Prevalence of Aflatoxin Biosynthesis Genes According to Aflatoxin Levels in Maize of Different Varieties in Kenya

Abstract

Aims: To determine aflatoxin biosynthetic genes in fungal isolates in relation to aflatoxin levels in maize grain varieties from epidemiologically aflatoxicosis hot spots and non-aflatoxin hot spots agro-ecological zones in Kenya. Study Design: Purposeful sampling technique was applied targeting regions of variable aflatoxicoses susceptibilities. Place and Duration of Study: Samples were sourced from Kitui/Kibwezi counties, an aflatoxin hot spot, during 2008-2010 Maize growing seasons. Comparative samples were from Uasin-Gishu county and Perkerra irrigation scheme in Baringo County, both with no previous acute aflatoxicosis and practicing commercial cultivation under rain-fed and irrigation farming systems, respectively. Methodology: Maize samples (n=295) and fungal isolates (n=61) were analyzed for aflatoxin contamination and presence of aflatoxin biosynthetic genes, respectively. Total aflatoxin quantification was by a commercial Enzyme Linked Immunosorbent Assay (ELISA) kits, Boratest®, while molecular characterization of Aspergillus flavus (n=40) and A. parasiticus (n=21) isolates applied Quadruplex Multiplex PCR technique encompassing four aflatoxin biosynthetic genes: nor-1, ver-1, omt-A and aflR. Findings from the study variables were analyzed according to maize variety, agro-ecological origin of maize samples and fungal species besides type of farming system. Results: Uasin-Gishu maize samples (n=158) assayed for aflatoxins belonged to six maize commercial varieties; H614, H629, H6213, H6210, H613 and H628 alongside an indigenous variety, Kipkaa. All Perkerra samples (n=61) also belonged to a commercial variety, H513. Contrastingly, all Kitui/Kibwezi samples (n=76) belonged to an indigenous variety, Kikamba (Kinyanya). The varieties H613, H628 and Kipkaa all had the samples (100%) within the Kenyan statutory safe total aflatoxin limit (≤10.0 ppb) whereas Kikamba and H513 varieties had 82.9% and 83.6% samples within safety limits, respectively. Similarly, the mean aflatoxin content for all the seven Uasin-Gishu varieties was only 1.62 ppb while Kikamba and H513 had means of 14.6 ppb and 15.6 ppb, respectively (P=0.05). Positive PCR amplification results were obtained in 96.3%, 84.2% and 80% for Kitui/Kibwezi, Perkerra and Uasin-Gishu isolates, respectively whereas regional distribution of amplicon spectrum was 6, 3 and 2 out of 8, respectively. A similar regional pattern was established regarding prevalence of PCR positive isolates and whose maize samples of origin also tested ELISA-aflatoxin positive, having been 81.5%, 52.6% and 26.7% for Kitui/Kibwezi, Perkerra and Uasin-Gishu. Interestingly, the only isolate PCR positive for all the four genes under assay and whose maize sample of origin had aflatoxins was coincidentally from Kitui/Kibwezi, an epidemiologically aflatoxicosis  hot spot agro-ecological zone.