Advances in Agriculture
Volume 2018 (2018), Article ID 3095096, 6 pages
https://doi.org/10.1155/2018/3095096

Research Article

Characterization of Mating Type Genes in Aspergillus flavus Populations from Two Locations in Kenya

Ouko Abigael,¹ Okoth Sheila,¹ Amugune Nelson,¹ and Vesa Joutsjoki²

¹School of Biological Sciences, University of Nairobi, P.O. Box 30197-00100, Nairobi, Kenya
²Natural Resources Institute Finland, FIN-31600, Jokioinen, Finland

Correspondence should be addressed to Ouko Abigael; abbykongete@uonbi.ac.ke

Received 31 July 2018; Revised 10 September 2018; Accepted 2 October 2018; Published 18 November 2018

Academic Editor: Tibor Janda

Copyright © 2018 Ouko Abigael et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

In this study, the possibility of sexual reproduction in sampled Aspergillus flavus strains was evaluated by assessing the distribution of mating type (MAT) genes, which are known to control sexual character among fungi, for two counties in Kenya. Forty-four isolates from Nandi and Makueni counties were genotyped by MAT using a multiplex polymerase chain reaction assay. The primer pair for the MAT1-1 amplified a 396 base pair (bp) fragment containing an α-box motif, and MAT1-2 primers targeted a 270 bp segment with a high mobility group protein. The MAT1-2 genes dominated in both regions although the frequency was higher in Nandi (75%) than in Makueni (54.17%). There were no MAT1-1 genes sampled in Nandi, and in Makueni their proportion was 15.91%. The percentage of isolates that amplified for both MAT genes in Makueni was 9.09%, while in Nandi it was 11.36%. Currently, use of aggressive aflatoxin non-producing A. flavus strains as biocontrol is the most promising preharvest aflatoxin control strategy in Kenya. However, we address the possibility of introduced biocontrol strains to breed with existing aflatoxin producing strains in nature, which could lead to the generation of A. flavus offspring capable of aflatoxin production while also being aggressive colonizers and possibly increasing the burden of aflatoxin exposure in food.

1. Introduction

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°C, but the fungus readily grows between temperatures of 25 and 42°C and will even grow at temperatures from 12 to 48°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. [6] 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. [8] reported high aflatoxin levels in Makueni compared to Nandi. Aflatoxin exposure through milk for children younger than 5 years was 4 × 10⁻⁴ and 1 × 10⁻⁴ µg/kg per day in Makueni and Nandi, respectively; the exposure of nursing children through breast milk was 6 × 10⁻³ and 1 × 10⁻⁶ µg/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. Non-aflatoxin producing A. flavus is introduced into the environment and outcompetes naturally occurring aflatoxin producing strains in the soil [10]. 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) [14]. 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 [14]. 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. [15] and Okoth et al. [16]. 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 [17].

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° and 0°34′′ North and longitude 34°44′′ and 35°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°C annually. Makueni area is 1218 m above sea level with average annual temperatures of 24°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 [16] and Nyongesa et al. 2015 [15] (are shown in Table 1), preserved on silica gel beads at 4°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°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.

Table 1: Aspergillus flavus isolates from Makueni and Nandi.

Strain identity
Location
Aflatoxin level (µg/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

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 µl reactions, which included 1µl of a 1:10 or 1:100 DNA dilution, 1 U REDTaq DNA polymerase (Sigma–Aldrich Company, Milan, Italy), 2 µl REDTaq buffer supplemented with 1.7 µl of 22 mM MgCl₂ for a final concentration of 3.0 mM, 10 mM deoxyribonucleotide triphosphates, 0.5% Bovine Serum Albumin (BSA), and 0.5 µM 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°C followed by 40 cycles of 30 s at 95°C, 60 s at 54°C, and 45 s at 72°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.

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

A concentration of 1% agarose gel was made. The agarose was boiled at 100°C for 5 minutes in a conical flask and left to cool to 55°C, and 0.3 µl 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 µl) was added to one well and DNA (4 µl) 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™ 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.

Figure 1: Mating types for A. flavus strains from Makueni and Nandi; L: molecular weight marker, Lane 1: IVM201365, 2: IVM100095, 3: IVM100130.

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 MAT1-2 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.

Table 3: Mating type distribution for A. flavus isolates sampled in two counties in Kenya.

Strain identity
Location
Aflatoxin level (µg/kg)
Mating type 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

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 [26]. 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 MAT1-1 IVM300566 (0 µg/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 [10] 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 [6].

4. Conclusion

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+MAT1-2 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.

Data Availability

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.

Authors’ Contributions

All the authors have contributed to the manuscript from the preparation to the submission stage.

Acknowledgments

This work was funded by Deans Grants Committee, University of Nairobi and Exchange Service (DAAD) In-Country Scholarship Programme.

Supplementary Materials

Mating types for A. flavus strains from Makueni and Nandi. (Supplementary Materials)

References

L. C. Grubisha and P. J. Cotty, “Genetic isolation among sympatric vegetative compatibility groups of the aflatoxin-producing fungus Aspergillus flavus,” Molecular Ecology, vol. 19, no. 2, pp. 269–280, 2010.

S. Okoth, “Improving the evidence base on aflatoxin contamination and exposure in Africa. Strengthening the Agriculture-Nutrition Nexus. Review,” CTA Working paper 16/13, 2016.

W. Felicia, N. Clarre, T. Marites, and L. Yan, “The health economics of aflatoxin. Global burden of disease,” Aflatoxin control, vol. 4, p. 20, 2011.

M. Paoletti, S. Elke, A. David, and D. Paul, “Sex and mating in Aspergillus; asexual or not? Aspergillus and Aspergillosis website,” Living with it working with it treating it, 2004.

B. W. Horn, G. G. Moore, and I. Carbone, “Sexual reproduction in Aspergillus flavus,” Mycologia, vol. 101, no. 3, pp. 423–429, 2009.

B. W. Horn, G. G. Moore, and I. Carbone, “Sexual reproduction in aflatoxin-producing Aspergillus nomius,” Mycologia, vol. 103, no. 1, pp. 174–183, 2011.

A. J. Sirma, E. O. Ouko, G. Murithi et al., “Prevalence of aflatoxin contamination in cereals from Nandi county, Kenya. International journal of agricultural sciences and veterinary medicine,” in Proceedings of the Prevalence of aflatoxin contamination in cereals from Nandi county, Kenya. International journal of agricultural sciences and veterinary medicine, pp. 2320–3730, 2015.

E. K. Kang’ethe, M. Gatwiri, A. J. Sirma et al., “Exposure of Kenyan population to aflatoxins in foods with special reference to Nandi and Makueni counties,” Food Quality and Safety, vol. 1, no. 2, pp. 131–137, 2017.

S. Okoth, L. J. Rose, A. Ouko et al., “Field evaluation of resistance to aflatoxin accumulation in maize inbred lines in Kenya and South Africa,” Journal of Crop Improvement, vol. 31, no. 6, pp. 862–878, 2017.

K. C. Ehrlich, “Non-aflatoxigenic Aspergillus flavus to prevent aflatoxin contamination in crops: Advantages and limitations,” Frontiers in Microbiology, vol. 5, 2014.

T. Ogunbayo, K. Callicott, C. Probst, and P. Cotty, “An atoxigenic vegetative compatibility group of Aspergillus flavuswidely adapted to maize production in Africa and North America,” Phytopathology, vol. 103, pp. 105-106, 2013.

J. Atehnkeng, P. S. Ojiambo, M. Donner et al., “Distribution and toxigenicity of Aspergillus species isolated from maize kernels from three agro-ecological zones in Nigeria,” International Journal of Food Microbiology, vol. 122, no. 1-2, pp. 74–84, 2008.

K. Hell, P. Fandohan, R. Bandyopadhyay et al., “Pre- and post-harvest management of aflatoxin in maize,” in Mycotoxins: Detection Methods, Management, Public Health and Agricultural Trade, J. F. Leslie, R. Bandyopadhyay, and A. Visconti, Eds., CABI, Wallingford, UK, 2008.

K. C. Ehrlich, G. G. Moore, J. E. Mellon, and D. Bhatnagar, “Challenges facing the biological control strategy for eliminating aflatoxin contamination,” World Mycotoxin Journal, vol. 8, no. 2, pp. 225–233, 2014.

B. W. Nyongesa, S. Okoth, and V. Ayugi, “Identification Key for <i>Aspergillus</i> Species Isolated from Maize and Soil of Nandi County, Kenya,” Advances in Microbiology, vol. 5, no. 4, pp. 205–229, 2015.

S. Okoth, B. Nyongesa, V. Ayugi, E. Kangethe, H. Korhonen, and V. Joutsjoki, “Toxigenic potential of Aspergillusspecies occurring on maize kernels from two Agro-Ecological zones in Kenya,” Toxins, vol. 4, no. 11, pp. 991–1007, 2012.

G. G. Moore, J. L. Elliott, R. Singh et al., “Sexuality Generates Diversity in the Aflatoxin Gene Cluster: Evidence on a Global Scale,” PLoS Pathogens, vol. 9, no. 8, 2013.

J. H. Ramirez-Prado, G. G. Moore, B. W. Horn, and I. Carbone, “Characterization and population analysis of the mating-type genes in Aspergillus flavus and Aspergillus parasiticus,” Fungal Genetics and Biology, vol. 45, no. 9, pp. 1292–1299, 2008.

R. Wada, J.-I. Maruyama, H. Yamaguchi et al., “Presence and functionality of mating type genes in the supposedly asexual filamentous fungus Aspergillus oryzae,” Applied and Environmental Microbiology, vol. 78, no. 8, pp. 2819–2829, 2012.

B. W. Horn, R. B. Sorensen, M. C. Lamb et al., “Sexual reproduction in aspergillus flavus sclerotia naturally produced in corn,” Journal of Phytopathology, vol. 104, no. 1, pp. 75–85, 2014.

J. Varga, G. Szigeti, N. Baranyi, S. Kocsubé, C. M. O’Gorman, and P. S. Dyer, “Aspergillus: Sex and Recombination,” Mycopathologia, vol. 178, no. 5-6, pp. 349–362, 2014.

B. W. Horn, R. M. Gell, R. Singh, R. B. Sorensen, and I. Carbone, “Sexual reproduction in aspergillus flavus sclerotia: Acquisition of novel alleles from soil populations and uniparental mitochondrial inheritance,” PLoS ONE, vol. 11, no. 1, 2016.

R. A. Olarte, B. W. Horn, J. W. Dorner et al., “Effect of sexual recombination on population diversity in aflatoxin production by Aspergillus flavus and evidence for cryptic heterokaryosis,” Molecular Ecology, vol. 21, no. 6, pp. 1453–1476, 2012.

M. L. Paret, G. E. Vallad, D. R. Averett, J. B. Jones, and S. M. Olson, “Photocatalysis: effect of light-activated nanoscale formulations of TiO₂ on Xanthomonas perforans and control of bacterial spot of tomato,” Journal of Phytopathology, vol. 103, no. 3, pp. 228–236, 2013.

J. Chepsergon, E. K. Kiprop, and L. A. Mwamburi, “Biocontrol of aflatoxins in corn using atoxigenic Aspergillus flavus,” in Proceedings of the International Journal of Science and research, vol. 12, pp. 1954–1958, 2014.

Y. Yin, L. Yan, J. Jiang, and Z. Ma, “Biological control of aflatoxin contamination of crops,” Journal of Zhejiang University, Science B, vol. 9, no. 10, pp. 787–792, 2008.