Identification of Atoxigenic Aspergillus flavus Isolates to Reduce Aflatoxin Contamination of Maize in Kenya

Probst, C., Bandyopadhyay, R., Price, L. E., and Cotty, P. J. 2011. Identification of atoxigenic Aspergillus flavus isolates to reduce aflatoxin contamination of maize in Kenya. Plant Dis. 95:212-218. Aspergillus flavus has two morphotypes, the S strain and the L strain, that differ in aflatoxin-producing ability and other characteristics. Fungal communities on maize dominated by the S strain of A. flavus have repeatedly been associated with acute aflatoxin poisonings in Kenya, where management tools to reduce aflatoxin levels in maize are needed urgently. A. flavus isolates (n = 290) originating from maize produced in Kenya and belonging to the L strain morphotype were tested for aflatoxin-producing potential. A total of 96 atoxigenic isolates was identified from four provinces sampled. The 96 atoxigenic isolates were placed into 53 vegetative compatibility groups (VCGs) through complementation of nitrate non-utilizing mutants. Isolates from each of 11 VCGs were obtained from more than one maize sample, isolates from 10 of the VCGs were detected in multiple districts, and isolates of four VCGs were found in multiple provinces. Atoxigenic isolates were tested for potential to reduce aflatoxin concentrations in viable maize kernels that were co-inoculated with highly toxigenic S strain isolates. The 12 most effective isolates reduced aflatoxin levels by >80%. Reductions in aflatoxin levels caused by the most effective Kenyan isolates were comparable with those achieved with a United States isolate (NRRL-21882) used commercially for aflatoxin management. This study identified atoxigenic isolates of A. flavus with potential value for biological control within highly toxic Aspergillus communities associated with maize production in Kenya. These atoxigenic isolates have potential value in mitigating aflatoxin outbreaks in Kenya, and should be evaluated under field conditions. Aflatoxins are a series of highly toxic polyketides produced by several species of Aspergillus (46,65). The most commonly occurring aflatoxin, aflatoxin B1, is a genotoxin known to be carcinogenic and teratogenic for both humans and animals (49,69) and, to date, the only mycotoxin classified as a group 1a human carcinogen by the International Agency for Research on Cancer (40,41). Crops infected by aflatoxin-producing fungi frequently become contaminated with aflatoxins. Aflatoxin contamination results in reduced crop value and diminished health of humans and domestic animals that consume the contaminated crops (72). The quantity of ingested aflatoxins determines whether health effects are chronic (e.g., immune suppression, impaired child growth, abnormal fetal development, and cancer) or acute (e.g., hepatitis and jaundice, abdominal swellings, and death) (11,32,36,43,71). To date, Kenya is the only nation with a population that has repeatedly experienced epidemics of acute aflatoxicosis (5,53,55). These episodes resulted from consumption of highly contaminated, homegrown maize and have extended over two decades, with the most recent occurring from 2004 through 2006, when several hundred Kenyans died from acute aflatoxin poisoning in several districts of the Eastern Province (12,54). During these periods, many thousands of individuals were exposed to unsafe aflatoxin levels (54,60). In 2010, another extensive epidemic occurred in Kenya, with high frequencies of harvested maize lots containing levels of aflatoxins unfit for human consumption (42). Aspergillus flavus is the most frequently implicated causal agent of aflatoxin contamination of maize (46). This species has several morphotypes (commonly called strains), among which the L and S strains are most studied. These strains differ in several characteristics, including production of sclerotia, conidia, and aflatoxins (14). L strain isolates produce few, large sclerotia (average >400 μm) and highly variable quantities of aflatoxins, with some isolates (called atoxigenic isolates) entirely lacking the ability to produce aflatoxins. In contrast, S strain isolates produce numerous, small sclerotia (average <400 μm) and, on average, higher levels of aflatoxins than L strain isolates (14). Each of the morphotypes is further subdivided into many vegetative compatibility groups (VCGs) delineated by a heterokaryon incompatibility system (57). There is also variability among VCGs in aflatoxin-producing ability. Thus, A. flavus exists in complex communities that vary widely in both strain and VCG composition and aflatoxin-producing ability. Fungal communities in Kenya associated with severe maize contamination and deaths in human populations have atypical structures, with the S strain of A. flavus highly dominant, and increasing incidence of the S strain associated with increasing contamination levels (59,60). The influence of aflatoxins on human populations in Kenya over the past decade demonstrates a clear need for tools to manage contamination of locally produced maize. A highly promising method for aflatoxin management has been the use of atoxigenic isolates of A. flavus to competitively exclude aflatoxin producers and, thereby, reduce aflatoxin concentration in a crop (21,25,29). Two atoxigenic isolates used commercially in the United States are very effective at inhibiting aflatoxin contamination by the S strain of A. flavus (35). Identification of atoxigenic isolates of A. flavus native to Kenya might provide an environmentally sound, ecologically adapted, native, biological resource useful in mitigating aflatoxin contamination of maize produced in Kenya. This study sought to determine whether atoxigenic isolates of A. flavus with potential value in biological control could be selected from highly toxic fungal communities in aflatoxin-contaminated maize produced in Kenya that had been associated with lethal aflatoxicosis. Materials and Methods Fungal isolation, maize samples, and fungal inoculum preparation. Isolates of A. flavus were collected during previous studies Corresponding author: P. J. Cotty, E-mail: [email protected] Accepted for publication 19 October 2010. doi:10.1094 / PDIS-06-10-0438 This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 2011.