensed aseptically into petri dishes.

Preparation of pure culture of fungal isolate
The young fungal colony were aseptically picked up and transferred to fresh sterile SDA and PDA plates to obtain pure culture. The pure cultures on SDA and PDA plates were grown at 25 ± 2 °C for 7days and kept under 4 °C in a refrigerator. The isolates were subculture to obtain young cultures for further studies (Klich, 2000).

Identification of the fungal isolate Cultural identification
Twelve isolates obtained from subculture were characterized and identified on the basis of their colonial and morphological characteristics which include macroscopic and microscopic examinations. Among the characteristics used were colonial characteristics such as size, surface, appearance, texture, and reversed pigmentation of the colonies of sporing structures. Appropriate references were done by using mycological identification keys and taxonomic description (Harrigan and McCance, 2006 In (%) = (Number of infected seeds) / (Total number of seeds) ×100.

RESULTS AND DISCUSSION
The incidence of the fungi was calculated and stated as indicated in Table 1. In this study, from findings stated in Table 2, there was more fungi incidences wi t h the potato dextrose agar (PDA) compared to the Sabouraud Dextrose Agar (SDA).

BR -Bwari, GZ-Goza and AB-Abaji
All maize seeds from the Abaji (AB Y and AB W) had no fungi incidences in both SDA and PDA, while all maize seeds from the experimental field (F Y and F W) show fungi incidences in both SDA and PDA. The yellow maize seed overall show more fungi incidence than the white maize seeds. On SDA, from Table 3 the maize color yellow and white had F (2,6) static values of 7.083 and 0.212 at p=0.129 and 0.941 respectively.

Macroscopic Characteristics of the Isolates on PDA
The colony characteristics of the isolates are shown in Fig. 2 at the inception, the isolates were seen to have mycelia white color. Figure 2 : Colony morphology of Aspergillus flavus isolates on PDA The isolates after three days were seen to produce olive and dark green conidia, which happen to be the predominant appearance of the colony. They look raised in the center but their edges appear to be flat and plain with wrinkled in pattern like a cerebri. Droplets of liquid that is brown or uncolored were produced by the isolates. Sclerotia that were deep brown in coloration were produced in the isolates. The colonies were encircled by a white border, and the colony diameter ranged between 65 and 75 mm. The undersides of the colonies were slightly pale Macroscopic Characteristics of the Isolates on SDA The attributes of the isolated colony are shown in Fig. 3. On the SDA the isol at e colony were at the inception white with a velvety soft surface. After four days of growth, a floccose was seen at the center with a raise.

Control
Aspergillus flavus Figure 3: Colony morphology of Aspergillus flavus isolates on SDA Yellowish-green and olive conidia were produced by the colony during sporulation. The whole surface of the colony was covered by conidia the edges, where border whitish in color were seen. On the sixth day of incubation, the produced sclerotia which were white initially became deep brown. No droplet of liquid known as exudates was produced.

Microscopic Characteristics of the A. flavus Isolates
The isolates were examined to ascertain their definitive identification, the microscopic attributes (conidiophores, conidia, metulae, phialides and vesicles) (Fig. 4). The conidiophores appeared uncolored, thick walled, roughened and vesicles bearing. Their diameter ranged between 800 and 1200 µm. Some isolates exhibit vesicles that were subglobose and globose in others with difference in diameter, ranging between 1800 and 2000 µm. There were uniseriate or biseriate or both kind of cells. The phialides were situated on the metuale with the biseriate cells, but attached to the vesicle, in uniseriate cells. The vesicles were covered with the metulae and radiated in all directions from the vesicles. Globose with thin wall with 250 and 450 µm range in diameter made up the conidia. According to Da Gloria (2011), both field and storage fungus contamination incidences in maize may vary among farms or producers in the same regions. This study share this view, for instance all the maize from Abaji-AB (yellow and white) show no fungal incidence in both PDA and SDA medium as illustrated i n Table 2. While the case of Gwagalada-GL and Kuje-KJ yellow side there was fungal incidence in both PDA and SDA. The environmental situations and conditions could be a major determinant in the varying occurrences of A. flavus in the various districts of Abuja indicated in this study. Warm climate play a significant role in a huge chance of infection by aflatoxin producing fungal in some regions and this infection occurs only when there is drought with increase in temperature (Cotty and Jaime-Garcia, 2007). Diba et al., (2007) examined the morphological characteristics of Aspergillus species from some specimens and they indicated that Aspergillus growth and conidia production maybe fasten if potato dextrose, malt extract, or likewise sporulation agars are used in growing them. This study used potato dextrose agar which from Table 2 show more incidence than SDA. Morphological methods as used in this study, had been reported to be used in identifying Aspergillus species from the soils in Fars and Kerman provinces of Iran (Mohammadi et al., 2009) and Larkana district in Pakistan (Afzal et al., 2013).

CONCLUSION
Aspergillus flavus was isolated in this study and the maize seeds from Abuja South Kuje district and the experimental field shows a high incidence records. All growth of the Aspergillus in the media were not significant at p>0.05. The maize seeds that recorded more incidences of Aspergillus flavus could be infected by higher levels of aflatoxins that cause some ill-health issue to humans, animals and plants. It will be wise then for fungus to be fight to its mini m um i n crops.