Maize is one of the staple foods for most of the population especially in areas adaptable for their production  . It is a major cereal crop for livestock feed and human nutrition     . Though, its production in Nigeria is constrained by biotic factors such as pests and diseases  .
Fusarium verticillioides (Sacc.) is a maize pathogen causing root, stalk and ear rots in Nigeria   . It is found in plant residue in almost every maize field at harvest which its symptoms vary widely and range from asymptomatic plants to severe rotting of all plant parts  . Symptomless infection exists in maize leaves, stems, roots and grains whereby its presence is ignored in many cases because it does not cause visible damage to the plant  . This suggests that some strains of F. verticillioides produce disease in maize while others do not  .
The knowledge of variability in pathogen is required by host resistance to de- velop effective disease management strategies  . Conventional markers used to study the variability in pathogens are based on the use of differential hosts, culture characteristics, morphological markers and biochemical tests   . These markers distinguished pathogens on the basis of the physiological characters such as pathogenicity and growth behaviours which are highly influenced by the host age, inoculum quality and environmental condition. The use of molecular markers is therefore necessary.
The genetic characterisation of Fusarium species using several molecular techniques has been adopted; they are RAPD (Random Amplified Polymorphic DNA), PCR- RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism), AFLP (Amplified Fragment Length Polymorphism)  . The application of AFLP in DNA fingerprinting of microbes particularly fungi had been reported   . This technique provides a high degree of flexibility due to different restriction enzymes, the nature and number of selective nucleotides that can be manipulated to limit amplification fragments generated in order to account for differences in genome size.
This study aimed to assess the genetic relatedness of F. verticillioides from maize in three agro-ecological zones of Southwest Nigeria.
2. Materials and Methods
2.1. Collection of Diseased Maize Samples
Six maize ear showing rot symptoms were randomly obtained from Derived Savanna (Ilora, Ijebu-Ode, Igboora and Eruwa), Rainforest (Ado-Ekiti, Akure, Ilesa and Ibadan) and Guinea Savanna (Saki, Igboho) of Southwest Nigeria between May to July, 2013.
2.2. Isolation of Fusarium verticillioides
Some infected and healthy grains obtained from the sampled maize ear were washed under running water, surface sterilized for one minute in 10% sodium hypochlorite solution, and then rinsed in three changes of sterile water, followed by drying with sterile paper towel. These were then placed on solidified potato dextrose agar in petri-dishes and incubated at room temperature (28˚C ± 2˚C) for 7 days. Observation was made daily on mycelial growth. Pure cultures were obtained by subculturing onto fresh plates.
2.3. Morphological Characteristic of Fusarium verticillioides
Morphological identification according to  and  was carried out at the pathology unit of International Institute of Tropical Agriculture (IITA) Ibadan. Morphological identification of F. verticillioides involved observable characters on the cultured plates (rate of growth, pigment production, presence or absence of mycelium and mycelium colour), while the microscopic identification involved distinctive structures and characters (phialides, chlamydospores, microconidia and macroconidia).
2.4. Extraction of Genomic DNA of Fusarium verticillioides
Genomic DNA was extracted using a DNeasy tissue kit according to the manufacturer’s instruction. DNA was eluted in 200 µl of elution buffer (buffer AE of the kit) and stored at −20˚C. DNA yield were measured using Nanodrop spectrophotometer, while the quality was assessed by agarose gel electrophoresis, restriction enzyme digestion and AFLP analyses using the procedure described by  .
2.5. Restriction and Ligation of Adapters
Two restriction endonucleases, EcoRI and Mse1 were used to digest 100 ng of genomic DNA in 10 µL reaction, which was incubated for 37˚C for 3 hours and inactivated at 70˚C for 15 minutes. Digested and undigested DNA were separated on 1% agarose gels in TBE buffer. The ligation reaction volume of 10 µl was prepared to contain of 5 U of T4 DNA ligase, ligase buffer 100× BSA, 5 µM EcoRI (Forward and reverse) and 50 µM MseI (forward and reverse). After combining 10 µL of ligation mix with the digested DNA fragment, ligation was allowed to proceed for 5 hours at 37˚C. The adapter-li- gated DNA (20 µL) was diluted 1:5 with autoclaved water to serve as template in the pre-selective PCR and was stored at −20˚C.
2.6. Pre Selective and Selective PCRs
The description of the adapters for pre-amplification and selective amplification primer sequence used in this study is shown in Table 1. Pre-selective amplification of the dilute template DNA was carried out with AFLP primer combinations (5 µM Pre EcoRI and 50 µM Pre MseI) without selective nucleotides and twenty microliter PCR reactions containing 8.0 µl of dilute template DNA, 2.0 µl of PreEcoRI (5 UM), 0.2 µl of PreMseI (5 UM) primer, 5 µ/Ul of Taq DNA polymerase, 2 µl of 10× Taq DNA polymerase buffer, 0.6 µl of 10 mM DNTPs and 0.6 µl of 50 mM of Mg2 were performed. Pre-ampli- fication reactions were performed for 20 cycles of: 30 s at 94˚C, 30 s at 94˚C, 56˚C at 30 s and 1 min at 72˚C. Following pre-amplification, the reactions were diluted 10 folds with autoclaved water and used as template for selective amplification. Selective amplification were performed using three (3) selected combination of primers with one selective nucleotides (EcoRI + T, EcoRI + G, EcoRI + C) and one MseI primers (MseI + C). The reactions were carried out in volumes of 20 µl of PCR buffer containing 6 µl of dilute preamplified template DNA, 0.2 µl of 10 ng EcoRI, 0.7 µl of 10 ng MseI, 0.3 µl of 10 mM dntps, 2 µl of reaction buffer and 0.3 µl of 50 nM. The program ran for 11 cycles at 94˚C for 30 s, 94˚C for 30 s, 72˚C for 60 s, 94˚C for 30 s, 56˚C for 30 s and 72˚C for 60 s. 7 µl of formaldehyde dye was added to 20 µl of each sample and the samples were
Table 1. Description of the primers used for amplified fragment length polymorphism.
Source:  .
denatured for 5 minutes at 95˚C. Each sample was loaded on a 6% polyacrylamide gel containing 400 µl ammonium persulphate (APS). Gels were cast using long and short plates with 0.25 mm thick spacers and comb. The long plates were treated with sigmacote, while the short plates were treated with a binding solution. Electrophoresis was performed at a constant power 60 W and a constant temperature of 47.5˚C for 3 h. Amplicons were revealed with silver nitrate stain.
2.7. Data Analysis
For each primer combination, all bands were manually recorded for presence (1) or absence (0) of each band in AFLP analysis. Bands of different electrophoretic mobility were assumed to be non-allelic, while bands of the same mobility were assumed to be allelic. A dendrogram was constructed from the similarity matrix using unweighted pair group method with arithmetic average (UPGMA) described by Sneath and Sokal (1973). Cluster analysis was performed using NTSYSpc (NTSYS-For Numerical Taxonomy System) version 2.1  .
A total number of 164 bands were produced from three primer combinations out of which 135 were polymorphic, and correspond to an average of 45 polymorphic bands per primer combination (Table 2). Most of the bands were polymorphic, while few were monomorphic. The maximum number of bands generated by EC + MC combination was 61 bands while the minimum obtained with EG + MC primer combination was 50 bands (Figure 1). The polymorphic information content (PIC) for each marker varied between 0.7734 and 0.8249, EG + MC and ET + MC primer combinations had the maximum with the same PIC values, while ET + MC primer combination had the lowest PIC value (Table 2).
The Fusarium verticillioides strains were grouped into five (5) clusters from three (3) Agro-ecological zones. The cluster three (3) had the highest number of F. verticillioides strains from Ilora (2, 3, 4, 5, 6), Ilesha (21), Akure (26, 27, 29), Ibadan (31, 32), Igbooho
Table 2. Characterisation of the Amplified Fragment Length Polymorphism (AFLP) primers based on Polymorphic Information Content (PIC).
NPB: Number of Polymorphic Bands, TNB: Total Number of Bands, PPB: Percentage Polymorphic Bands, AN: Allele Number.
Figure 1. Amplified Fragment Length Polymorphism (AFLP) Products from Fusarium verticillioides strains using EcoRI-G/MseI-C Primer Combination.
(38, 41, 42), Ado-Ekiti (43, 44, 45) and Saki (53), while cluster I recorded the least number of F. verticillioides strains (Table 3).
The genetic relationships of F. verticillioides strains were also represented in the dendrogram (Figure 2). The coefficient of similarity ranged from 0.67 to 0.97. There were five main clusters constituting all samples with similarity coefficient of 0.72. Group 1 had two subgroups consisting of six strains; 1 (ILR), 10 (ERW), 49 (SAK), 25 (AKR), 33 (IBD) and 24 (ILH). 24 (ILH) had the highest genetic distance within the group. The group II in the dendrogram had 2 sub-groups of 10 F. verticillioides strains of which the highest occurrence were from Eruwa (ERW), though, strains 17 (IJB) was the most distanced within the group. Group I and II had coefficient similarity at 0.87, while Group III with 5 sub-groups had the highest number of strains. Strains 2 and 3 (ERW) formed coefficient similarity with strains 4 and 5 (ERW) at 0.876, while strains 6 (ERW) and 29 (AKR) were the most distanced within the group. The fourth group had 5 subgroups containing 16 isolates in which strain 54 from SAK was the most distanced within the group. Strains from IBD (34, 36), IGH (39), EKT (46, 47), SAK (52) and IGB (57, 58, 59, and 60) were distinctly separated from other strains at 0.67 similarities. The closest strains are 19 and 20 in cluster II from ILH, with the highest pairwise similarity coefficient of 0.97 (Figure 2).
Understanding the diversity of pathogens is necessary to predict the control measures, which include; the use of resistant cultivar, fungicides application or the use of biological agents  -  . The detected 164 AFLP bands in which 135 were polymorphic (82% polymorphic rate) indicates high genetic diversity in accordance with the findings of  and  who reported genetic similarity between and among the isolates. Genetic similarity between the isolates which ranged from 0.67 to 0.97 for similar coefficient index is an indication of genetic variation as similarly reported by  .
It was shown that polymorphism was present in the PCR products from the F. verticillioides strains with the AFLP primers used in characterization. There was no association between AFLP patterns and agro-ecological origin of the tested strains, again, the
Table 3. Cluster groups of Fusarium verticillioides strains from the three agro-ecological zones.
Figure 2. Dendrogram showing Fusarium verticillioides strains using three (3) AFLP Primer Combination. Samples: 1-6 (Eruwa), 7-12 (Akure), 13-18 (Ibadan), 19-24 (Ilesa), 25-30 (Igbooho), 31-36 (Saki), 37-42 (Ekiti), 43-48 (Igboora), 49-54 (Ijebu), 55-60 (Ilora).
biology and epidemiology of this pathogen may provide some insight as to during favourable condition. F. verticillioides causes many diseases such as root, stalk and ear rots, which is responsible for the decrease in nutritive value, loss in germination, discoloration, increase in fatty acid and secretion of mycotoxins which results in losses of maize production      .
The dendrogram based on the genetic similarity coefficient established the interrelationship among the isolates from different locations in the three agroecological zones. The clustering pattern of the isolates, irrespective of the source implies that the genetic distance is not correlated with the agro-ecological distance as similarly reported by  . The genetic similarity obtained through AFLP analysis showed level of genetic variation among the strains. The strains do not depicts close relatedness with each other, and strains from different location showed a common clad which supports that migration event are common in the population of F. verticillioides as similarly observation was reported by  .
Random mating population displays a high degree of genotypic diversity due to difficulties in the management of disease as a result of constant appearance of new strains which increase the variability features like fungicides resistance, higher aggressiveness and better fitness in the population  . Other factors that could be responsible for increase in genetic diversity are mutation, translocation, chromosomal deletion are common in fungi as well as difference in climatic condition where the isolates were collected   .
In conclusion, molecular characterisation of Fusarium verticillioides was investigated to give the genetic variability. Amplified fragment length polymorphism markers should be encouraged in studying the genetic variation in natural population of F. verticillioides which can help pathologists to detect the pathogenic potential of this pathogen as well as helping to develop an effective control strategy.
Conflict of Interests
There is no any kind of conflict of interests with any trademark mentioned in this paper, competitive interest, or secondary interest that could have influenced the research. This declaration is carried out by all the authors of the work presented.
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
 Ofor, M.O., Ibeawuchi, I.I. and Oparaeke, A.M. (2009) Crop Protection Problems in Production of Maize and Guinea Corn in Northern Guinea Savanna of Nigeria and Control Measures. Nature and Science, 7, 8-14.
 Akande, S.R. and Lamidi, G.O. (2006) Performance of Quality Protein Maize Varieties and Disease Reaction in the Derived-Savanna Agro-Ecology of South-West Nigeria. African Journal of Biotechnology, 5, 1744-1748.
 Olawuyi, O.J., Odebode, A.C., Alfar, A., Olakojo, S.A. and Adesoye, A.I. (2010) Performance of Maize Genotypes and Arbuscular Mycorrhizal Fungi in Samara District of South West Region of Doha-Qatar. Nigerian Journal of Mycology, 3, 86-100.
 Olowe, O.M., Olawuyi, O.J. and Odebode, A.C. (2015) Response of Maize Genotypes to Fusarium verticillioides Strains from Two Agro Ecological Zones in Southwest Nigeria. International Journal of Pure and Applied Sciences and Technology, 27, 77-86.
 Olawuyi, O.J., Babatunde, F.E., Akinbode, O.A., Odebode, A.C. and Olakojo, S.A. (2011) Influence of Arbuscular Mycorrhizal and N.P.K Fertilizer on the Productivity of Cucumber (Cucumis sativus). International Journal of Organic Agriculture Research & Development, 3, 22-31.
 Sobowale, A.A., Odebode, A.C., Cardwell, K.F. and Bandyopadhyay, R. (2009) Suppression of Growth of Fusarium verticillioides Niren. Using Strains of Trichoderma harzianum from Maize (Zea mays) Plant Parts and Its Rhizosphere. Journal of Plant Protection Research, 49, 452-459.
 Haque, S., Park, R.F., Keiper, F.J., Bariana, H.S. and Wellings, C.R. (2008) Pathogenic and Molecular Variation Support the Presence of Genetically Distinct Clonal Lineages in Australian Populations of Puccinia graminis f. sp. avenae. Mycological Research, 112, 663-673.
 Zhang, J., Torriani, S.F.F. and McDonald, B.A. (2007) Significant Difference in Pathogenicity between MAT1-1 and MAT1-2 Isolates in the Wheat Pathogen Mycosphaerella graminicola. Fungal Genetics and Biology, 44, 339-346.
 Mitter, N., Srivastava, A.C., Ahamad, R.S., Sarbhoy, A.K. and Agarwal, D.K. (2001) Characterization of Gibberellin Producing Strains of Fusarium moniliforme Based on DNA Polymorphism. Mycopathologia, 153, 187-193.
 Prom, L.K., Perumal, R. and Erattaimuthu, S.R. (2011) Virulence and Molecular Genotyping Studies of Sporisorium reilianum Isolates in Sorghum. Plant Disease, 95, 523-529.
 Leslie, J.F. and Summerell, B.A. (2006) The Fusarium Laboratory Manual. Blackwell, Oxford, 388 p.
 Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Freijters, A., Pot, J., Peleman, J., Kuiper, M. and Zabeau, M. (1995) AFLP: A New Concept for DNA Fingerprinting. Nucleic Acids Research, 21, 4407-4414.
 Ramasamy, P., Frederiksen, R.A., Prom, L.K. and Magill, C.W. (2007) Head Smut. In: Thakur, R., Reddy, B. and Mathur, K., Eds., Screening Techniques for Sorghum Diseases, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, 58-63.
 Heydari, A. (2007) Biological Control of Turfgrass Fungal Diseases. In: Pessarakli, M., Ed., Turfgrass Management and Physiology, CRC Press, Boca Raton, 223-235.
 Huang, R., Galperin, M., Levy, Y. and Perl-Treves, R. (1997) Genetic Diversity of Fusarium moniliforme Detected by Vegetative Compatibility Groups and Random Amplified Polymorphic DNA Markers. Plant Pathology, 46, 871-881.
 Odebode, A.C., Ladoye, A.O. and Osonubi, O. (1995) Influence of Arbuscular Mycorrhizal Fungi on Disease Severity of Pepper and Tomato Caused by Sclerotium rolfsii. Journal of Science Research, 2, 49-52.
 Olawuyi, O.J., Odebode, A.C., Oyewole, I.O., Akanmu, A.O. and Afolabi, O. (2013) Effect of Arbuscular Mycorrhizal Fungi on Pythium aphanidermatum Causing Foot Rot Disease on Pawpaw (Carica papaya L.) Seedlings. Archives of Phytopathology and Plant Protection, 47, 185-193.
 Sobowale, A.A., Cardwell, K.F., Odebode, A.C., Bandyopadhyay, R. and Jonathan, S.G. (2005) Growth Inhibition of Fusarium verticillioides (Sacc.) Nirenberg by Isolates of Trichoderma pseudokoningii Strains from Maize Plant Parts and Its Rhizosphere. Journal of Plant Protection Research, 45, 249-266.
 Mishra, A.K., Sharma, K. and Misra, R.S. (2010) Isozyme and PCR-Based Genotyping of Epidemic Phytophthora colocasiae Associated with Taro Leaf Blight. Archives of Phytopathology and Plant Protection, 43, 1367-1380.
 Nath, V.S., Sankar, M.S., Hegde, V.M., Jeeva, M.L., Misra, R.S. and Veena, S.S. (2012) Analysis of Genetic Diversity in Phytophthora colocasiae Using RAPD Markers. Asian and Australasian Journal of Plant Science and Biotechnology, 6, 38-43.
 Prabhu, H.V., Adiver, S.S., Bhat, R., Narayana, Y.D., Jahagirdar, S. and Parameshwarappa, K.G. (2012) Genetic Variability in Macrophomina phaseolina (Tassi.) Goid., Causal Agent of Charcoal Rot of Sorghum Karnataka. The Journal of Agricultural Science, 25, 72-76.
 Abd-Elsalam, K.A., Khalil, M.S., Aly, A.A. and Asran-Amal, A. (2002) Genetic Diversity among Fusarium oxysporum f. sp. vasinfectum Isolates Revealed by UP-PCR and AFLP Markers. Phytopathologia Mediterranea, 41, 252-258.
 Kiprop, E.K., Baudoin, J.P., Mwang’ombe, A.W., Kimani, P.M., Mergeai, G. and Maquet, A. (2002) Characterization of Kenyan Isolates of Fusarium udum from Pigeon Pea (Cajanus cajan (L.) Millsp.) by Cultural Characteristics, Aggressiveness and AFLP Analysis. Journal of Phytopathology, 150, 517-527.
 Machon, P., Pajares, J.A., Diez, J.J. and Alves-Santos, F.M. (2006) Influence of the Ectomycorrhizal Fungus Laccaria laccata on Pre-Emergence, Post-Emergence and Late Damping-Off by Fusarium oxysporum and F. verticillioides on Stone Pine Seedlings. Symbiosis, 49, 101-109.
 Mohmed, A.A., Mohmed, S.K., Mohmed, I.N., Kamel, A.A. and Joseph, A.V. (2003) Molecular Phylogeny of Fusarium Species by AFLP Fingerprint. African Journal of Biotechnology, 2, 51-55.
 Yates, I.E., Arnold, J.W., Hinton, D.M., Basinger, W. and Walcott, R.R. (2003) Fusarium verticillioides Induction of Maize Seed Rot and Its Control. Canadian Journal of Botany, 81, 422-428.
 Shekhar, M., Sharma R.C., Lokendra, S. and Ram, D. (2006) Genetic Variability of Macrophomina phaseolina (Tassi) Goid., Incitant of Charcoal Rot of Maize in India. Indian Phytopathology, 59, 453-459.
 Singh, P.K. and Hughes, G.R. (2006) Genetic Similarity among Isolates of Pyrenophora tritici-repentis, Causal Agent of Tan Spot of Wheat. Journal of Phytopathology, 154, 178-184.