Development of Aflatoxin Resistance Screening Tools

Several screening tools have been developed and used to facilitate corn breeding for developing germplasm resistant to fungal growth and/or aflatoxin contamination (King and Scott 1982). Inoculation methods employed with corn include the pinbar inoculation technique (for inoculating kernels through husks with A. flavus conidia), the silk inoculation technique, and infesting corn ears with insect larvae infected with A. flavus conidia. (King and Scott 1982; Tucker et al. 1986). Two resistant inbreds (Mp420 and Mp313E; Scott and Zummo 1988; Windham and Williams 1998) were discovered and tested in field trials at different locations, using the pinbar technique, and released as sources of resistant germplasm.

A rapid laboratory kernel screening assay (KSA) was developed and used to study resistance to aflatoxin production in mature kernels (Brown et al. 1993; 1995). The results of this study indicated the presence of two levels of resistance: at the pericarp and at the subpericarp level. The subpericarp level of resistance was shown to require a viable embryo (Brown et al. 1993). KSA studies further demonstrated a role for pericarp waxes in kernel resistance (Guo et al. 1995; 1996) and highlighted quantitative and qualitative differences in pericarp wax between resistant and susceptible genotypes (Gembeh et al. 2001; Russin et al. 1997). This research was all based on the prior identification, during field studies, of a resistant corn breeding population, GT-MAS:gk (Widstrom et al. 1987).

The KSA also confirmed sources of resistance among 31 inbreds tested in Illinois field trials (Brown et al. 1995; Campbell and White 1995), thus demonstrating that the KSA can be used, at least initially, to rank corn for its field resistance to aflatoxin contamination. Subsequently, the KSA was used as a preliminary screen for resistance to aflatoxin contamination in kernels of maize inbreds selected for ear rot resistance in West and Central Africa (Brown et al. 2001a). The KSA has advantages over traditional field screening techniques (Brown et al. 1995), mainly because of the rapidity of the assay. However, field trials are irreplaceable for confirmation of resistance.

Recently, the KSA was improved by including a method to quantify fungal biomass using the ^-glucuronidase (GUS) or green fluorescent protein (GFP) (Du et al. 1999; Windham and Williams 1998; Windham et al. 1999) reporter gene-containing A. flavus tester strains. A. flavus tester strains were genetically engineered with a gene construct consisting of the GUS reporter gene linked to an A. flavus b-tubulin gene promoter for monitoring fungal growth (Brown et al. 1995; 1997) or with the reporter gene linked to an aflatoxin biosynthetic pathway gene which could also provide a quick and economical way to indirectly measure aflatoxin levels

(Brown-Jenco et al. 1998; Payne 1997). Thus, it is now possible to accurately assess fungal infection levels and to predict the corresponding aflatoxin levels in the same kernels, as a result of fungal infection. This technology might be applied to screening for resistance to mycotoxin contamination by other mycotoxigenic fungi. Recently, an F. verticillioides strain, containing a GUS reporter gene was used in the KSA to demonstrate that this fungus is inhibited in aflatoxin-resistant genotypes (Brown et al. 2001b). This indicates that some resistance mechanisms may be generic for ear rotting/mycotoxigenic fungi.

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