Morphological Characterization and Determination of Aﬂatoxigenic and Non-Aﬂatoxigenic Aspergillus ﬂavus Isolated from Sweet Corn Kernels and Soil in Malaysia

: This study aimed to morphologically characterize and determine the aﬂatoxigenic and non-aﬂatoxigenic Aspergillus ﬂavus isolates. Forty isolates of A. ﬂavus were obtained from sweet corn kernels and soil samples collected from Kampong Raja, Rose Valley, Kea, and Klebang farms in Malaysia. They were cultured on potato dextrose agar (PDA), dichloran rose-bengal chloramphenicol (DRBC), Aspergillus ﬂavus and Aspergillus parasiticus agar (AFPA), and coconut cream agar (CCA). Macromorphological characteristics were determined by observing the colony color and texture, while the micromorphological characteristics were determined by examining the spore color, size, structure, conidiophore structure, and vesicle shape. The production of aﬂatoxin was determined by direct visualization of the UV ﬂuorescence of A. ﬂavus colonies on CCA. Aﬂatoxin was qualitatively detected in 18 (45%) isolates of A. ﬂavus using UV ﬂuorescence screening while the remaining 22 (55%) isolates did not exhibit any aﬂatoxin production. The highest incidence of A. ﬂavus (30%) and aﬂatoxin production (15%) was recorded in samples from Kampong Raja. On the other hand, isolates from Rose Valley (17%) and Kea (12%) were non-aﬂatoxigenic. Klebang recorded a 25% incidence of A. ﬂavus in which 15% were aﬂatoxigenic while 10% were non-aﬂatoxigenic. The occurrence of aﬂatoxin-producing A. ﬂavus emphasizes the need for the measure to eradicate their presence in food crops. A biological control treatment utilizing the non-aﬂatoxigenic strains to compete with the aﬂatoxigenic ones is underway. Validation of aﬂatoxin production through high performance liquid chromatography is also ongoing.


Introduction
Living in the present time is dangerous, not simply because of accidents or ecological threats, but also because of the foodstuff that we consume in our routine life. Most of the foods we consume are contaminated by microorganisms as well as their toxins. The intake of food materials affected by toxigenic fungi causes the induction of mycotoxicosis (toxic syndrome). Mycotoxicosis is a disease February to May. The average temperature of Cameron Highlands ranges from 25 • C to 28 • C with the highest being recorded from June to August (33 • C) [25].

Sweet Corn Kernels
A total of 80 sweet corn cobs (20 cobs from each farm) were collected, kept in sterile plastic bags, and sealed properly. The cobs were kept at room temperature and examined the day after collection. The sweet corn kernels were sampled from different parts of the cob using closed spear driven through to the top and sides to obtain 300 g of incremental samples [26]. All the samples were randomly sampled.

Soil Sampling
The intermittent design was followed across the sweet corn farm to obtain the points where the soil samples were collected using a trowel. Firstly, 9 m was diagonally measured from one corner, which was the first point from where the topsoil up to 5 cm deep was collected. Then, 9 m horizontally and 9 m vertically were measured, from where the second sample was collected. The process was continued until 16 samples, giving a composite soil sample from 50 to 60 g collected in sterilized plastic bags. Finally, the soil samples were kept at room temperature and analyzed the day after collection.

Isolation and Identification of Aspergillus flavus
Several growth media including potato dextrose agar (PDA; general medium), dichloran rose bengal chloramphenicol (DRBC; enumeration medium) agar, and Aspergillus flavus and Aspergillus parasiticus agar (AFPA; selective medium) were used for the isolation of A. flavus isolates from sweet corn and soil samples.

Preparation of Growth Media
The growth media (PDA, 39 g/L dH 2 O, DRBC, 31.5 g/L dH 2 O, AFPA, 45.5 g/L dH 2 O) were prepared following the manufacturer's instruction (PDA; Oxoid, UK, DRBC and AFPA; Hardy Diagnostics, USA) supplemented with chloramphenicol (Fisher, UK) and autoclaved (Hirayama, Japan) at 121 • C and 15 psi for 15 min. The sterilized media were left to cool for 20 min and transferred into 90 mm Ø Petri plates. The Petri plates were kept at 4 • C until inoculation.

Aspergillus flavus from Sweet Corn Samples
One gram of each sample (sweet corn) was washed with distilled water and sterilized with sodium hypochlorite solution. The samples were then inoculated on PDA plates supplemented with 0.1 mg of chloramphenicol to prevent bacterial contamination. Three replicates for each sample were made. The uninoculated agar plate served as a negative control while the plate inoculated with A. flavus American Type Culture Collection (ATCC) 200026 (synonym: NRRL 3357; Northern Regional Research Laboratory) served as a positive control. The uninoculated and inoculated plates were incubated at 28 • C for 7 d and examined daily for growth and spore formation. Following 7 d of incubation, the fungal colonies were sub-cultured on freshly prepared PDA and incubated for 7 d. Aspergillus section Flavi colonies were observed under a stereo-binocular microscope (Magnus M24). Representative isolates of Aspergillus section Flavi were then single spored as described by Ho and Ko [27], Choi, Hyde and Ho [28], and Singh, Kumar and Bandyopadhyay [29] to obtain axenic cultures. The axenic cultures of Aspergillus section Flavi were sub-cultured on PDA and incubated for 7 d to observe the morphological characteristics [30,31].

Aspergillus flavus from Soil Samples
In a 7 mL sterile polystyrene tube, 1 g of soil was suspended in 3 mL of distilled water, mixed for 20 min on a Roto-Shaker prior to plating onto DRBC [32]. Then, 1 mL of soil solution was plated onto the surface of the agar. The uninoculated agar plate served as negative control while the plate inoculated with ATCC 200026 (NRRL 3357) served as a positive control. The uninoculated and inoculated plates were incubated at 28 • C for 7 d. Aspergillus section Flavi colonies were observed under a stereo-binocular microscope (Magnus M24). The representative isolates of Aspergillus section Flavi were then single spored as described by Ho and Ko [27], Choi, Hyde and Ho [28], and Singh, Kumar and Bandyopadhyay [29] to get pure cultures. Isolates of A. flavus were identified by the colony morphology and morphological keys as described by Klich [33].

Morphological Confirmation of Aspergillus flavus
To confirm the identification of A. flavus by colony reverse color, the isolates were cultured on AFPA as described by Pitt, Hocking and Glenn [34] and incubated at 28 • C for 2-3 d.

Determination of Aflatoxigenic Potential of Aspergillus flavus
Coconut cream agar (CCA) was used to screen the ability of A. flavus to produce aflatoxins, thus distinguishing the aflatoxigenic from the non-aflatoxigenic isolates. CCA was prepared in Petri dishes as described by Davis, Iyer and Diener [35] and Fente et al. [36]. Spores from 7-d old A. flavus cultures were suspended in dH 2 O comprising 0.025% Tween 80. Then, 10 µL of spore suspension was aseptically inoculated at the center of the Petri plates containing CCA and 0.1 mg chloramphenicol [35]. The Petri plates were incubated at 28 • C for 7 d. Following incubation, the plates were examined under UV light (365 nm) for the fluorescence screening of aflatoxins, and the results were recorded.

Aspergillus flavus from Soil Samples
In a 7 mL sterile polystyrene tube, 1 g of soil was suspended in 3 mL of distilled water, mixed for 20 min on a Roto-Shaker prior to plating onto DRBC [32]. Then, 1 mL of soil solution was plated onto the surface of the agar. The uninoculated agar plate served as negative control while the plate inoculated with ATCC 200026 (NRRL 3357) served as a positive control. The uninoculated and inoculated plates were incubated at 28 °C for 7 d. Aspergillus section Flavi colonies were observed under a stereo-binocular microscope (Magnus M24). The representative isolates of Aspergillus section Flavi were then single spored as described by Ho and Ko [27], Choi, Hyde and Ho [28], and Singh, Kumar and Bandyopadhyay [29] to get pure cultures. Isolates of A. flavus were identified by the colony morphology and morphological keys as described by Klich [33].

Morphological Confirmation of Aspergillus flavus
To confirm the identification of A. flavus by colony reverse color, the isolates were cultured on AFPA as described by Pitt, Hocking and Glenn [34] and incubated at 28 °C for 2-3 d.

Determination of Aflatoxigenic Potential of Aspergillus flavus
Coconut cream agar (CCA) was used to screen the ability of A. flavus to produce aflatoxins, thus distinguishing the aflatoxigenic from the non-aflatoxigenic isolates. CCA was prepared in Petri dishes as described by Davis, Iyer and Diener [35] and Fente et al. [36]. Spores from 7-d old A. flavus cultures were suspended in dH2O comprising 0.025% Tween 80. Then, 10 µL of spore suspension was aseptically inoculated at the center of the Petri plates containing CCA and 0.1 mg chloramphenicol [35]. The Petri plates were incubated at 28 °C for 7 d. Following incubation, the plates were examined under UV light (365 nm) for the fluorescence screening of aflatoxins, and the results were recorded.

Macroscopic Characteristics of Aspergillus flavus on PDA
The morphology of A. flavus colonies on PDA is presented in Figure 2. In the beginning, the mycelial color of A. flavus was white. Following 3 d of incubation, the A. flavus colony formed olive-green conidia that dominated the colony's appearance. Generally, the colonies were flat at their borders while raised in the middle. On PDA, all the A. flavus isolates produced exudates (droplets) that are brown in color or colorless. In some isolates, a compact mass of fungal mycelia (sclerotia) was produced, which were dark brown. Moreover, the colony diameter was ranged from 65 to 75 mm, surrounded by a white circle. The reverse of the colonies was pale in color.

Macroscopic Characteristics of Aspergillus flavus on PDA
The morphology of A. flavus colonies on PDA is presented in Figure 2. In the beginning, the mycelial color of A. flavus was white. Following 3 d of incubation, the A. flavus colony formed olivegreen conidia that dominated the colony's appearance. Generally, the colonies were flat at their borders while raised in the middle. On PDA, all the A. flavus isolates produced exudates (droplets) that are brown in color or colorless. In some isolates, a compact mass of fungal mycelia (sclerotia) was produced, which were dark brown. Moreover, the colony diameter was ranged from 65 to 75 mm, surrounded by a white circle. The reverse of the colonies was pale in color.  The morphology of A. flavus colonies on PDA is presented in Figure 2. In the beginning, the mycelial color of A. flavus was white. Following 3 d of incubation, the A. flavus colony formed olivegreen conidia that dominated the colony's appearance. Generally, the colonies were flat at their borders while raised in the middle. On PDA, all the A. flavus isolates produced exudates (droplets) that are brown in color or colorless. In some isolates, a compact mass of fungal mycelia (sclerotia) was produced, which were dark brown. Moreover, the colony diameter was ranged from 65 to 75 mm, surrounded by a white circle. The reverse of the colonies was pale in color.   produced a bright orange or golden color on the reverse due to the reaction between aspergillic acid with ferric ammonium citrate contained in the medium.
Agriculture 2020, 10, x FOR PEER REVIEW 6 of 13 produced a bright orange or golden color on the reverse due to the reaction between aspergillic acid with ferric ammonium citrate contained in the medium.

Microscopic Characteristics of Aspergillus flavus
The microscopic characteristics of A. flavus are shown in Figure 5. Under the microscope, the conidiophores of A. flavus isolates were colorless, thick walled, roughed, and bearing vesicles. The diameter of the conidiophores ranged from 800 to 1200 µm. The vesicle shape of A. flavus isolates was globose to sub-globose. The diameter of the vesicles ranged from 1800 to 2000 µm. The cells were uniseriate or biseriate. In the biseriate cells, the phialides grew on the metulae whereas, in uniseriate

Microscopic Characteristics of Aspergillus flavus
The microscopic characteristics of A. flavus are shown in Figure 5. Under the microscope, the conidiophores of A. flavus isolates were colorless, thick walled, roughed, and bearing vesicles. The diameter of the conidiophores ranged from 800 to 1200 µm. The vesicle shape of A. flavus isolates was globose to sub-globose. The diameter of the vesicles ranged from 1800 to 2000 µm. The cells were uniseriate or biseriate. In the biseriate cells, the phialides grew on the metulae whereas, in uniseriate cells, they grew on the vesicles. The metulae enclosed the vesicles' surface and emitted in all directions. The conidia were globose, thin walled, slightly roughed, and ranged from 250 to 450 µm in diameter.
Agriculture 2020, 10, x FOR PEER REVIEW 6 of 13 produced a bright orange or golden color on the reverse due to the reaction between aspergillic acid with ferric ammonium citrate contained in the medium.

Microscopic Characteristics of Aspergillus flavus
The microscopic characteristics of A. flavus are shown in Figure 5. Under the microscope, the conidiophores of A. flavus isolates were colorless, thick walled, roughed, and bearing vesicles. The diameter of the conidiophores ranged from 800 to 1200 µm. The vesicle shape of A. flavus isolates was globose to sub-globose. The diameter of the vesicles ranged from 1800 to 2000 µm. The cells were  Figure 6 represents the findings obtained from the UV light screening of A. flavus, displaying the fluorescent and non-fluorescent isolates of A. flavus on CCA. It was found that 18 (45%) out of 40 isolates fluoresced blue on CCA once they were exposed to UV light (365 nm), thus indicating the ability of aflatoxin production (Table 1). On the other hand, the remaining 22 (55%) isolates did not exhibit blue fluorescence in UV light, thus classified as non-aflatoxigenic isolates of A. flavus. The distribution of the aflatoxigenic and non-aflatoxigenic isolates of A. flavus in four different farms is presented in Figure 7. Of the 12 isolates from Kampong Raja, six isolates (15%) were tested positive for aflatoxin production while the remaining six (15%) were tested negative for aflatoxin production. For Rose Valley, aflatoxin was detected in three isolates (7.5%) and the remaining seven (17.5%) isolates did not produce aflatoxin. Similarly, of the eight isolates of Kea, three (7.5%) isolates were aflatoxigenic while 12% (five) of the isolates were non-aflatoxigenic. Finally, six (15%) out of 10 isolates of Klebang were aflatoxigenic whereas the remaining four (10%) isolates were found to be non-aflatoxigenic.

Prevalence of Aspergillus flavus across Sweet Corn Farms in Malaysia
In Malaysia, sweet corns are grown in different environments. In unfavorable environmental conditions, sweet corn can be infected by various fungi. A. flavus has been recognized as among the most important fungal pathogens of corn. A. flavus contaminates corn both in the field as well as in storage, and the rate of infection could be different amid farms of the same region [38,39]. In this study, 40 isolates of A. flavus were identified from the fungal cultures formerly isolated from the sweet corn and soil of Kampung Raja, Rose Valley, Kea, and Klebang farms in Malaysia.
The highest occurrence (30%) of A. flavus was found in the sweet corn and soil of Kampung Raja while the lowest (20%) incidence of A. flavus was recorded for Kea farm. Rose Valley and Klebang farms comprised 25% and 25% incidence of A. flavus, respectively. The difference in the occurrence of A. flavus in the different farms could be owed to the diversity in environmental conditions between those farms. Crops grown in hot environments are more susceptible to aflatoxin-producing A. flavus and in some areas, contamination simply takes place as soon as the temperature rises with high humidity [39]. Kampung Raja is characterized by temperatures of about 25 to 33 • C with 80% humidity, which favors aflatoxin infection of sweet corn by A. flavus. While the other three farms (Rose Valley, Kea, and Klebang) are characterized by temperatures of 12-21 • C with 46-60% humidity.

Morphological Characterization of Aspergillus flavus
The morphological characterization of fungi via observing the main macro and micromorphological features cultured on diverse media is an extensively used method for fungal identifications. In this study, the morphological characterization was done to give importance to such basic identification methodologies for the fast screening of isolates in most of the developing countries, where the accessibility of unconventional tools is a great challenge. Consistent identification of A. flavus isolates was done by observing the key morphological characteristics. The physical characteristics of A. flavus including the colony color, texture and edges, as well as the exudates, soluble pigmentations, and sclerotia were studied on four different cultural media.
Overall, the A. flavus had olive-green, yellowish-green, or dark green colonies surrounded by a white circle that was eventually covered by conidia. The colonies' textures were usually velvety or woolly with a floccose center. The A. flavus colonies produced exudates on two of the four culture media utilized in this study. Similarly, sclerotia were detected in the majority of A. flavus isolates, as it is a specific characteristic of aflatoxigenic A. flavus. In the interpretation of the taxonomic descriptions by Klich [33], the isolates' colony morphology in this study bore a resemblance to A. flavus as shown in Figures 2 and 3. On the other hand, some other Aspergillus species have the same phenotypical characteristics and which makes it difficult to differentiate them from A. flavus. It is known to have the same colony color as A. oryzae. This renders the need for comprehensive scrutiny using microscopic and macroscopic characteristics for accurate identification and classification. However, there is a consensus that, even microscopically, some characteristics are common among Aspergillus isolates [40]. The microscopic features of A. flavus such as conidiophore, vesicles, sterigmata and conidia were examined in this study.
The key microscopic features of A. flavus were rough and thick-walled conidiophores, globose vesicles with radiated sterigmata and thin walled finely rough conidia. The macroscopic and microscopic characteristics presented in Figures 2-5 are similar to that of A. flavus characteristics described by Rodrigues et al. [41] and Diba et al. [42]. The use of PDA, DRBC, and AFPA media in this study allowed for sufficient growth and sporulation that permitted a satisfactory examination of the macroscopic and microscopic features of A. flavus. A review of the recent literature presented that these selective growth media have frequently been used for the identification of A. flavus [34]. Many A. flavus isolates isolated from corn in North-Eastern China have been identified after their morphological characterization on SZA and MEA [43].
Likewise, Diba et al. [42] characterized the Aspergillus species from medical and environmental samples through observing their macroscopic and microscopic features and stated that the utilization of PDA, DRBC, AFPA, and other growth media may stimulate the growth and sporulation process of Aspergillus species. Additionally, morphological characterization approaches have also been used on Aspergillus species in the Fars and Kerman provinces of Iran [44] and the Larkana district of the Sindh province, Pakistan [45]. Based on the size of sclerotia, A. flavus can be divided into L-strain and S-strain. The L-strain of A. flavus produces large-sized sclerotia while the S-strain A. flavus produces small sclerotia.
The relationship between the sclerotial size and aflatoxin production has been studied but the published data are unreliable. Cotty and Mellon [46] have reported a close relationship between small sclerotia and aflatoxin production. On the other hand, Abbas et al. [47] found the highest level of aflatoxins in isolates having large sclerotia. Some studies did not even find any correlation between the sclerotial size and aflatoxin production [48,49]. Thus, the sclerotial size and aflatoxin quantity have not been calculated, due to which no correlation between aflatoxin production and sclerotial type could be made.

Discrimination between Aflatoxigenic and Non-aflatoxigenic Aspergillus flavus
The screening of A. flavus isolates, based on their fluorescence characteristics under UV light at 365 nm was carried out. Once the aflatoxigenic isolates of A. flavus were exposed to UV light, they showed a blue fluorescence on the reverse of the colonies, while the non-aflatoxigenic A. flavus did not show any fluorescence (Figure 7). In this study, the use of CCA media made it possible to distinguish the aflatoxigenic A. flavus from non-aflatoxigenic ones very rapidly, albeit just qualitatively. Fluorescence was found in 18 out of 40 A. flavus (45%), thus demonstrating their aflatoxin-producing potential. In most of the developing nations, where aflatoxin determination may be delayed due to the unavailability of advanced detection instruments, culture-based techniques could be beneficial for the rapid screening of isolates.
Although the culture-based techniques are comparatively simple and economical, for aflatoxin detection however, these techniques are usually employed in combination with chromatographic methods [47]. For the precise quantification of aflatoxin, a more advanced method such as high-performance liquid chromatography equipped with fluorescence detection (HPLC-FLD) would be useful. Similarly, it has been reported that strains of A. flavus could produce B and G aflatoxins [50], a recent classification of Aspergillus section Flavi noted that such strains, though morphologically similar to A. flavus, were completely novel species [51,52]. These outcomes pointed out the fact that modern molecular approaches are mandatory to be used for A. flavus isolates' characterization.

Conclusions
In conclusion, both aflatoxigenic and non-aflatoxigenic strains of A. flavus were present in the sweet corn kernels and soil of Cameron Highlands, Malaysia. Cameron Highlands being a primary source of agricultural produce could take measures to ensure that aflatoxin contamination is minimized. Non-aflatoxigenic strains indigenously present could be further harnessed into a biological control agent that can reduce the dependency on chemically synthesized pesticides. Culture media and morphology are rapid tools for the characterization of A. flavus in this study. The morphological characteristics of these isolates were similar to those explained in the published identification manuals. The screening of A. flavus isolates for aflatoxin detection under UV light (365 nm) is a fast and reliable method to distinguish the aflatoxigenic and non-aflatoxigenic isolates. Although the culture-based techniques are comparatively simple and economical, however, for the precise quantification of aflatoxin, a more advanced method such as high-performance liquid chromatography equipped with fluorescence detection (HPLC-FLD) would be useful. This further reinforces the need for measures to prevent the fungal contamination of sweet corn and emphasizes the importance of field interventions aimed at curbing agricultural soil contamination by these fungi.
Author Contributions: F.M.G., supervision, project administration, funding acquisition; R.K., methodology, writing-original draft preparation; N.I.P.S., conceptualization, and methodology assistance; N.A.M., review, resources, and data curation. All authors have read and agreed to the published version of the manuscript.