Maize (Zea mays L.) is one of the famous food crops in Kenya. It is always contaminated by fungi, Fusarium spp. producing mycotoxins, The most common mycotoxin is Fumonisin FB1. Whose intake above 2.0 mg/kg body weight/day has a role in development of Oesophangeal cancer. No data is readily available on the genetic diversity of Fusarium species in various maize genotypes in Kenya. This study was to determine diversity of Fusarium fungi infecting maize in Nakuru County-Kenya. A Purposive sampling was used in collecting maize grains from 277 farmers. Fusarium species were isolated and confirmed using molecular techniques. DNA sequencing of Translation elongation factor 1-alpha (TEF-1) gene was done. The data was edited using vector NTI software and blasted in NCBI data base. Fusarium was identified using spore morphology on potato dextrose Agar (PDA). DNA sequence analysis revealed presence of F. temperatum, F. boothii, and F. verticillioides. The finding was to enlighten; the public, farmers, and agricultural officers on the infections of maize by specific Fusarium sp., hence the need to grow less infected maize genotypes H614, H6218 and H6210.
Research Article
Diversity of Fusarium Species Infecting Maize Grown in Nakuru County, Kenya
https://doi.org/10.21203/rs.3.rs-5306045/v1
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Kenya's primary food crop is maize (Zea mays L.), with a production of 39.0 million (90 kilogram) bags in 2014 (Ministry of Agriculture and Livestock Economic Review 2015). Nakuru County used an area of 86,504 hectares of land to produce 1,765,714 90 kg bags in total the year (Economic Review of Agriculture 2015). A number of factors limit Kenya's ability to produce maize, including soil acidification from the continuous use of Di-ammonium Phosphate (DAP) fertilizer, inability to obtain improved and certified seeds, and presence of diseases such as maize ear rot and maize lethal necrosis disease (MLND) among others.
Maize productivity is severely limited by the ear rot fungus Fusarium, Aspergillus, Penicillium, and Sternocarpella in particular (Olanya et al., 1997; Hefny et al., 2012). Studies by Duan et al. (2016) documented the severity of maize ear rot largely caused by F. verticillioides and F. graminearum species. China reported three complexes species of F. graminearum, F. meridionale, and F. boothii, using TEF-1α gene sequence (Duan et al., 2016).
Fusarium verticillioides can infect maize by a variety of mechanisms, including roots, insect pest-caused lesions in the seed, and silk produced by flying conidia (Bacon et al., 2008; Cao et al., 2014). When an infection spreads throughout the roots of maize seedlings, it first shows no symptoms. However, as the crop matures, the infection spreads throughout the whole plant, resulting in leaf blight, stalk rot, and ear rot. Corn kernels become infected when contaminated silk is used (Munkvold and Desjardins, 1997; Munkvold, 2003). The endosperm and embryo are the two main tissues that Fusarium verticillioides colonizes. In the endosperm, it results in increased levels of FB1 (Shim et al., 2003). It has been found that in tropical and subtropical climates, Fusarium verticillioides produces large levels of fumonisin (Shephard et al., 2005; Marasas, 2001; Reddy et al., 2009). In addition to products derived from maize, fumonisins have also been found in rice (Omurtag, 2008; Huong et al., 2016). Maize may also contain citrinin, cyclopiazonic acid, penicillic acid, ochratoxin A, zearalenone, trichothecenes (T-2), and deoxynivalenol (DON or vomitoxin) (Logrieco et al., 2002).
Identification of Fusarium species has been done using morphological characterization (Hafizi, et al., 2013). By using morphological approaches, Hafizi and his fellow researchers were able to determine diversity of the Fusarium species. Morphological characterization has been based on single-spore isolate, colour on the media and macrospore characteristics (Nelson et al., 1983; Britz et al., 2002; Leslie et al., 2006; Rahjoo et al., 2008).
Morphological identification of F. verticillioides carried out by Leslie et al. (2006) was based on the colony formed on the media and microscopic examination of the macro and microspores. Fusarium verticillioides produces abundant single-celled micro-conidia in distinctive long chains (Leslie et al., 2006; Aoki et al., 2014). In a normal conidium development of a wall building zone (WBZ) assists in the development of the conidium (Glenn et al., 2004). It was reported that besides the use of morphological characteristics, F. thapsinum from F. verticillioides and F. graminearum are effectively differentiated by use of molecular techniques (Glenn et al., 2004).
Fusarium temperatum, has been identified and characterized in Spain, Poland, Argentina, France and China and was found to be pathogenic in maize seedlings and stalks (Boutigny et al., 2011). Fusarium temperatum was distinguished from F. subglutinans within Fusarium fujikuroi species complex (FFSC) by use of translation elongation factor-1 gene (tef-1α). Studies of mating compatibility and metabolite profiling have also been used in identification of F. temperatum. Isolates of F. temperatum were described after isolation from maize in Argentina (Fumero et al., 2015; Gromadzka et al., 2019). Other countries where F. temperatum had been isolated include; Spain, France and Poland (Varela et al., 2013; Boutigny et al., 2017). It had also been isolated in China and proved to be pathogenic and able to produce fumonisins in infected maize ears (Wang et al., 2014).
Using DNA sequence-based identification provides a much stronger inference. The strength of the inference depends on the sequences available in the databases. An exact sequence match with a known isolate in the database is considered very close to unambiguous species identification. However, this is not always the case. A query sequence that differ by one or few bases may be an allelic variant not present in FUSARIUM-ID from a known species. It may also be that the sequence is from a new species that is not represented in FUSARIUM-ID or it corresponds to a species that is currently poorly defined. Molecular identification of F. verticillioides strains have been carried out at the gene level using polymerase chain reaction (Patino et al., 2004).
Maize samples collection
During sample collection, maize grains, 12 weeks Harvest Time Points After Physiological Maturity (HTPAPM) according to Alakonya et al. (2009) were collected from each farmer. Bulk samples were made consisting of similar maize genotypes collected from each of the farmers in Molo Sub-County. Similarly the same maize genotypes collected from each farmer in Njoro Sub-County were bulked. This gave a total of 13 bulk samples, since five maize genotypes which were previously given to farmers in Njoro, formed five bulks samples; while eight maize genotypes previously distributed to the farmers in Molo formed eight bulk samples. In each bulk sample, an eighth of a kilogram of maize grain of each genotype from each of the farmers in the study area was collected, figure 1.
Isolation and identification of Fusarium species
Symptomless maize kernels collected from the farmers stores were taken to Kenyatta University, Microbiology laboratory for mycological isolation of Fusarium spp. The media, Pentachloronitrobenzene (PCNB) agar, which is a selective media for promotion of growth of only Fusarium fungi by inhibiting growth of other fungi species was used. Data on the incidence was first normalized and the percentage incidence of Fusarium sp. in each maize genotypes was computed as;
Five grains of each genotype from each bulk sample, surface sterilized by immersion in 70 % ethanol for five seconds followed by 1.0 % NaOCl for 90 seconds. The kernels were rinsed in three changes of sterile distilled water for 20 seconds, blot dried between two sterile filter papers and inoculated on petri dishes containing Pentachloronitrobenzene agar (PCNB) medium. This was replicated five times. The inoculated petri dishes were incubated at 25 °C for 5 days to allow growth of the fungi and the samples analyzed for percentage incidence of Fusarium spp. infection. Each colony formed from each of the kernels was sub-cultured by transferring to potato dextrose agar (PDA) slants for identification.
Morphological identification of Fusarium isolates
The first identification was based on colour morphology of the mycelium according to procedure by Nelson et al. (1983) after 10 days growth on PDA slant. In the second step, carnation leaf agar cultures were used for identification based on morphology of the micro and macroconidia according to Leslie et al. (2006). Microscopic observation of the spores was carried out using Leica compound microscope (Leica DM4, P DM 2700, Leica microsystems, Buffalo groove, U.S.A) which was mounted in-build camera. This microscope uses a computer Programme LAS EZ version 1.6.0 which enabled documentation of macrospores and microspores observed.
Molecular identification of Fusarium species infecting various maize genotypes
Extraction of the DNA from each Fusarium isolate as a representative sample was carried out using procedure described by Matny (2014) for plant DNA extraction. This was achieved by culturing the fungus directly in an Eppendorf tube and suppressing the phenolization step. In this method, a 1.5 ml Eppendorf tube was filled with a 500 µl of liquid Potato-dextrose broth medium. The culture was started by inoculating a single hyphal thread of each of the Fusarium fungal isolate and incubated for 72 hours at 25 °C. The mycelial mat from each of the isolate was pelleted by centrifugation for 5 minutes at 13,000 rpm in a microfuge, washed with 500 µl of TE buffer and pelleted again. The TE buffer was then decanted and 300 µl of extraction buffer added. The mycelium was crushed with a conical grinder (Treff AG, Degersheim, Switzerland), fitting the tube and centrifuged by hand potter at 200 rpm for 10 minutes. After that, 150 µl of 3.0 M sodium acetate, (pH 5.2) was added and tubes placed at -20 °C for ten minutes. Tubes were then centrifuged in a microfuge at 13,000 rpm and the supernatant transferred to another tube. An equal volume of isopropanol was added. After 5 minutes, the precipitated DNA was pelleted by centrifugation in a microfuge at 13,000 rpm. This was therefore washed using 70 % ethanol and then air dried on paper towel for 20 minutes. The pellet was re-suspended in 50 µl of TE for assessment of the DNA quality by running in a 1.0 % agarose gel at 80 volts for 30 minutes together with a known size, 1 kb universal gene ruler (Biolab).
PCR amplification and sequencing
Fusarium isolates were identified by sequencing the translation elongation factor-1 alpha gene (α-TEF gene) region. DNA was extracted by CTAB method (Stępień et al., 2013). The PCR was carried out using forward primer, TEF 1-α gene, EF1, 5’GTGGGGCATTTACCCCGCC3’ and reverse primer EF2, 5’ACGAACCCTTACCCACCTC3’. A PCR reaction mixture (25 μl) for TEF 1-α gene consisted of 12.8 μl milli Q H2O, 2.5 μl 10x buffer, 2.5 μl MgCl2, and 0.5 μl dNTP mix (10 mM each). A mount of 0.8 μl forward primer EF1 (10 mM), 0.8 μl reverse primer EF2 (10 mM), 0.1 μl Taq (Biotech) and 5 μl DNA sample was used. The PCR conditions were ; initial denaturation at 95 oC for 75 seconds, 35 cycles of denaturation at 95 oC for 15 seconds, annealing temperature at 53 oC for 30 seconds and a primer extension at 72 oC and final extension at 72 oC.
Sequencing alignment and phylogenetic analysis
Using Molecular Evolutionary Genetic Analysis software, (MEGA 7; http://www.megasoftware.net), the ITS sequences were edited and aligned. To assess the relationships between genetic traits of the identified Fusarium species, ITS sequences were used to generate phylogenetic tree by grouping the isolates into clusters. The aligned sequences were therefore deposited in the GenBank database.
The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model (Tamura et al., 2012). The tree with the highest log likelihood (-1474.6907) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Joining and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree was drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 25 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA 7 (Kumar et al., 2018).
Data management
Molecular DNA sequence data obtained was edited and aligned using vector NTI software and further BLAST on the NCBI sequence database used to find best matches for each obtained sequence for every isolate. Phylogenic and molecular evolutionary analysis was carried out using Mega version 7 (Tamura, 1992; Tamura et. al., 2013). Neighbour joining approach for geographical representation of pairwise nucleotide sequence divergence among the fungi strains was used.
Result of enumeration of Fusarium spp. identified using mycelia colour
Eight different Fusarium spp. identified using colour of the mycelium on PDA media were;F. dimerum, F. equseti, F. solani, F. verticillioides, F. oxysporum, F. culmorum, F.graminearum and unknown Fusarium species. Number of Fusarium species that was recorded in the different maize genotypes varied. The result showed that H6213 maize genotype was infected by six out of the eight Fusarium species (75.0 %), Pioneer, H626 and H614 were infected by 37.5 % of the Fusarium species. H6210 and H624 maize genotypes were only infected by F. verticillioides as shown in Table 1.
Table 1: Fusarium species infecting maize genotypes
|
Maize genotype |
Number of Fusarium species (n = 8) |
Percent |
|
H6218 |
4 (F. solani, F. verticillioides, F. oxysporum, Unknown F. species) |
50.0 |
|
H6213 |
6 (F. solani, F. verticillioides, F. oxysporum, F. culmorum, F. graminearum, Unknown F. species) |
75.0 |
|
H6210 |
1 (F. verticillioides,) |
12.5 |
|
H614 |
3 (F. verticillioides, F. oxysporum, F. graminearum,) |
37.5 |
|
H626 |
3 (F. verticillioides, F. graminearum, Unknown F. species) |
37.5 |
|
H624 |
1 (Unknown F. species) |
12.5 |
|
H629 |
4 (F. equiseti, F. verticillioides, F. graminearum, Unknown F. species) |
50.0 |
|
Pioneer |
3 (F. dimerum, F. culmorum, Unknown F. species) |
37.5 |
Fusarium species spore morphology
Spore identification of 10 day old Fusarium cultures grown on CLA medium showed 8 different Fusarium sp. Microscopic observations of the macrospores were as shown in Figure 4. The Fusarium sp. of interest i.e. Fusarium verticillioides macrospores were slender, uniform in size and thick-walled. The macrospores were straight to moderately curved with the ventral straight surface and smooth dorsal side. They had 5 – 6 septate which are distinct as shown in Figure 4B. Figure 4A shows a F. temperatum isolate macroconidia having a typical four‐septa with a foot‐shaped basal cell, figure 2.
Molecular identification of Fusarium species infecting maize
On gel electrophoresis, it was observed that Fusarium species’ DNA extracted from the fungal mycelium had a high molecular weight, pure DNA. The DNA samples were quantified on 1.0 percent Agarose gel electrophoresis using 100 ng/μl of 1kb standard Lambda DNA. The DNA quantification revealed that the concentration ranged from 10 to 30 ng/μl of isolated DNA, Figure 3.
Figure 3. shows images of quality DNA bands on 1.0 % agarose gel. It shows sizes of high molecular weight, genomic DNA of 10 samples in relation to Molecular marker 1kb. Lane 1, UnknownFusarium species; Lane 2, F. solani; Lane 3, F. dimerum; Lane 4, F. Culmorum; Lane 5, unknown B; Lane 6, F. oxysporum; Lane 7, F. graminearum; Lane 8, F. equiseti; Lane 9, unknown Fusarium species; Lane 10, F. verticillioides,
Fusarium species identified using molecular DNA sequences
Based on the Fusarium DNA sequences obtained, the result confirmed that; sample L10-EF-1 was Fusarium verticillioides isolate with an NCBI data base, which showed a percentage similarity of 97.2 % to F. verticillioides isolate KF562141. Sample L7_EF-1 was Fusarium boothii which showed a similarity of 96.1 % to isolate KX269067 in the NCBI data base. Samples L1_EF-1, L2_EF-1, L4_EF-1 and L6_EF-1 were strains of F. temperatum which showed percentage similarity of between 96.5 – 99.1 % as shown in Table 2.
Table 2: DNA sequences using Mega 7 Programme
|
Sample |
Bit score |
Grade (%) |
Hit start |
Hit end |
Close NCBI Accession |
Description |
|
L1_EF1 |
1175.59 |
97.5 |
656 |
8 |
KC964112 |
F. temperatum, |
|
L2_EF1 |
1201.44 |
98.2 |
666 |
14 |
KC964117 |
F. temperatum, |
|
L4_EF1 |
1166.36 |
99.1 |
649 |
7 |
KC964119 |
F. temperatum, |
|
L6_EF1 |
1160.36 |
96.5 |
649 |
14 |
KC964119 |
F. temperatum, |
|
L7_EF1 |
1125.73 |
96.1 |
38 |
667 |
KX269067 |
F. boothii |
|
L10_EF1 |
1188.52 |
97.2 |
13 |
655 |
KF562141 |
F. verticillioides |
Phylogenetic tree showing diversity of Fusarium sp.
Figure 4 shows a phylogenetic tree based on TEF-1α gene showing three Fusarium isolate clusters (cluster I, II and III). Number shown at the nodes in the dendrogram indicates the percentage bootstrap support values for 1000 interactions. Cluster I comprised of the isolates L1_EF1, L2_EF1, L4_EF1 and L6_EF1. Cluster II comprised of L10_EF1 while cluster III had L7_EF1. Samples marked in red colour are the research samples while the rest were sequences from the Genebank NCBI accessions. GenBank accession numbers for each mitogenomic sequences are shown in parentheses. The tree confirmed mainly three groups; group I (F. temperatum), group II (F. verticillioides) and group III which had F. boothii.
Figure 4 shows the genetic relationship between F. verticillioides (sample L10_EF-1), F. boothii (sample L7_EF-1) isolates and F. temperatum (sample L1_EF-1, L2_EF-1, L4_EF-1 and L6_EF-1). Among the group of F. temperatum, sample L1_EF-1 was much closer to L2_EF-1 while sample L4_EF-1 and L6_EF-1 formed a sub-group.
Morphological identification of Fusarium spp.
In the present study, the first identification of Fusarium species carried out using morphological characteristics separated different Fusarium fungi on PDA slants as shown in figure 2. Similar identification had been reported by Nelson et al. (1983). This was followed by microscopic identification that was carried out on the spores formed by the different Fusarium species in-vitro on CLA media indicated in figure 2 using the procedure by Leslie et al. (2006). These two morphological methods of identification relied on cultural characteristics and were dependent on visual assessment by the eye based on the colony formed on the media and microscopic examination of the macro and the microspores. Research by Leslie et al. (2006) and Nelson et al. (1983) used different methods of identification of Fusarium species. Using the two methods of identification, F. verticillioides produced abundant single-celled micro-conidia in distinctive long chains (Leslie et al., 2006). Glenn et al. (2004) used powerful microscopes to view the small hyaline micro-conidia, a rising from morphologically simple phialides, as distinct characteristics. Studies have shown that in a normal conidium development, a specific zone at the phialide apex, referred to as wall building zone (WBZ) was present to initiate and assists in the development of the conidium (Glenn et al., 2004). Morphological identification of F. temperatum was based on the macro-conidia usually having four‐septa with a foot‐shaped basal cell. This makes a distinct difference from its most closely related species F. subglutinans and others found within Gibberella fujikuroi complex species. In the present study, seven morphologically identified Fusarium species and one unknown Fusarium sp. were isolated from the eight maize genotypes. These Fusarium species were therefore subjected to further identification using DNA sequencing.
Molecular analysis using DNA sequences
Molecular identification of Fusarium spp. in this study, which was carried out using DNA sequence comparisons of the translation elongation factor 1-α gene regions, clearly showed that the Fusarium isolates infecting maize in Nakuru County were;F. verticillioides, F. temperatum and F. boothi. Strains of F. temperatum are closely related to F. subglutinans and F. boothi which belongs to F. graminearum species complex (FGSC) as had been reported previously by Aoki et al. (2012). Findings from the present study therefore reveal that F. temperatum is reported for the first time in maize samples in Kenya. This result indicated that, besides the use of morphological techniques in identification (Leslie et al., 2006), the most accurate method of identification is by using molecular DNA sequence analysis. In the present study, the five Fusarium species that were identified using morphological characteristics were only three (F. temperatum, F. verticillioides and F. boothii) by using DNA sequences analysis as shown in phylogenetic tree Figure 4.
Visually, ear rot symptomless maize kernels used for human consumption in Nakuru County, Kenya, were infected by more than one Fusarium spp. The most dominant Fusarium species in ear rot symptomless maize genotypes in Nakuru County was F. verticillioides which had 75.0 % dominance of the maize genotypes. Using morphological characteristics, the lowest dominating Fusarium sp. was F. dimerum and F. equiseti which recorded 12.5 % dominance in the maize genotypes.
Molecular analysis in identification of Fusarium spp. is the most accurate method in genetic diversity determination and it revealed presence of a novel F. temperatum and F. boothii.
In an in vitro culture, the use of the colour of the mycelium and pigmentation of the medium, is the preliminary method for the identification of Fusarium species. After preliminary identification, further authentication of the various Fusarium species by using DNA sequencing techniques gives the accurate results to confirm Fusarium species identity.
ACKNOWLEDGEMENTS
I wish to acknowledge Kenyatta University where the research was carried out. My gratitude to Dr. J.F. (Hanneke) Alberts and mycotoxicology research group, Cape Peninsula University of Technology, South Africa, for the provision of Fumonisin FBs standards used. Thanks to Prof. Steven Runo, Kenyatta University for DNA sequence analysis. I appreciate Njoro and Molo Sub-County Agricultural officers and farmers.
DATA AVAILABILITY STATEMENT
Data used for this study were primary data obtained by the researcher. The data is available as already presented in the document. Other detailed information is with the Author.
- Alakonya A. E. (2004). Ear rot fungi and fumonisin B1 levels in selected maize varieties at different harvest intervals in Western Kenya. MSc thesis, Kenyatta University, Nairobi, Kenya.
- Alberts, J. F., Van Zyl, W. H., and Gelderblom, W. C. (2016). Biologically based methods for control of fumonisin-producing Fusarium species and reduction of the fumonisins. Frontiers in Microbiology, 7, 548.
- Alexander, N. J., Proctor, R. H., and McCormick, S. P. (2009). Genes, gene clusters, and biosynthesis of trichothecenes and fumonisins in Fusarium. Toxin reviews, 28(2-3); 198-215.
- Aoki, T., O’Donnell, K. and Geiser, D. M. (2014). Systematics of key phytopathogenic Fusarium species: current status and future challenges. Journal of General Plant Pathology, 80(3), 189-201.
- Aoki, T., Ward, T. J., Kistler, H. C. and O'Donnell, K., (2012). Systematics, phylogeny and trichothecene mycotoxin potential of Fusarium head blight cereal pathogens. Mycotoxins, 62, 91–101.
- Bacon, W. C., Glenn, A. E. and Yates I. E. (2008). Fusarium verticillioides: managing the endophytic association with maize for reduced fumonisins accumulation. Toxin Reviews, 27; 1 -36
- Bacon, C. W., Yates, I. E., Hinton, D. M., and Meredith, F. (2001). Biological control of Fusarium moniliforme in maize. Environmental Health Perspectives, 109 (suppl 2); 325-332.
- Bojja, R. S., Cerny, R. L., Proctor, R. H., and Du, L. (2004). Determining the biosynthetic sequence in the early steps of the fumonisin pathway by use of three gene-disruption mutants of Fusarium verticillioides. Journal of Agricultural and Food Chemistry, 52(10), 2855-2860.
- Boutigny, A. L., Scauflaire, J., Ballois, N., and Ioos, R. (2017). Fusarium temperatum isolated from maize in France. European Journal of Plant Pathology, 148(4), 997-1001.
- Britz, H., Steenkamp, E. T., Coutinho, T. A., Wingfield, B. D., Marasas, W. F., and Wingfield, M. J. (2002). Two new species of Fusarium section Liseola associated with mango malformation. Mycologia, 94(4), 722-730.
- Cao, A., Santiago, R., Ramos, A. J., Souto, X. C., Aguín, O., Malvar, R. A., and Butrón, A. (2014). Critical environmental and genotypic factors for Fusarium verticillioides infection, fungal growth and fumonisin contamination in maize grown in northwestern Spain. International Journal of Food Microbiology, 177, 63-71.
- Dall'Asta, C., Falavigna, C., Galaverna, G., and Battilani, P. (2012). Role of maize hybrids and their chemical composition in Fusarium infection and fumonisin production. Journal of Agricultural and Food Chemistry, 60(14), 3800-3808.
- Desjardins, A. E., and Proctor, R. H. (2007). Molecular biology of Fusarium mycotoxins. International Journal of Food Microbiology, 119(1-2), 47-50.
- Desjardins, A. E., Plattner R.D., and Proctor R. H. (1996) Linkage among genes responsible for fumonisin biosynthesis in Gibberellafujikuroi mating population A. Applied Environmental Microbiology. 62, 2571–2576.
- Dohnal, V., Ježková, A., Polišenská, I., and Kuca, K. (2010). Determination of fumonisins in milled corn grains using HPLC-MS. Journal of chromatographic science, 48(8), 680-684.
- Duan, C., Qin, Z., Yang, Z., Li, W., Sun, S., Zhu, Z., and Wang, X. (2016). Identification of pathogenic Fusarium spp. causing maize ear rot and potential mycotoxin production in China. Toxins, 8(6), 186.
- Economic Review of Agriculture (2015). Ministry of Agriculture Livestock and fisheries, Kenya.
- Fandohan, P., Gnonlonfin, B., Hell, K., Marasas, W. F. O., and Wingfield, M. J. (2005). Natural occurrence of Fusarium and subsequent fumonisin contamination in pre-harvest and stored maize in Benin, West Africa. International Journal of Food Microbiology, 99(2), 173-183.
- Fumero, M. V., Reynoso, M. M., and Chulze, S. (2015). Fusarium temperatum and Fusarium subglutinans isolated from maize in Argentina. International Journal of Food Microbiology, 199, 86-92.
- Glenn, A. E., Richardson, E. A., and Bacon, C. W. (2004). Genetic and morphological characterization of a Fusarium verticillioides conidiation mutant. Mycologia, 96(5), 968-980.
- Gromadzka, K., Chełkowski, J., Basińska-Barczak, A., and Lalak-Kańczugowska, J. (2019). Diversity and mycotoxin production by Fusarium temperatum and Fusarium subglutinans as causal agents of pre-harvest Fusarium maize ear rot in Poland. Journal of Applied Genetics, 60(1), 113-121.
- Hafizi, R., Salleh, B., and Latiffah, Z. (2013). Morphological and molecular characterization of Fusarium. solani and F. oxysporum associated with crown disease of oil palm. Brazilian Journal of Microbiology, 44(3), 959-968.
- Hefny, M., Attaa, S., Bayoumi, T., Ammar, S., and Bramawy, M. E. (2012). Breeding maize for resistance to ear rot caused by Fusarium moniliforme. Pakistan Journal of Biological Sciences, 15(2), 78.
- Huong, B. T. M., Do, T. T., Madsen, H., Brimer, L., and Dalsgaard, A. (2016). Aflatoxins and fumonisins in rice and maize staple cereals in Northern Vietnam and dietary exposure in different ethnic groups. Food Control, 70, 191-200.
- Leslie, J. F., Summerell, B. A., and Bullock, S. (2006). The Fusarium. Laboratory Manual, 388.
- Li, C., Wu, Y. L., Yang, T., and Huang-Fu, W. G. (2012). Rapid determination of fumonisins B1 and B2 in corn by liquid chromatography–tandem mass spectrometry with ultrasonic extraction. Journal of chromatographic science, 50 (1), 57-63.
- Ling, S., Pang, J., Yu, J., Wang, R., Liu, L., Ma, Y., and Wang, S. (2014). Preparation and identification of monoclonal antibody against fumonisin B1 and development of detection by Ic-ELISA. Toxicon, 80, 64-72.
- López-Errasquín, E., Vázquez, C., Jiménez, M., and González-Jaén, M. T. (2007). Real-time RT-PCR assay to quantify the expression of FUM1 and FUM19 genes from the fumonisin-producing Fusarium verticillioides. Journal of Microbiological Methods, 68(2), 312-317.
- Marasas, W. F. O. (1995). Fumonisins: their implications for human and animal health. Natural Toxins, 3, 193–198.
- Marin, S., Ramos, A. J., Cano-Sancho, G., and Sanchis, V. (2013). Mycotoxins: Occurrence, toxicology and exposure assessment. Food and Chemical Toxicology, 60, 218-237.
- Munkvold, G. P. (2003). Epidemiology of Fusarium diseases and their mycotoxins in maize ears. European Journal of Plant Pathology, 109 (7), 705-713.
- Munkvold, G. P. (2003). Cultural and genetic approaches to managing mycotoxins in maize. Annual Review of Phytopathology, 41(1), 99-116.
- Munkvold, G. P., and Desjardins, A. E. (1997). Fumonisins in maize: can we reduce their occurrence? Plant Disease, 81(6), 556-565.
- Mutiga, S. K., Hoffmann, V., Harvey, J. W., Milgroom, M. G. and Nelson, R. J. (2015). Assessment of aflatoxin and fumonisin contamination of maize in Western Kenya. Phytopathology, 105(9), 1250-1261.
- Mwalwayo, D. S., and Thole, B. (2016). Prevalence of aflatoxin and fumonisins (B1+ B2) in maize consumed in rural Malawi. Toxicology reports, 3, 173-179.
- Nelson, P. E, Toussoun, T. A. and Marasas, W. F. O. (1983). Fusarium species: an illustrated manual for identification. Pennsylvania State University Press, University Park.
- Olanya, O. M., Hoyos, G. M., Tiffany, L. H., and McGee, D. C. (1997). Waste corn as a point source of inoculum for Aspergillus flavus in the corn agroecosystem. Plant Disease, 81(6), 576-581.
- Oliveira, M. S., Diel, A. C., Rauber, R. H., Fontoura, F. P., Mallmann, A., Dilkin, P. and Mallmann, C. A. (2015). Free and hidden fumonisins in Brazilian raw maize samples. Food Control, 53, 217-221.
- Omurtag, G. (2008). Fumonisins, trichothecenes and zearalenone in cereals. International Journal of Molecular Sciences, 9 (11), 2062-2090.
- Patino, B., Mirete, S., Gonzalez-Jaen, M. T., Mule, G., Rodriguez, M. T., and Vazquez, C. (2004). PCR detection assay of fumonisin-producing Fusarium verticillioides strains. Journal of Food Protection, 67(6), 1278-1283.
- Pascale, M., Visconti, A., and Chelkowski, J. (2002). Ear rot susceptibility and mycotoxin contamination of maize hybrids inoculated with Fusarium species under field conditions. In Mycotoxins in Plant Disease (pp. 645-651). Springer, Dordrecht.
- Pretorius, A. (2018). Elucidating the Fusarium graminearum species complex on maize in South Africa. Doctoral dissertation, North-West University.
- Proctor, R. H., Brown, D. W., Plattner. R. D. and Desjardins, A. E., (2003). Co. expression of 15 continuous genes delineates a fumonisin biosynthetic gene cluster in Gibberella monilliformis. Fungal Genetics and Biology, 38: 237-249.
- Reddy, K. R. N., Abbas, H. K., Abel, C. A., Shier, W. T., Oliveira, C. A. F. D., and Raghavender, C. R. (2009). Mycotoxin contamination of commercially important agricultural commodities. Toxin Reviews, 28(2-3), 154-168.
- Rossi, V., Scandolara, A., and Battilani, P. (2009). Effect of environmental conditions on spore production by Fusarium verticillioides, the causal agent of maize ear rot. European Journal of Plant Pathology, 123 (2), 159-169.
- Seo, J. A., Proctor, R. H., and Plattner, R. D. (2001). Characterization of four clustered and coregulated genes associated with fumonisin biosynthesis in Fusarium verticillioides. Fungal Genetics and Biology, 34(3), 155-165.
- Shephard, G. S. (2008). Risk assessment of aflatoxins in food in Africa. Food Additives and Contaminants, 25(10), 1246-1256.
- Shephard, G. S., Van Der Westhuizen, L., Gatyeni, P. M., Katerere, D. R., and Marasas, W. F. (2005). Do fumonisin mycotoxins occur in wheat?.Journal of Agricultural and Food Chemistry, 53 (23), 9293-9296.
- Shim, W. B., Flaherty, J. E., and Woloshuk, C. P. (2003). Comparison of fumonisin B1 biosynthesis in maize germ and degermed kernels by Fusarium verticillioides. Journal of food protection, 66 (11), 2116-2122.
- Tamura K. (1992). Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G + C-content biases. Molecular Biology and Evolution 9: 678-687.
- Tamura, K., Battistuzzi, F. U., Billing-Ross, P., Murillo, O., Filipski, A., and Kumar, S. (2012). Estimating divergence times in large molecular phylogenies. Proceedings of the National Academy of Sciences, 109(47): 19333-19338.
- Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., and Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular biology and evolution, 28(10) : 2731-2739.
- Tamura K., Stecher G., Peterson D., Filipski A., and Kumar S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution 30: 2725-2729.
- Thiel, P. G., Shephard, G. S., Sydenham, E. W., Marasas, W. F., Nelson, P. E., and Wilson, T. M. (1991). Levels of fumonisins B1 and B2 in feeds associated with confirmed cases of equine leukoencephalomalacia. Journal of Agricultural and Food Chemistry, 39(1), 109-111.
- USDA Foreign Agricultural Services Gain Report (2016). Global Agricultural Information network
- van Egmond, H. P., Schothorst, R. C., and Jonker, M. A. (2007). Regulations relating to mycotoxins in food. Analytical and Bioanalytical Chemistry, 389(1), 147-157.
- Varela, C. P., Casal, O. A., Padin, M. C., Martinez, V. F., Oses, M. S., Scauflaire, J., and Vázquez, J. M. (2013). First report of Fusarium temperatum causing seedling blight and stalk rot on maize in Spain. Plant Disease, 97(9), 1252-1252.
- Visconti, A. (1996). Fumonisins in maize genotypes grown in various geographic areas. In Fumonisins in food (pp. 193-204). Springer, Boston, MA.
- Wagacha, J. M., Steiner, U., Dehne, H. W., Zuehlke, S., Spiteller, M., Muthomi, J., and Oerke, E. C., (2010). Diversity in mycotoxins and fungal species infecting wheat in Nakuru District, Kenya. Journal of Phytopathology, 158(7-8), 527-535.
- Wang, E., Norred, W. P., Bacon, C. W., Riley, R. T., and Merrill, A. H., Jr. (1991). Inhibition of sphingolipid biosynthesis by fumonisins. Implications for diseases associated with Fusarium moniliforme. Journal of Biological Chemistry, 266, 14486-14490.
- Wang, J. H., Zhang, J. B., Li, H. P., Gong, A. D., Xue, S., Agboola, R. S., and Liao, Y. C. (2014). Molecular identification, mycotoxin production and comparative pathogenicity of Fusarium temperatum isolated from maize in China. Journal of Phytopathology, 162(3), 147-157.
The authors declare no competing interests.