Diversity of Fusarium Species Infecting Maize Grown In Nakuru County, Kenya

Kenya's primary food crop is maize (Zea mays L.), with a production of 39.0 million (90 kilogram) bags in 2014 [1]. Nakuru County used an area of 86,504 hectares of land to produce 1,765,714 90kg bags in total that year [1]. A number of factors have limited Kenya's ability to produce maize, including soil acidification from the continuous use of Di-ammonium Phosphate (DAP) fertilizer, the inability to obtain improved and certified seeds, and presence of diseases such as maize ear rot and Maize Lethal Necrosis Disease (MLND) among others. Table 1 shows common maize varieties. 

Table 1: Maize genotypes recommended by the Kenya seed company for Nakuru County. 

Note: Source: Kenya Seed Bulletin (2016). 

Maize productivity is severely limited by the ear rot fungus Fusarium, Aspergillus, Penicillium, and Sternocarpella in particular [2,3]. Studies by Duan et al. [4], documented the severity of maize ear rot, which is largely caused by F. verticillioides and F. graminearum species. China reported three species complexes of F. graminearum, F. meridionale, and F. boothii, along with the TEF-1α gene sequence [4]. 

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 [5,6]. When an infection spreads throughout the roots of maize seedlings, it first shows no symptoms. However, as the crop matures, the infection spreads throughout practically the whole plant, resulting in leaf blight, stalk rot, and ear rot. Corn kernels become infected when contaminated silk is used [7,8]. The endosperm and embryo are the two main tissues that Fusarium verticillioides colonizes in maize. In the endosperm, it results in increased levels of FB1 [9]. It has been found that in tropical and subtropical climates, Fusarium verticillioides produces large levels of fumonisin [10-12]. Fumonisins have been found in rice in addition to products derived from maize [13,14]. Maize may also contain other mycotoxins such as citrinin, cyclopiazonic acid, penicillic acid, ochratoxin A, zearalenone, trichothecenes (T-2), and Deoxynivalenol (DON or vomitoxin). 

Kenya's National Government and the corresponding County governments in corn-growing regions have started taking action to boost maize production. Among these steps include providing farmers with alternate fertilizers and certified seedlings. In the North Coast region, the Kenyan government is also carrying out a second pilot phase of the Galana/Kulalu irrigation project. A additional 500,000 hectares of maize were predicted to be produced in this second phase in MY 2016-2017. Furthermore, in order to minimize postharvest losses through reducing aflatoxin contamination, the Kenyan national government has constructed drying and storage facilities in the corn-growing regions. Under the Kenya Cereal Enhancement Programme (KCEP), farmers in Nakuru County received gunny bags for storage after receiving maize seeds for planting. 

Identification of Fusarium species has been done using morphological characterization [15]. Using morphological approaches, Hafizi and his fellow researchers were able to determine the morphological diversity of the Fusarium species. Morphological identification has been based on single-spore isolate coloured on the media and macrospore characteristics [16-18]. Using a single spore ensures that only one strain is examined, since natural sources contain many morphologically distinct Fusarium strains. 

Morphological characteristics, identification of F. verticillioides has been done based on cultural practices according to Leslie et al. [18]. This was done by looking at the colony formed on the media and microscopic examination of the macrospores and microspores. Fusarium verticillioides produce abundant single-celled micro-conidia in distinctive long chains [18,19]. In a normal conidium development, Wall Building Zone (WBZ) is present to initiate and assists in the development of the conidium [20]. It was reported that besides use of morphological characteristics, F. thapsinum from F. verticillioides and F. graminearum are effectively differentiated by use of molecular markers. 

Fusarium temperatum, was identified and characterized in Spain, Poland, Argentina, France and China. It was found to be pathogenic in maize seedlings and stalks [21]. 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 was described after isolation from maize in Argentina [22,23]. Other countries where F. temperatum had been isolated include; Spain, France and Poland [21,24]. It had also been isolated in China and proved to be pathogenic and able to produce fumonisins in infected maize ears [25]. 

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 is different 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 [26].

Maize Samples Collection

During sample collection, maize grains which were 12 weeks Harvest Time Points After Physiological Maturity (HTPAPM) according to Alakonya et al. [27], 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 was collected from each of the farmers.

 Figure 1: Nakuru County map (2016). 

Note: Source: https://cgspace.cgiar.org/bitstream/handle/10568/80458/Nakuru.pdf?sequence

Isolation and Identification of Fusarium Species from Maize Kernels

Symptomless maize kernels collected from the farmers stores were taken to Kenyatta University, Microbiology laboratory for mycological isolation of Fusarium spp. The media used was, Pentachloronitrobenzene (PCNB) agar media, which is a selective media for promotion of growth of only Fusarium fungi by inhibiting growth of other fungi species. Data on the incidence was first normalized and the percent incidence of Fusarium sp. on the maize genotypes was computed as;

A total of fifteen grams of each maize genotype (about 25-30 maize grains) showing no symptoms of fungal infection were collected from each bulk sample. Five grains of each genotype were picked 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 of each of the maize genotype in each petri dish was 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 Using Colour

The first identification was based on colour morphology of the mycelium according to procedure by Nelson et al. [16], 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. [18]. Microscopic observation of the spores was carried out at the Department of Veterinary Anatomy, University of Nairobi, 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. Percentage incidence of each Fusarium species in the different maize genotypes was computed.

DNA Extraction

Extraction of the DNA from each Fusarium isolate as a representative sample was carried out using procedure described 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 1.5ml Eppendorf tube was filled with a 500µl 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. This method is faster and less prone to contamination procedure. The mycelial mat from each of the isolate was pelleted by centrifugation for 5 minutes at 13,000rpm in a microfuge, washed with 500µl of TE buffer and pelleted again. The TE was then decanted and 300µl of extraction buffer added. The mycelium was crushed with a conical grinder (Treff AG, Degersheim, Switzerland), fitting exactly the tube and centrifuged by hand potter at 200rpm for 10 minutes. After that, 150µl of 3.0M sodium acetate, (pH 5.2) was added and tubes placed at -20°C for ten minutes. Tubes were then centrifuged in a microfuge at 13,000rpm and the supernatant transferred to another tube. An equal volume of isopropanol was added. After 5 minutes at room temperature, the precipitated DNA was pelleted by centrifugation in a microfuge at 13,000rpm. This was therefore washed using 70% ethanol and then air dried on paper towel for 20 minutes. The pellet was re-suspended in 50µl of TE for assessment of the DNA quality was determined by running in a 1.0% agarose gel at 80 volts for 30 minutes together with a known size, 1kb universal gene ruler (Biolab). 

1 X TE Buffer, pH 8.0 (1mM Na₂ EDTA and 10mM Tris base) was made as 50X stock solution and diluted as required for use. To prepare 1 litre of 50X stock solution: In a 2 litre beaker, 700ml of double-distilled water was added, 60.6g of Tris base (i.e 500mM) and 18.6 g of Na₂ EDTA (i.e 50mM). This was stirred vigorously to dissolve on a stir plate. After the Tris and the EDTA were both in solution, obtained pH was adjusted to pH 8.0 using concentrated Hydrochloric Acid (HCl) and the final volume made to 1 litre with double-distilled water. 

Lysis bufferwas used at (pH 8.0), 50mM Tris, 50mM EDTA, 3% SDS, 1% β-mercaptoethanol, and 0.1mg/ml Proteinase K were mixed. To obtain 500ml of buffer; to a 1L beaker was added 350ml of double-distilled water, 25ml of 1M Tris-HCl (pH 8.0) stock. Tris was allowed to dissolve and mix. EDTA (Na₄EDTA·2H₂O; 50ml of 0.5 M EDTA stock) was added and continued mixing (pH≥8 for the EDTA to stay in solution). After which 150g SDS (sodium dodecyl sulfate (Sigma cat. no. L-6026) was added and continued to mix until dissolved. Solution was transferred to a graduated cylinder and a final volume adjusted to 500ml by adding distilled water. β-mercaptoethanol at 1.0 % (v/v) and Proteinase K (Sigma cat. no. P-6556) at 0.1mg/ml was therefore added to the solution just before use.

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. 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 (25μl) for TEF 1-α gene consisted of 12.8μl milli Q H₂O, 2.5μl 10x buffer, 2.5μl MgCl₂, and 0.5μl dNTP mix (10mM each). A mount of 0.8μl forward primer EF1 (10mM), 0.8μl reverse primer EF2 (10mM), 0.1μl Taq (Biotech) and 5μl 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 [28]. 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.

Data Analysis 

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 [29,30]. Neighbour joining approach for geographical representation of pairwise nucleotide sequence divergence among the fungi strains was used.

Fusarium Fungi Identified Using Colour on PDA Slants

Visual observation of Fusarium sp. from each maize grains isolated on each petri dish which contained PCNB agar and thereafter sub-cultured onto Potato dextrose Agar slants, revealed differences in the colour of Fusarium mycelium on the media (Figure 2(A and B). Figure 3(A and B) is showing F. verticillioides on petri dish revealing a rapidly growing white, becoming tinged with purple aerial mycelium.

 Figure 2: Macrospores (x400). (A): F. temperatum; (B): F. verticillioides.

 Figure 3: F. verticillioides on PDA slant. A: Front view; B: Back view.

Note: Arrows showing white aerial mycelium.

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 2.

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 3B. Figure 3A shows a F. temperatum isolate macroconidia having a typical four-septa with a foot-shaped basal cell, figure 3. 

Using morphological identification, Fusarium species found infecting maize varieties were as shown in table 2.

Table 2: Fusarium species infecting maize genotypes.

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 100ng/μl of 1kb standard Lambda DNA. The DNA quantification revealed that the concentration ranged from 10 to 30ng/μl of isolated DNA, figure 4.

 Figure 4: DNA bands in 1.0 % Agarose gel. 

Note: Lanes L₁-EF₁ to L₁₀ -EF₁, Fusarium spp. Isolates; M, Gene ruler.

Figure 4, shows images of quality DNA bands on 1.0% agarose gel. It shows the sizes of high molecular weight, genomic DNA of 10 samples in relation to Molecular marker 1kb. Lane 1, Unknown Fusarium 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 and which showed percentage similarity of between 96.5-99.1% as shown in table 3.

Table 3: DNA sequences using Mega 7 Programme.

Phylogenetic Tree Showing Diversity of Fusarium sp.

Figure 2 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. The figure is drawn to scale, with bootstrap values showing number of substitutions per site. 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 5 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.

 Figure 5: Fusarium species’ phylogenetic tree based on TEF-1α gene sequences.

Branch bootstrap values indicating the number of times out of 1000 (in this case), the same branch was obtained when repeating the phylogenetic reconstruction on a re-sampled set of the data. Evolutionary analyses were conducted in MEGA 6 and show the tree with the highest log likelihood.

Fusarium spp. Speciation

Six out of the ten cleaned DNA samples amplified during PCR reaction. These were sample L1_EF-1, L2_EF-1, L4_EF-1, L6_EF-1, L7_EF-1 and sample L10_EF-1. The remaining four (Sample L3_EF-1, L5_EF-1, L8_EF-1 and L9_EF-1) did not amplify during PCR amplification. Results of the six Fusarium spp. samples’ DNA sequences based on the National Center for Biotechnology Information (NCBI) database searching using individual sequences confirmed that sample L10_EF-1 isolate belong to F. verticelloides and had 99.69 % identity with 93 % query cover. Four of the DNA sequences (sample L1_EF-1, L2_EF-1, L4_EF-1, and L6_EF-1) were Fusarium temperatum, which is within the Gibberella fujikuroi species complex and closely related to Fusarium subglutinans. Sample L1_EF-1; F. temperatum had 98.93% identity with 94% query cover, Sample L2_EF-1; F. temperatum had 99.96% with 94 % query cover and, sample L4_EF-1 and L6_EF-1; F. temperatum had identity of 98.72 % with 80 % query cover. Sample L7_EF-1 was Fusarium boothii (formerly F. graminearum) and had identity of 98.51 % with 86 % query cover (Figure 2). 

Morphological Identification of Fusarium spp. 

In the present study, the first identification of Fusarium species was carried out using morphological characteristics. Identification was first based on colour differences of Fusarium fungi on PDA slants as shown in figure 2. Similar identification had been reported by Nelson et al. [16]. 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 3 using the procedure by Leslie et al. [18]. These two morphological methods of identification relied on cultural characteristics and were dependent on visual assessment by the eye. This is by looking at the colony formed on the media and microscopic examination of the macrospores and the microspores. Research by Leslie et al. [18], and Nelson et al. [16], 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 [18]. Glenn et al. [20], 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 [20]. Morphological identification of F. temperatum was based on the macro-conidia usually having four-septate 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 a molecular technique of 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. temperatumare 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. [31]. 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 [18], 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 confirmed to be only three (F. temperatum, F. verticillioides and F. boothii) by using DNA sequences analysis as shown in phylogenetic tree figure 5.

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.

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 support by Njoro and Molo Sub-County Agricultural officers together with farmers.

This work was done with the knowledge of the author and no organization made any contradiction on the findings of the research. There was no conflicting opinion on publication of this research.

Data used for this study were primary data obtained by the researcher. The data can be made available as already presented in the document and any other detailed information is available with the Author.

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