EVALUATION OF DIFFERENT FERTILIZER SOURCES FOR THE MANAGEMENT OF AFLATOXIN CONTAMINATION IN GROUNDNUT (ARACHIS HYPOGAEA L.) IN THE SOUTHERN GUINEA SAVANNAH AGRO-ECOLOGICAL ZONE OF NIGERIA

Eche C.O. 1 , Vabi. M. B 2 , Ekefan. E. J 1 , Ajeigbe. H. A. 2 and Ocholi F. A 1 . 1. Plant Pathology Research Group, Department of Crop and Environmental Protection, Federal University of Agriculture, Makurdi, Benue State, Nigeria. 2. International Crops Research Institute for the Semi-Arid Tropics, Kano, Nigeria. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


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Gypsum + NPK should be promoted since their use was associated with reduced levels of aflatoxin contamination and better pod and haulm yields.
Groundnut is an important component of the diets of most Nigerians, because of its high protein contents and carbohydrate contents (Ya'u, 2016).It is also rich in calcium, potassium, phosphorus, magnesium and vitamins B and E. Groundnut meal, a by-product of oil extraction, is an important ingredient in livestock feed.Groundnut haulms (stem and leaves) are very nutritious and widely used for feeding livestock across the drylands of West and Central Africa.According to Young (1996), oil extracted from groundnut is composed of mixed glycerides, and contains a high proportion of unsaturated fatty acids, in particular Oleic (50-56%) and Linoleic (18-30%) acids.The leaves are used in some West African countries as vegetable.Groundnut oil is the most important outcome of processing groundnut, which is used both for domestic and industrial purposes.About 75% of the world's groundnut production is used in extraction of edible oil.With the recent thrust on bioenergy development, options are being explored to use groundnut as a bio-diesel crop, because the crop produces more oil per hectare than many other food crop ((Ya'u, 2016).In most, if not all countries of Sub-Saharan Africa, groundnut production, marketing and trade are important sources of employment, cash incomes and foreign exchange earners (Ntare et al., 2005).Nigeria mainly exports its nuts to Indonesia and countries of the European Union.However, groundnuts produced in Sub-Saharan Africa (SSA) are reported to be highly susceptible to attacks by the Aspergillus group of soil-borne fungi that produce toxic secondary metabolites known as aflatoxins (Payne, 1998).
The genus Aspergillus is a member of the phylum Ascomycota and comprises over 185 known species.Members of Aspergillus section Flavi are characterized by their ability to produce flavine-derived secondary metabolites which include aflatoxins.Aflatoxins are mycotoxins associated with hepatotoxicity, mutagenicity and carcinogenicity in human beings and cattle (Hesseltine, 1965;Ainsworth and Austwick, 1973).Species capable of producing aflatoxins include Aspergillus flavus, Aspergillus parasiticus, and several less common taxa such as Aspergillus nomius, Aspergillus tamarii, A. pseudotamarii, A. minisclerotigenes and A. bombycis (Cotty, 1994;Klich et al., 2009).Other Aspergillus outside of the section Flavi are also known to produce aflatoxins and species with this ability are known to be more diverse than previously thought (Cotty et al., 1999).Toxigenic strains of Aspergillus contaminate groundnuts and other crops along the value chain including post-harvest handling.Contamination varies from year to year and is particularly high when plants are exposed to stress toward the end of the growing season (Waliyar et al., 2015).Pre-harvest infections of aflatoxin usually occur when the plant is exposed to moisture and heat stress during pod development, when pods are damaged by insects or nematodes or when they are mechanically damaged during farm operations (Waliyar et al., 2007).As farming is primarily rain-fed in most countries of SSA, but also recognizing current climatic variability, conditions favouring the development of aflatoxin contamination occur more frequently.Hence the management of aflatoxin contamination in the face of climate change has become an increasing concern.
Quite a few approaches for the management of aflatoxin contamination have been reported.Cases for the use of aflatoxin resistant varieties (Upadhyaya et al., 2002;Nigam, 2004;Upadhyaya et al., 2004), use of biocontrol agents such as Trichoderma [T.viride (Tv 47), T. harzianum (Th 23), T. harzianum (Th 20), T. koningii (Tk 83)], geocarposphere bacterial strains of Pseudomonas [P.aeruginosa CDB35, P. cepacia and P. fluorescens] and Actinomycetes [strain CDA19)] in reducing groundnut seed colonization by competitive exclusion/inhabitation of 969 Aspergillus species (Thakur and Waliyar, 2005) have also been advocated.Efforts are also being made to introduce 13S LOX (lipoxygenase) gene, which has been shown to deregulate aflatoxin production, and/or RNAi technology to knock-out 9S Lox gene that enhances the sporulation and aflatoxin production in groundnut (Anjaiah et al., 2006;Hameeda et al., 2006;Sharma et al., 2006).The use of soil amendments techniques such as gypsum, and cereal crop residues applied either singly or in various combinations at different cropping stages have also been reported (Waliyar et al., 2006).The advent of readily available fertilizers has brought about the reduction or termination of many pathogenic diseases through improved plant resistance, disease escape, altered pathogenicity, or microbial interactions (Alenyorege et al., 2015).Since aflatoxin resistant varieties, biocontrol agents and transgenic plants are not readily available to farmers with limited resource endowments in Nigeria, efficient fertility programs can indirectly enhance groundnut resistance to toxigenic strains of Aspergillus by reducing the impact of environmental stress, and increasing the quality of groundnut and groundnut-based products.This is the hypothesis upon which this study was conducted.The objectives of these trials were (i) to determine the effects of various combinations of Farmyard Manure (FYM) and inorganic fertilizers on aflatoxin contamination in the Southern Guinea Savannah, of Nigeria alongside while assessing the agronomic performance of SAMNUT 24; a recently released groundnut variety which is extra early maturing, high yielding, rosette resistant and market/farmer preferred (FMARD, 2014).

Materials and Methods:-
Experimental Site:-Two experiments were conducted, each during the 2016 and 2017 cropping seasons at the Teaching and Research Farms, Federal University of Agriculture, Makurdi located at latitude 07º45'N to 07º50'N, longitude 08º45'E to 08º50'E, 98m above sea level.The site falls within the Southern Guinea Savannah agro-ecological zone of Nigeria where the rainfall is bi-modally distributed with the highest peak in July/August.The raining season commences between March/April and terminates in October/November.The total annual rainfall is about 2000-2500 mm and maximum and minimum temperatures of 37ºC and 21ºC, respectively, while the relative humidity is about 70-80%.The land used was previously under intercropping with cassava (Manihot esculenta) and maize (Zea mays L.).The surface soil (0-15 cm depth) was characteristic loamy sandy with 80.8% sand, 8.2% silt and 11.0% clay contents.The soil pH (soil:H 2 O) was 6.01 and contained 1.09% organic carbon, 0.09% total N, 8.70 mg/kg available P, while Ca, Mg, K and Na contents were 3.79, 1.3, 0.06 and 0.12 cmol/kg, respectively.The ECEC of the soil sample was 6.68 cmol/kg, exchangeable acidity 0.36 cmol/kg and base saturation was 20.0%.The nutrient profile of the farmyard manure used was 69.2% organic carbon, 5.2% total N, 37 mg/kg available P, while calcium, magnesium, potassium and sodium concentrations were 4.8, 3.0, 0.17 and 0.10 cmol/kg, respectively.The ECEC and base saturation of the manure were 19.2 cmol/kg and 40%, respectively respectively.

Experimental Design:-
The experiment evaluated various combinations of organic [Farmyard manure (FYM)] and inorganic [Gypsum, Single superphosphate (SSP) and NPK (15:15:15)] fertilizers applied at recommended rates.Farmyard manure was applied at the rate of 5 tons/ha while Gypsum, SSP and NPK were applied at 2.24 tonsha -1 , 60 kg P 2 O 5 and and 120 kg NPK kgha -1 respectively.A total of eleven treatments were used in the experiment and included; FYM, Gypsum, SSP, NPK, FYM+Gypsum, FYM+SSP, FYM+NPK, Gypsum+SSP, Gypsum+NPK, SSP+NPK and Control (untreated plots).The experiments in both 2016 and 2017 were laid-out in Randomized Complete Block Design (RCBD) with four replications of 79m x 26m (2054 m 2 ) with plot sizes of 5 m x 4 m (20 m 2 ).The experimental site was cleared and cultivated manually.Farmyard manure was incorporated into randomly selected plots two weeks before planting while other inorganic fertilizers were applied two weeks after planting (WAP).The groundnut seeds (SAMNUT 24) were sown at a spacing of 10 cm within rows and 75 cm between rows, planting two seeds per hole.Weeding was carried-out at 3, 6 and 9 WAP.

Enzyme Linked Immunosorbent Assay (ELISA):-Sample Extraction:-
One hundred grams of groundnut seeds were collected from each treatment and blended separately each time, thoroughly washing and rising the blender with sodium hypochlorite (NaOCl) to avoid cross contamination.Twenty (20g) of each of the blended groundnut seeds were ground into fine powder and triturated with 100 ml of 70% methanol (v/v 70 ml absolute methanol in 30 ml distilled water) containing 5g potassium chloride in Waring Commercial blender up until homogeneity was reached.The extract was transferred into a 250 ml of properly labelled conical flasks and shaken on a Benchamark orbital (Model ORBI-Shaker) shaker for 30 minutes.Filtration was done using Whatman filter paper number 41 and the sample was later diluted in 1:10 phosphate buffer saline in 970 Tween-20 (1 ml of extract and 9 ml of buffer).The setup was left standing for 10 hours after which analysis of each sample began.

Sample Analysis:
-AFB 1 -BSA antigen was coated unto an ELISA plate.Specific antibodies available in the sample or standard competed with the bound AFB 1 -BSA antigen with the help of immune-globulins.Para Nitrophenylphosphate substrate was added which helped in colour development.AFB 1 levels were quantified using a spectrophotometer by giving optical density values at a 405 nm wavelength.Linear regression curve was plotted for optical density values and a standard curve was extrapolated with a known correlation coefficient thereby giving AFB 1 concentrations in parts per billion.1.
Data Analysis:-Agronomic data were subjected to analysis of variance using GenStat 17 th Edition (VSN International Ltd, 2014) software.Statistically significant differences were reported at p < 0.05.If the overall F-test was significant (p < 0.05), then Fisher's Least Significant Difference (LSD) test was used to compute the smallest significant difference between two means.

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The application of gypsum consistently reduced concentration levels of AfB 1 when applied solely or in combination with NPK or SSP.When compared to the untreated plot, contamination in groundnut grown on gypsum-solely treated plots was reduced from 15.17 ppb and 20.57ppb in the untreated plots in 2016 and 2017 respectively, to 3.76 ppb and 5.7 ppb representing over 70% reduction in both years.

Effect of Fertilizer Combinations on Growth of Groundnut:-
Table 2 shows the effects of different fertilizer combinations on groundnut Plant Height, Number  3. Treatment effect was statistically significant for all parameters except 100 Seed Weight.Groundnut plants grown on plots treated with SSP + NPK consistently had the highest Fresh Pod Yield (1.46 t/ha and 1.71 t/ha) and Dry Pod Yield (1.64 t/ha and 1.66 t/ha) in 2016 and 2017.The least Fresh and dry pod yields were recorded on plants grown on the untreated plots.Plants grown on plots treated with FYM + SSP outperformed the remaining fertilizer treatments in terms of Fresh Pod Yield (1.44 t/ha and 1.65 t/ha), Dry Pod Yield (1.38 t/ha and 1.56 t/ha) in 2016 and 2017.Plots treated with FYM and Gypsum + SSP had fresh pod yield values which were above the grand mean (1.30 t/ha) in 2016.In 2017 however, plots treated with FYM, SSP, NPK and Gypsum + SSP had higher fresh pod yield values above the grand mean (1.51 t/ha).

Discussions:-
Aflatoxin contamination constitutes one of the most challenging problems in food safety in developing countries.This is majorly due to its health implications but also negative effect on trade across country borders.In a country like Nigeria which is the largest producer of groundnut in Africa, it is pertinent that exposure to aflatoxin contamination is highly reduced if the country must continue in exportation of groundnut and ensure the health of its citizenry.In order to curb this menace of aflatoxin, it is therefore necessary to explore options that are not only effective but also affordable to the vast majority of resource-limited farm families.

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This study focused on elucidating the effects of different fertilizer combinations on aflatoxin contamination levels on an improved high yielding, extra early maturing and rosette reistant variety groundnut variety under the Southern Guinea Agroecology of Nigeria.Of all the fertilizer sources evaluated in the study, gypsum was the most effective in reducing aflatoxin contamination.This finding corroborates previous work by Waliyar et al. (2008), who reported that the application of lime or any calcium source fertilizer alone reduced aflatoxins contamination by 72%.Furthermore, the study showed that although, the application of gypsum alone reduced aflatoxin contamination levels, it did not significantly improve growth characteristics (plant height and number of branches) and yield compared to the other fertilizer combinations.
Contrastingly, the use of farmyard manure significantly increased aflatoxin contamination levels of over the two years, especially during the 2017 cropping season.In their report, Nakhro and Dkhar (2010) noted that organically treated plots recorded the maximum microbial population counts (fungal and bacterial) and microbial biomass carbon, followed by the inorganically treated plots.Similar findings were reported by Kumar et al. (2010).This study, however showed that farmyard manure significantly improved growth characteristics and biomass of groundnut.In fact, all combinations of farmyard manure such as FYM+Gypsum, FYM+NPK, FYM+SSP had higher levels of aflatoxin contamination compared to fertilizer combinations without farmyard manure with the least been FYM+Gypsum.
The use of farmyard manure is very popular among resource-limited farmers who use it as a primary source of fertilizer.The implication of such a practice as revealed by this study is that mitigating aflatoxin contamination becomes difficult (if not impossible) if this practice is not adequately controlled.Therefore, the source of fertilizer is a critical factor in groundnut production as it enhances the possibility of aflatoxin contamination.However, considering that the ultimate aim of agronomic venture is crop yield, combining gypsum with NPK, or SSP in groundnut production should be advocated.As demonstrated from this experiment, groundnut grown under these conditions had lower levels of aflatoxin contamination with a corresponding higher yield than solely gypsum-treated plots.
Levels of aflatoxin contamination was generally higher in 2017 compared to 2016; this could be linked to the higher rainfall and temperatures experienced in Benue State in 2017.It is known that Moisture content plays an important role in microbial activity in the soil.Indeed, high moisture contents enhances microbial activities and is even higher in an organically amended soil.Madge (1965) documented marked effects of moisture on the number of soil fungi.Moisture content and temperature gradients are chiefly responsible for the colonization of microorganisms (Marinari et al., 2000;Zaller and Koepke, 2004).
The Standards Organization of Nigeria (SON) sets limits on several food commodities, taking into account global standards as well as national production and target export markets.While it is generally recognized that there is no safe level of aflatoxin exposure, SON has set the maximum acceptable limit for groundnut at 4 ppb for total aflatoxins and 2 ppb for aflatoxin B1.Although none of the fertilizer combinations reduced contamination below levels to those stipulated by SON, groundnut grown in gypsum and gypsum-combined fertilizer treated-plots showed reduced levels.With respect to products for human consumption, the current action level is 20 ppb total aflatoxin in finished products and findings from this study clearly showed that all gypsum-treated plots reduced aflatoxin levels below 20 ppb and recommended for use in groundnut production systems.Nonetheless, research is still required to quantify toxigenic Aspergillus fungal population and determine optimal rates of Gypsum+NPK and Gypsum+SSP fertilizer combinations that will increase yields while reducing AfB 1 contamination in groundnut.

Figure 1 :
Figure 1:-Mean aflatoxin levels of groundnut grown under soils treated with difference combinations of fertilizers in 2016

Figure 2 :
Figure 2:-Mean aflatoxin levels of groundnut grown under soils treated with difference combinations of fertilizers in 2017 Agronomic data collected included Plant Height at 4, 8 and 12 WAP measured in centimeters, Number of Branches at 4, 8 and 12 WAP, Number of Days to 50 % Flowering, Fodder Weight/plot (g), Fresh Pod Weight/plot (g), Dry Pod Weight/plot (g) and 100 Seed weight/plot (g).Meteorological data for 2016 and 2017 Cropping Seasons were obtained from the Nigerian Meteorology Headquarter, Tactical Command, Makurdi Airport, Benue State, Nigeria as shown in Table

Table 1 :
-Meteorological Data of Makurdi, Benue State Nigerian Meteorological Agency (NiMet) 1(21.2 ppb and 30.3 ppb) representing 28.4% and 32.1% increase over the untreated plots in 2016 and 2017, respectively.In 2016, there was a reduction in AfB 1 concentrations when FYM was combined with SSP, NPK and gypsum by 65.5%, 44.3% and 59.4% respectively; similar results were obtained in 2017.A combination of FYM with SSP, NPK and gypsum, resulted in 18.4%, 26.5% and 50.3% resulting in a reduction in AfB 1 concentrations, respectively.

Table 2 :
-Effect of Different Fertilizer Sources and Combinations on Growth of Groundnut in 2016 and 2017

Table 3 :
-Effect of Different Fertilizer Sources and Combinations on Groundnut Yield in 2016 and 2017 Fertilizer Combination