F. verticillioides is known to pose a severe threat to human and animal health due to its potent mycotoxigenic and carcinogenic characteristics    . Generally, the pathogen is known to produce different mycotoxins which pose severe threats to both human and animal health. Such mycotoxins include fusaric acid, trichothecenes, moniliformins, zearalenones, fumonisin, amongst others  -  . The toxicity of the corn contaminated by F. verticillioides had been well-documented for more than a hundred years, but the fumonisins were discovered only recently.
The fumonisins are a group of structurally related secondary metabolites, which has been found to contain fumonisin B1 (FB1) and B2 (FB2) with cancer-promoting activity in rat liver and they have been found to occur naturally in maize-based feed samples associated with field outbreaks of equine leukoencephalomalacia (ELEM), a fatal brain disease in horses, donkeys, mules, and rabbits     . The FB1 was reported to cause liver cancer in rats  , ELEM in horses,  and porcine pulmonary edema (PPE) in pigs in which the principal lesions are not in the brain but in the lungs   . Because F. verticillioides infects maize worldwide, fumonisins contamination of maize from all geographic regions is not unlikely. In maize grown in temperate regions, F. verticillioides and F. proliferatum represent the greatest threat for fumonisin production  . In the experiment conducted by  , Trichoderma viride isolated from the roots of corn plants was able to suppress the radial colony extension of Fusarium verticillioides on agar as well as the fumonisin B1 (FB1) on corn kernels.
Trichoderma species are known to be commonly used as biocontrol agents against several pathogens  . The genus Trichoderma had actually produced tested fungal antagonists against several pathogens of many crops  . The experiment was therefore conducted to examine the fumonisin content of maize (Z. mays) seeds whose stems had received dual inoculations of F. verticillioides and some Trichoderma species.
2. Materials and Methods
2.1. Planting and Inoculation in the Field
Maize seeds (DMR-LSRW) were planted in the field in three replicates, with each replicate representing a block of 30 rows (14.5 m by 15 m). There were 25 plant stands per row and 90 rows in all the three replicates, experimental design being randomized complete block (RCBD). Seven weeks after planting, sterile 1.8 mm-diameter nails on wooden handles were used to puncture each stem on either sides at the 3rd internode  . Four Trichoderma species viz., Trichoderma harzianum strain 2, T. hamatum, T. pseudokoningii strains 2 and 11 were obtained from the Pathology Unit, International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria and used for the experiment. These were among the Trichoderma species employed as antagonists in  . The pathogen (F. verticillioides) employed was isolated from soil from maize field and identified in the pathology laboratory, International Institute of Tropical Agriculture, Ibadan, Nigeria. Toothpicks that have been dressed separately with pathogen and the antagonists were inserted into the holes on either side of the stems using toothpick method as employed by  . Three pairing methods were used for the inoculation of pathogen (F. verticillioides) and the antagonists into the stem of the maize (Z. mays) plant, viz., “Pathogen inoculated before Antagonist”, “Antagonist inoculated before Pathogen”, and also “Antagonist and Pathogen inoculated simultaneously”. Controls include “Inoculation of Pathogen alone”, “Inoculation of Antagonist alone”, and “Inoculation of sterile toothpicks alone. There were thus 6 treatments in a block of 30 rows (5 rows per treatment) while border rows were not inoculated.
2.2. Fumonisin Assay
Five weeks after inoculation (12 weeks of planting), seeds from maize (Z. mays) plants whose stems received the various treatment combinations of pathogen (F. verticillioides) and the four antagonists, Trichoderma harzianum strain 2, T. hamatum, T. pseudokoningii strains 2 and 11 in the field, were collected and subjected to fumonisin analysis. The Veratox for fumonisin test kit was used, this being a direct competitive enzyme-linked immunosorbent assay (ELISA) for the quantitative analysis of fumonisin in such commodities as corn, corn meal and rice. The kit was stored refrigerated at 2˚C - 8˚C.
2.3. Maize Sample Preparation and Fumonisin Extraction
The maize samples for each treatment were ground to a fine blend and thoroughly mixed. All the samples were stored at 2˚C - 8˚C until analysis was completed. 70% methanol was used for the extraction processes. 25 grams of the ground sample was blended with 125 ml of 70% methanol for 2 minutes in a high-speed blender. The extract was filtered by passing 10 ml through a Whatman #1 filter and the filtrate was collected in beaker. The filtrate sample was then subjected to analysis using the standard fumonisin test kit (VERATOX). After analysis, the bottoms of the microwells were wiped with dry towels and later read in a microwell reader using a 650 nm filter. Air bubbles were eliminated as they could affect analytical results. The result was read within 20 minutes of completion of the test. The results were calculated with Neogen’s Log/Logit Software and the data obtained were subjected to ANOVA (p ≤ 0.05) using the GLM procedure of SAS (version 9.2).
Table 1 shows the summary of the fumonisin analysis done on the maize seeds from the plants that received treatment combinations of the pathogen and the selected antagonists. The seeds from the maize plants whose stems received treatments of T. pseudokoningii strain 5 alone and T. harzianum strain 2 alone had the highest mean fumonisin content (6.90 and 6.88 respectively) though not significantly higher than those of the controls (“Inoculating pathogen alone” and “sterile toothpick alone”) which had mean fumonisin content of 5.40 and 5.30 respectively. On the other hand, seeds from plants that received treatments containing the same T. pseudokoningii strain 5 and T. harzianum strain 2 (i.e. “Inoculating T. pseudokoningii strain 5 and pathogen simultaneously” and also “Inoculating T. harzianum strain 2 before pathogen”) were also among the ones with the lowest fumonisin content. However seeds from plants that received treatments involving “Inoculation of T. hamatum alone”, “Inoculation of T. pseudokoningii strain 5 and pathogen simultaneously”, and “Inoculation of T. harzianum strain 2 before pathogen” were significantly low in fumonisin content (i.e. 2.48, 2.69 and 2.91 respectively) compared to those from most of the other treatments. There was a high significance among treatments and within the blocks (replicates), i.e. there were varying levels of fumonisin occurrence among the treatments and varying Fusarium occurrences within the blocks (replicates) (Table 2).
The fumonisin analysis showed that the maize seeds from all the maize plants that received the treatments most likely contained fumonisin though at varying levels. This might be part of the reasons for the arguments of   that F. verticillioides is perhaps the best known ear rot pathogen. It might also be part of the reasons for the conclusion of  that F. verticillioides is almost a constant companion of maize infecting the roots, stalk and kernels. This also agreed with the belief of close relationship between maize plant and F. verticillioides by  . They submitted that because of the close relationship, technology is not available to ensure that all maize-based foods and feeds are completely free of fumonisins. It is such a serious case that, according to them, there cannot be complete elimination without banning maize as a food and feed ingredient in the United States, an economically and politically unrealistic option.
The high fumonisin content in the seeds of the plants that received treatments involving “Inoculation of T. pseudokoningii strain 5 alone” and “Inoculation of T. harzianum strain 2 alone”, as well as “Inoculation of sterile toothpicks” (control) were in line with the conclusion of  . In other words the high fumonisin content is likely indicative of the resident Fusarium verticillioides in the seeds. This is because  opined that F. verticillioides and F. proliferatum represent the greatest threat for fumonisin production in maize. The symptomless expression of almost all the seeds (even though they contained fumonisin) corroborates the findings of  , which was of the opinion that F. verticillioides is not only the most common pathogen of maize, but also the most common fungi found colonizing symptomless maize plants. The high significance within the blocks might be due to the ubiquitous nature of fumonisin-producing Fusarium as concluded by  which naturally will be at varying levels in different places.
Table 1. Fumonisin concentrations in seeds of maize plant that received treatment combinations of pathogen and the selected antagonists.
Table 2. ANOVA Table for Fumonisin analysis of maize seeds whose stems received treatment combinations of pathogen and selected antagonists.
Even though T. harzianum strain 2 seemed to be the most effective antagonist against the pathogen within the stem tissues in another experiment  , seeds from the plants that received “Inoculation of T. harzianum strain 2 alone” and two other different treatment combinations of the fungus (i.e. “Inoculation of pathogen before T. harzianum strain 2” and “Inoculation of T. harzianum strain 2 and pathogen simultaneously”) contained a comparatively high content of fumonisin. The same could be said of two of the treatment combinations of T. hamatum (i.e. “Inoculating pathogen before T. hamatum” and “Inoculating T. hamatum and pathogen simultaneously”) even though “Inoculating T. hamatum alone” gave the lowest fumonisin concentration in the seeds. Even though this might be interpreted that the treatments applied on the stem had little or no effect on the fumonisin content and hence Fusarium occurrence in the seeds, this, however could not be said to be totally true. This is because seeds with the lowest fumonisin content were also obtained from plants whose stems received treatments involving the same fungi (i.e. “Inoculation of T. hamatum alone” and “Inoculation of T. harzianum strain 2 before pathogen”). This low fumonisin content might not be unconnected with the presence of the two Trichoderma species. The antagonists might have moved up the stem through the parenchyma tissue into the cob as concluded by  thereafter reducing Fusarium occurrence in the seeds. Perhaps T. hamatum and/or T. harzianum strain 2 isolated from maize plant parts might be used, just like T. viride employed by  to suppress the radial colony extension of Fusarium verticillioides on agar as well as the fumonisin B1 (FB1) on corn kernels. In line with their suggestions for T. viride, perhaps T. hamatum and/or T. harzianum strain 2 isolated from the maize plant parts might be useful in biological control as a pre-harvest agent (used separately or combined) to prevent disease during plant development and/or as a post-harvest agent to suppress FB1 production during seed storage. This will be an adventurous challenge going by the opinions of  that there will be many obstacles to successful fumonisin reduction by biological control because of the numerous pathways for infection by the fungi. They added that the ubiquitous nature of naturally occurring, fumonisin-producing Fusarium strains will make this approach extremely challenging and Environmental Protection Agency (EPA) approval of such practice may also pose an obstacle.
 Cecile, L.G., Sylviane, B., Didier, K. and Jean-Denis, B. (2015) Assessing Risk of Fumonisin Contamination in Maize Using Near-Infrared Spectroscopy. Journal of Chemistry, 2015, Article ID: 485864.
 Queiroz, V.A.V., Vieira, Q., da Costa, R.V., et al. (2012) Occurrence of Fumonisins and Zearalenone in Maize Stored in Family Farm in Minas Gerais, Brazil. Food Control, 28, 83-86.
 Muthomi, J.W., Njenga, L.N., Gathumbi, J.K. and Chemining’wa, G.N. (2009) The Occurrence of Aflatoxin in Maize and Distribution of Mycotoxin-Producing Fungi in Eastern Kenya. Plant Pathology Journal, 8, 113-119.
 Humpf, H. and Voss, K.A. (2004) Effects of Thermal Food Processing on the Chemical Structure and Toxicity of Fumonisin Mycotoxins. Molecular and Nutritional Food Research, 48, 255-269.
 Porter, J.K., Rimando, A.M., Bacon, C.W. and Voss, K.A. (1995) Identification of Fusaric Acid in the Stomach Colostrum of Neonate Rats and Effects on Growth. In: Chelkowski, J., Ed., Fusarium, Mycotoxins, Taxonomy and Pathogenicity, Elsevier Science, Amsterdam.
 Julian, A.M., Wareing, P.W., Philips, S.I., Medlock, V.F.P., MacDonald, M.V. and Del Rio, L.E. (1995) Fungal Contamination and Selected Mycotoxins in Pre- and Postharvest Maize in Honduras. Mycopathologia, 129, 5-16.
 Lazzaro, I., Falavigna, C., Galaverna, G., Dall’Asta, C. and Battilani, P. (2013) Cornmeal and Starch Influence the Dynamic of Fumonisin B, A and C production and Masking in Fusarium verticilloides and F. proliferatum. International Journal of Food Microbiology, 166, 21-27.
 Gelderblom, W.C.A., Jaskiewicz, K., Marasas, W.F.O., Thiel, P.G., Horak, R.M., Vleggaar, R. and Kriek, N.P.J. (1988) Fumonisins-Novel Mycotoxins with Cancer Promoting Activity Produced by Fusarium moniliforme. Applied and Environmental Microbiology, 54, 1806-1811.
 Thiel, P.G., Shephard, G.S., Sydenham, E.W., Marasas, W.F.O., Nelson, P.E. and Wilson, T.M. (1991) Levels of Fumonisin B1 and B2 in Feeds Associated with Confirmed Cases of Equine Leukoencephalomalacia. Journal of Agricultural and Food Chemistry, 39, 109-111.
 Gelderblom, W.C.A., Kriek, N.P.J., Marasas, W.F.O. and Thiel, P.G. (1991) Toxicity and Carcinogenicity of the Fusarium moniliforme Metabolite, Fumonisin B1 in Rats. Carcinogenesis, 12, 1247-1251.
 Harrison, L.R., Colvin, B.M., Greene, J.T., Newman, L.E. and Cole, J.R. (1990) Pulmonaryedema and Hydrothorax in Swine Produced by Fumonisin B1, a Toxic Metabolite of Fusarium moniliforme. Journal of Veterinary Diagnostic Investigation, 2, 217-221.
 Yates, I.E., Meredith, F., Bacon, C.W. and Jaworski, A.J. (2000) Trichoderma viride Suppresses Fumonisin B1 Production by Fusarium moniliforme. Journal of Food Protection, 62, 1326-1332.
 Paavanen-Huhtala, S., Avikainen, H. and Yli-Mattila, T. (2000) Development of Strain-Specific Primers for a Strain of Gliocladium catenulatum Used in Biological Control. European Journal of Plant Pathology, 106, 187-198.
 Etebarian, H.R., Scott, E.S. and Wicks, T.J. (2000) Trichoderma harzianum T39 and T. virens DAR 74290 as Potential Biological Control Agents for Phytophthora erythroseptica. European Journal of Plant Pathology, 106, 329-337.
 Sobowale, A.A., Cardwell, K.F., Odebode, A.C., Bandyopadhyay, R. and Jonathan, S.G. (2007) Persistence of Trichoderma Species within Maize Stem against Fusarium verticillioides. Archives of Phytopathology and Plant Protection, 40, 215-231.
 Lawrence, E.B., Nelson, P.E. and Ayers, J.E. (1981) Histopathology of Sweet Corn Seed and Plants Infected with Fusarium moniliforme and F. oxysporum. Phytopathology, 71, 379-386.