Identification and culturing of fungi
Aspergillus species used in this study were isolated from maize kernels collected in Eastern Kenya aflatoxicosis hot-spot [29]. The fungi were sub-cultured following standard protocols then identified to species level following morphological and colony characteristics using Aspergillus specific keys [30]. The selection of the fungi in this study was based on their isolation frequency from the maize samples. This gave a total of 16 most frequently isolated Aspergillus species. Molecular identification of the fungi was carried out using the Internal Transcribed Spacer (ITS1–5.8S-ITS2 cluster) regions of the ribosomal DNA gene cluster [4]. The fungi were then sub-cultured on Czapek Dox Agar medium (HiMedia Laboratories Pvt. Ltd), then incubated at 28°C for five days. The fungi were coded depending on the season and area of collection and maize sample number: A. flavus were coded as: [1EM1901, 1EM1201, 1EM2606, 1EM2901, 1EM4501, 1EM4501, 1EM4503, 2EM0501, 2EM0502, 2EM0602, 2EM1201, 2EM3503, 2EM3506, and 2EM6103] and A. parasiticus coded as: [2EM0402 and 2EM0601].
Dichlorvos-ammonia (DV-AM) test for aflatoxigenicity
For the DV-AM method, the fully grown isolates initially cultured on the Czapek Dox Agar medium were sub-cultured on aflatoxin-inducing Yeast Extract Sucrose (YES) medium (amended with 50mg/L streptomycin sulfate and penicillin, Zhonghuo Pharmaceuticals, China) and maintained for seven days at 28°C. The culturing and testing for aflatoxigenicity of the isolates on the aflatoxin-inducing YES media was sequentially done following the protocols for the DV-AM method as described by [25]. The method briefly involved: (i) Dichlorvos diluted with methanol in 250 fold ratio (Amiran Kenya Ltd) was premixed with the media prior to solidification, (ii) the isolates were incubated in darkness at 28°C for 5 days, (iii) 0.2 mL of ammonium hydroxide solution was poured onto the inside of the lid of the petri dish plate used to set up the DV-AM method after the 5-day incubation period, (iv) the isolates were categorized as either aflatoxigenic or non-aflatoxigenic depending on their ability to produce aflatoxins (shown by a characteristic red coloration at the underside of the plates), and (v) the plate images of the aflatoxigenic isolates were further analyzed with ImageJ 1.x software for enhanced detection (Figure 1 C, F).
Identification of aflatoxin-associated gene clusters
The fungal isolates used for molecular identification were grown in Potato Dextrose Agar medium (PDA; pH 6.0) and maintained at 28 °C for five days. Afterwards, the periphery of the exponentially growing fungal mycelium (50-100 mg) was excised aseptically using a sterile scalpel. DNA extraction was done using Quick-DNA™ Fungal/Bacterial Kit (Zymo Research). Finally, 100 µL of DNA was eluted for amplification. Four aflatoxin-specific primers (Inqaba Biotechnical Industries (Pty) Ltd (South Africa)) were used to amplify genes (aflD, aflQ, and aflP) encoding for enzymes involved in the aflatoxin biosynthesis and regulation (aflR) of the pathway (Table 3). The four genes selected for this study were chosen based on their direct role in the final production of aflatoxins as reported in the literature (Table 2).
Table 3 Aflatoxin-associated primer-pair combinations for Aspergillus species
Primer code
|
Target gene
|
Primer Sequences
|
References
|
Tm
|
Purification
|
Modifications
|
AflD-1for
AflD-2rev
|
aflD (nor-1)
|
Nor1-F (5′-ACC GCT ACG CCG GCA CTC TCG GCA C-3′)
Nor1-R (5′-GTT GGC CGC CAG CTT CGA CAC TCC G-3′)
|
[22]
|
74.42
72.78
|
Standard
Standard
|
None
None
|
AflQ-1for
AflQ-2rev
|
aflQ (ordA)
|
Ord1-gF (5′-TTA AGG CAG CGG AAT ACA AG-3′)
Ord1-gR (5′-GAC GCC CAA AGC CGA ACA CAA A-3′)
|
[23]
|
58.35
64.54
|
Standard
Standard
|
None
None
|
AflP-1for
AflP-2rev
|
aflP (omtA)
|
AflP-1for (5′-AGC CCC GAA GAC CAT AAA C-3′)
AflP-2rev (5′-CCG AAT GTC ATG CTC CAT C-3′)
|
[9]
|
60.16
60.16
|
Standard
Standard
|
None
None
|
AflR-1for
AflR-2rev
|
aflR
|
AflR-1for aflR (5′-AAG CTC CGG GAT AGC TGT A-3′)
AflR-2rev (5′-AGG CCA CTA AAC CCG AGT A-3′)
|
[9]
|
60.16
60.16
|
Standard
Standard
|
None
None
|
During the reaction, a total reaction volume of 25 µL consisting of 12.5 µL of One Taq® 2X Master Mix (New England Biolabs), 0.2 µL of DNA template (< 1000 ng), 0.5 µL (0.2 µM) of each (forward and reverse) of the primers and 9.5 µL of nuclease-free water. The mixture was gently mixed by priming pipettor at least 4 times and spun prior to PCR. PCR tubes were transferred to a preheated thermocycler at 94 °C for 3 minutes followed 30 cycles consisting of 1 minute of denaturation at 94 °C, 1 minute of annealing at 57 °C and 1 minute of extension at 72 °C. A final extension of 10 minutes at 72 °C and a storage temperature of 4 °C were considered. The PCR products were then subjected to gel electrophoresis using 1 % agarose gel pre-cast with Ethidium Bromide. The optimized conditions for the process involved a constant supply of voltage (50 V (41mA)) for 75 minutes in a Tris-borate-EDTA (TBE) buffer electrophoretic chamber. The gel was visualized using Automatic Gel Imaging System (Peiqing Science and Technology Co., Ltd).
Statistical analysis
All experiments were replicated thrice and data is presented in percentages for the parameters analyzed. ImageJ 1.x software was used for color enhancements in plates with fungal colonies.