Effects of Botanicals and Biocontrol Agents on Growth and Aflatoxin Production by Aspergillus Flavus Infecting Maize in Some Parts of Nigeria

Abstract

In a comprehensive study to assess the effects of botanicals and biocontrol agents on growth and aflatoxin (AF) production by Aspergillus flavus (A. flavus) infecting maize, a total of 1143 maize samples, collected in eighteen batches of five maize varieties (yellow, white, pop, variegated and mixed) from northern and southern parts of Nigeria were investigated between June 2011 to December 2013. Samples collected from field, 414 (36.2%) and stored batches, 729 (63.8%) were cultured on to mycological and bacteriological media after surface sterilization using 0.1 % sodium hypochlorite solution (NaOCl). Fungal isolates 358 (31.3%) and bacterial isolates 111 (9.71%) were identified using standard methods. Aspergillus niger 72 (20.1%) ranked highest followed by A. flavus 62 (17.3%) amongst the fungal isolates while Staphylococcus aureus 48 (43.2%) followed by Escherichia coli 32 (28.8%) were the most predominant bacterial isolates. Fungal isolates, 220 (61.5%) were more in the field samples than in the stored samples 138 (38.5%) and the difference was statistically significant (p < 0.05). Bacterial isolates were more in stored samples, 75 (67.6 %) than in the field samples 36 (32.4 %) (p < 0.05). A seasonality pattern was observed favouring the rainy season, 245 (68.4 %) than the dry season, 113 (31.6%) for fungal distribution. The reverse was observed for bacteria with 75 (67.6 %) and 36 (32.4%) for the dry and rainy seasons respectively during the study period (p < 0.05). Of the total of 62 (17.3 %) A. flavus isolates, 26 (41.9 %) were found to be aflatoxigenic strains and was more in the field 17 (65.4 %) than in the stored 9 (34.6%) samples (p < 0.05). Generally, maize were more contaminated by fungi from the southern 290 (83.1%) than the northern samples 59 (16.9%) which was statistically significant (p < 0.05). Similar pattern was observed for bacterial isolates with values of 57.7 % and 42.3 % from the South and North respectively though the difference was not statistically significant (p > 0.05). Detection of the total natural aflatoxin (AF) levels in the eigthteen maize batches studied showed a mean range of 2450 to 125300 ng/kg. The highest detectable mean level of AF was from stored samples (125300 ng/kg) which were statistically significant from the highest detectable mean level from field samples (112000 ng/kg) (p < 0.05). This total natural aflatoxin were reduced to various levels using various edible plant extracts (Annona muricata and Moringa oleifera) and essential oils (EOs) (Cymbopogon citratus and Ocimum gratissimum) with a range of percentage reduction ranging from 0 to 91.8 % for Cymbopogon citratus, 7.1 % to 88.3 % for Ocimum gratissimum, 0 to 77.9 % for Moringa oleifera and 23.3 % to 90 % for Annona muricata showing the highest effect. There were statistically significant differences in the percentage reduction amongst these plant treatments (p < 0.05) using ANOVA. Similarly, in in-vitro studies using plate experiments, the various plant extracts also showed significant reductions in counts/ mean clearance zone diameter according to the extract concentration on the various fungi/ bacteria studied. Co-inoculation studies using co-bacteria or co-fungal pairings with A. flavus also showed varying inhibitions. Saccharomyces cerevisiae, Geotrichum candidum and Aspergillus niger significantly inhibited production and detoxification when co-administered with A. flavus. Among the bacteria tested Bacillus subtilis and Pseudomonas aeruginosa showed significant reduction in AF production and detoxification when co-administered with A.flavus. However, these bacteria are potentially pathogenic and their virulence need to be genetically altered before they can be used together with the fungi mentioned above as biocontrol agents.