Characterization of Mating Type Genes in Aspergillus flavus Populations from Two Locations in Kenya.
Aspergillus spp. belongs to phylum Ascomycota and family Trichocomaceae. Species in the fungus genus' section Flavi have potential to infect maize, peanuts, cotton, and tree nuts among other crops and may contaminate with aflatoxins, which are Group 1 carcinogens according to International Centre for Cancer Research [1, 2]. The growth of A. flavus is usually favored by hot dry conditions with optimum temperature of 37[degrees]C, but the fungus readily grows between temperatures of 25 and 42[degrees]C and will even grow at temperatures from 12 to 48[degrees] C. Its ability to grow at such high temperatures contributes to its pathogenicity in humans [2, 3].
Many fungi have the potential to reproduce both sexually and asexually. Successful sexual reproduction requires the presence of functional and compatible mating type genes (MAT1-1 and MAT1-2 genes). In homothallic fungi, self-fertility is possible when both mating type genes are present and functional within the same organism, either on different chromosomes or adjacent to the same chromosome. In heterothallism, only one mating type gene is present in a strain, so it is considered self-infertile and requires the presence of a compatible strain having a functional gene representative of the opposite mating type [4, 5] (Horn et al., 2013). Aspergillus flavus is considered functionally heterothallic, and it is uncommon for strains to contain both MAT genes. Horn et al.  reported A. nomius strain that contained both MAT genes, which were self-infertile but functionally bisexual, meaning they could mate with both MAT1-1 and MAT1-2 strains.
Since 1981, yearly cases of aflatoxicosis have been reported in the Eastern parts of Kenya following consumption of maize contaminated with A. flavus and aflatoxins. Agriculture is the economic backbone in Rift Valley and Eastern Kenya with maize being the staple food, although aflatoxicosis has not yet been reported in the Rift Valley. Makueni is one of the areas in Eastern Kenya that has reported history of aflatoxicosis [2, 7-9]. Despite Nandi being a maize growing area, cases of aflatoxicosis have not been recorded, although oesophageal cancer cases have been reported [7, 8]. In young children, between 0 and 5 years of age, Kangethe et al.  reported high aflatoxin levels in Makueni compared to Nandi. Aflatoxin exposure through milk for children younger than 5 years was 4 x [10.sup.-4] and 1 x [10.sup.-4] [micro]g/kg per day in Makueni and Nandi, respectively; the exposure of nursing children through breast milk was 6 x [10.sup.-3] and 1 x [10.sup.-6] [micro]g/kg per day in Makueni and Nandi, respectively. Children below 30 months of age in Makueni had 1.4 times higher aflatoxin levels in their urine than those of the same age in Nandi.
The use of biological control method is presently the most favorable strategy for lowering preharvest contamination of cereals, groundnuts, and tree nuts with aflatoxin. Nonaflatoxin producing A. flavus is introduced into the environment and outcompetes naturally occurring aflatoxin producing strains in the soil . The technology is under trial in Kenya in the aflatoxin hot spots [11-13]. However, A. flavus populations show high genetic variations due to its potential to outcross by sexual recombination under special conditions in the soil. When such outcrossing occurs, the biocontrol strain could recombine with the indigenous populations of aflatoxigenic strains and generate offspring that not only inherit the aggressiveness of the non-aflatoxigenic parent, but could also inherit the ability to produce one or more serious mycotoxins, such as aflatoxin B1 or cyclopiazonic acid (CPA) (Horn et al., 2013) . Therefore, there is a need to understand the potential of introduced biocontrol strains to outcross with the existing aflatoxin producing A. flavus in the soil . This study aimed to determine the distribution of MAT genes for A. flavus strains originally isolated from soil and maize samples from Makueni and Nandi as described by Nyongesa et al.  and Okoth et al. . This is important to be assessed for two reasons: (1) to ascertain the level of recombination in the field population, which is relative to the proportions of MAT genes present, and (2) to better select a candidate biocontrol strain that will lessen the opportunity for recombining with the indigenous population .
2. Materials and Methods
2.1. Source of Fungal Isolates. Forty-four Aspergillus flavus isolates used in this study were originally isolated from maize and soil samples collected from Eastern (Makueni) and Rift Valley (Nandi) regions of Kenya and were recorded to have capacity to produced aflatoxins [15, 16]. The culture collection was maintained at Mycology Laboratory, University of Nairobi, Kenya.
Nandi lies within latitudes 0[degrees] and 0[degrees] 34" North and longitude 34[degrees]44" and 35[degrees]25" East, altitude between 1300 m and 2500 m above sea level. The area receives approximately 1200-2000 mm of rainfall and average temperatures of 20[degrees]C annually. Makueni area is 1218 m above sea level with average annual temperatures of 24[degrees]C and rainfall between 200 and 1200 mm. The rainfall is unreliable with frequent droughts [2, 7-9].
2.2. Molecular Characterization of Aspergillus Isolates
2.2.1. Extraction of DNA. Fungal isolates from Eastern and Rift Valley parts of Kenya, with determined aflatoxin accumulation levels in yeast extract sucrose (YES) according to Okoth et al. 2012  and Nyongesa et al. 2015  (are shown in Table 1), preserved on silica gel beads at 4[degrees]C were used. Two to three silica gel beads were transferred on potato dextrose agar (PDA) plates under sterile conditions. Each A. flavus strain was cultured in replicates. The isolates were grown under 37[degrees]C for 7 days, and DNA was extracted using a Zymo Research fungal/Bacterial DNA Mini Prep Kit (Epigenetics, Hatfield, South Africa) according to the manufacturer's instructions.
2.3. Analysis of Aspergillus flavus Isolates by Mating Type. The MAT genes were established by a diagnostic polymerase chain reaction (PCR) using primers M1F and M1R for the MAT1-1 gene and M2F and M2R for the MAT1-2 gene [18,19] (see Table 2). The PCR was performed in 20 [micro]l reactions, which included of a 1:10 or 1:100 DNA dilution, 1 U REDTaq DNA polymerase (Sigma-Aldrich Company, Milan, Italy), 2 [micro]l REDTaq buffer supplemented with 1.7 [micro]l of 22 mM Mg[Cl.sub.2] for a final concentration of 3.0 mM, 10 mM deoxyribonucleotide triphosphates, 0.5% Bovine Serum Albumin (BSA), and 0.5 [micro]M of each of the 4 primers (M1F, M1R, M2F, and M2R) [18, 19]. Reactions were run in a Mastercycler ep gradient (Bio-Rad, California, USA) with a thermal profile of 5 min at 95[degrees]C followed by 40 cycles of 30 s at 95[degrees]C, 60 s at 54[degrees]C, and 45 s at 72[degrees]C. The amplified DNA was electrophoresed in 1.5% (w/v) Tris-acetate-Ethylenediaminetetraacetic acid (EDTA) agarose gels, and amplicons were designated as MAT1-1 and MAT1-2 using a 100 bp DNA ladder (exACTGene, Fisher Scientific International) as a size standard.
A concentration of 1% agarose gel was made. The agarose was boiled at 100[degrees]C for 5 minutes in a conical flask and left to cool to 55[degrees]C, and 0.3 [micro]l of ethidium bromide was added while swirling the flask to enable the gel mix with ethidium bromide. The mixture was poured into a gel tank with the combs on and left to solidify. Molecular marker (2 [micro]l) was added to one well and DNA (4 [micro]l) to the other wells and the arrangements were noted. The gel was run for 45 minutes at 80 voltage and viewed under gel doc (Bio-Rad, Molecular Imager Gel Doc[TM] XR-CLASS, Imaging System, California, USA).
3. Results and Discussion
Among the 44 isolates, six strains were MAT1-1 (Figures 1, 2, 3, and 5) and 29 strains had MAT1-2 (Figures 3,4 and 5) while nine strains amplified both MAT genes (Figures 1,2, 3,4, and 5). Figures 2,3,4, and 5 are in the supplemental figures.
In this study, sampled A. flavus strains from Makueni and Nandi include those that contain at least fragments of both MAT genes and those that contain a single MAT gene. Fungi that produce functional MAT1-1 and MAT1-2 genes on the same thallus are self-fertilizing while those with a single MAT gene require compatible MAT1-1 or MAT1-2 nuclei from two different individuals. There were 20.45% isolates with fragments of both MAT genes though Nandi had 11.36%, while in Makueni, the percentage was lower (9.09%). In both regions, MAT1-2 isolates frequency was dominant (61.36%), although the frequency in Nandi was higher (75%) than in Makueni (54.17%). The isolates from Nandi had no MAT1-1 genotypes, while in Makueni their percentage was 15.91%.
Laboratory crosses between strains of A. flavus of opposite mating type genes have successfully been applied to induce sexual recombination between MAT1-1 and MAT12 isolates [20-22]. In our study, most of the isolates from Makueni were either MAT1-1 or MAT1-2, indicating that they are likely heterothallic. Four of the isolates sampled in Makueni contained fragments of both MAT genes (Table 3), indicating the potential for a homothallic existence. The nearly equal distribution of single mating types, as well as the presence of isolates exhibiting evidence of two MAT genes, in this region could be an indication that there is active recombination occurring in the fields where these isolates were sampled. Five of the Nandi isolates exhibited evidence for containing both MAT genes. Of the isolates that contained one or the other MAT gene, no MAT1-1 isolates were identified (Table 3). From this we can infer that recombination is not occurring at a rate comparable to Makueni. Mating tests would allow us to confirm or refute the functionality of these loci.
We may be able to infer a correlation between mating type distribution and aflatoxin outbreaks based on our findings. For example, aflatoxin outbreaks in in Makueni (having a more equal distribution of mating types) have been reported since 1981, while no aflatoxin outbreaks have been reported in Nandi, which has a predominance of MAT1-2 genes [7-9] (Okoth et al., 2016). Sexual recombination between toxigenic and non-aflatoxigenic A. flavus strains in the soil is viewed as a major cause of diversity enabling some of the A. flavus progenies to inherit the ability to produce aflatoxin [5,14,17, 20, 23-25].
Currently, biocontrol is the most favorable technique for lowering preharvest aflatoxin contamination. The strategy includes spreading non-aflatoxigenic A. flavus strain spores, which result in greatly reduced levels of aflatoxin. Both aflatoxin and non-aflatoxin producing strains occupy the same niches in the soil . In our study, the 44 A. flavus strains characterized are agricultural isolates [15, 16]. Existence of a nearly equal distribution of MAT genes in Makueni indicates potential for sexual recombination (Table 3). Atoxigenic MAT1-1IVM300566 (0 [micro]g/kg) as biocontrol could outcross with toxigenic MAT1-2 in the two regions which could facilitate recombination. Therefore, it is advisable to search for an atoxigenic MAT1-2 strain as a candidate biocontrol strain. Ehrlich  suggests that it is important to test frequency of genetic recombination in agricultural environments where non-aflatoxin producing biocontrol has been introduced. Based on our findings and extrapolation to the field, it is important to assess this distribution for a field prior to release of fertile biocontrol strains that can recombine with the indigenous population. We must also verify the fecundity of strains that contain both MAT genes to determine if they are functionally bisexual as Horn et al. did with A. nomius .
This study underscores the importance of investigating the mating type distribution in the field, prior to biocontrol selection and release, to minimize the potential for sexual recombination while promoting efficacy of the biocontrol strain. Our findings indicate that A. flavus strains in Kenya have the potential to harbour both MAT genes, although we are uncertain of their functionality. Also, we show that not all fields will have the same distribution of mating types. The Makueni field population may have higher genetic and chemotype diversity, and potential for sexual recombination, due to the observed distribution of mating types. Selection of a naturally infertile atoxigenic strain as biocontrol would be better here. For Nandi, the distribution is less diverse, and if the MAT1-1?2 isolates are incapable of self-fertilizing or outcrossing, then use of MAT1-2 biocontrol strain would be effective. Another characteristic that would be helpful would be to ensure that the biocontrol strain lacks aflatoxin cluster genes since it might be more difficult to inherit the entire cluster during recombination.
The data used to support the findings of this study are included within the article.
Conflicts of Interest
The authors have no conflicts of interest rgarding the publication of this manuscript.
All the authors have contributed to the manuscript from the preparation to the submission stage.
This work was funded by Deans Grants Committee, University of Nairobi and Exchange Service (DAAD) In-Country
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Ouko Abigael,(1) Okoth Sheila ID), (1) Amugune Nelson, (1) and Vesa Joutsjoki (2)
(1) School of Biological Sciences, University of Nairobi, P.O. Box 30197-00100, Nairobi, Kenya
(2) Natural Resources Institute Finland, FIN-31600, Jokioinen, Finland
Correspondence should be addressed to Ouko Abigael; email@example.com
Received 31 July 2018; Revised 10 September 2018; Accepted 2 October 2018; Published 18 November 2018
Academic Editor: Tibor Janda
Caption: Figure 1: Mating types for A. flavus strains from Makueni and Nandi; L: molecular weight marker, Lane 1: IVM201365, 2: IVM100095,3: IVM100130.
Table 1: Aspergillus flavus isolates from Makueni and Nandi. Strain identity Location Aflatoxin level ([micro]g/kg) 40245BMIG Nandi 116666 40041BM38Y6 Nandi 80664 60801BSIYG Nandi 23883 40224BSIYG Nandi 22203 40296BSY Nandi 12803 60756 Nandi 11292 50400BM2YG Nandi 247 40243BMIB Nandi 151 40013BM126 Nandi 145 40267BSIYG Nandi 96 60661BMIYG Nandi 91 504820BMIYG Nandi 77 50536BMIYG Nandi 77 60690BMIYG Nandi 57 40143BM112G Nandi 46 60572 BM2Y Nandi 46 60795BM8BJ Nandi 22 40025BSIYG Nandi 22 50444BSIYG Nandi 0 1VM201365 Makueni 152965 1VM40018 Makueni 97184 1VM100095 Makueni 92249 1VM100130 Makueni 88878 2M2002LG Makueni 74367 2M1983G Makueni 67350 1VM250WY Makueni 47653 2VM964 Makueni 29484 1VM132G Makueni 23069 2VM882G Makueni 19038 1VM414 Makueni 13574 1VM100957 Makueni 11971 1VM201122 Makueni 11292 1VM130LG Makueni 10199 1VM100079 Makueni 9089 1VM350G Makueni 2912 3VM566G Makueni 1850 2M1983DG Makueni 1165 2M1002LG Makueni 962 1VM195 Makueni 785 1VM131LG Makueni 602 3VM566 Makueni 332 1VM201204 Makueni 22 1VM300566 Makueni 0 Table 2: Sequences of nucleotide primers used in the study. Primer code Target gene Primer sequence PCR product size M1F MAT1-1 ATTGCCCATTTGGCCTTGAA 396 base pairs M1R MAT1-1 TTGATGACCATGCCACCAGA 396 base pairs M2F MAT1-2 GCATTCATCCTTTATCGTCAGC 270 base pairs M2R MAT1-2 GCTTCTTTTCGGATGGCTTGCG 270 base pairs Table 3: Mating type distribution for A.flavus isolates sampled in two counties in Kenya. Strain identity Location Aflatoxin level Mating type ([micro]g/kg) identity using PCR multiplex assay 1VM201365 Makueni 152965 MAT1-1 & MAT1-2 1VM40018 Makueni 97184 MAT1-1 & MAT1-2 1VM100095 Makueni 92249 MAT1-1 1VM100130 Makueni 88878 MAT1-1 2M2002LG Makueni 74367 MAT1-2 2M1983G Makueni 67350 MAT1-1 1VM250WY Makueni 47653 MAT1-2 2VM964 Makueni 29484 MAT1-2 1VM132G Makueni 23069 MAT1-2 2VM882G Makueni 19038 MAT1-2 1VM414 Makueni 13574 MAT1-2 1VM100957 Makueni 11971 MAT1-2 1VM201122 Makueni 11292 MAT1-1 1VM130LG Makueni 10199 MAT1-1 1VM100079 Makueni 9089 MAT1-2 1VM350G Makueni 2912 MAT1-1 & MAT1-2 3VM566G Makueni 1850 MAT1-2 2M1983DG Makueni 1165 MAT1-2 2M1002LG Makueni 962 MAT1-2 1VM195 Makueni 785 MAT1-1 1VM131LG Makueni 602 MAT1-2 3VM566 Makueni 332 MAT1-2 1VM201204 Makueni 22 MAT1-1 & MAT1-2 1VM300566 Makueni 0 MAT1-1 40245BMIG Nandi 116666 MAT1-2 40041BM38Y6 Nandi 80664 MAT1-2 60801BSIYG Nandi 23883 MAT1-2 40224BSIYG Nandi 22203 MAT1-2 40296BSY Nandi 12803 MAT1-1 & MAT1-2 60756 Nandi 11292 MAT1-2 50400BM2YG Nandi 247 MAT1-2 40243BMIB Nandi 151 MAT1-1 & MAT1-2 40013BM126 Nandi 145 MAT1-2 40267BSIYG Nandi 96 MAT1-2 60661BMIYG Nandi 91 MAT1-2 504820BMIYG Nandi 77 MAT1-1 & MAT1-2 50536BMIYG Nandi 77 MAT1-2 60690BMIYG Nandi 57 MAT1-2 40143BM112G Nandi 46 MAT1-1 & MAT1-2 60572 BM2Y Nandi 46 MAT1-2 60795BM8BJ Nandi 22 MAT1-2 40025BSIYG Nandi 22 MAT1-2 50444 BSIYG Nandi 0 MAT1-1 & MAT1-2
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|Title Annotation:||Research Article|
|Author:||Abigael, Ouko; Sheila, Okoth; Nelson, Amugune; Joutsjoki, Vesa|
|Publication:||Advances in Agriculture|
|Date:||Jan 1, 2018|
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