Abebe Menkir Robert L Brown Ranajit Bandyopadhyay Zhiyuan Chen and Thomas E Cleveland

Abstract

Ear-rot-causing fungi, including Aspergillus and Fusarium spp., are common in maize in West and Central Africa. These fungi contaminate maize with mycotoxins that pose serious potential health hazards to humans in these areas. A collaborative germplasm screening project was initiated between the International Institute of Tropical Agriculture (IITA) and the Southern Regional Research Center (SRRC) of the USDA's Agricultural Research Service in 1998 to develop maize germplasm with resistance to aflatoxin contamination. In a laboratory screen, some IITA inbred lines had potential levels of resistance to aflatoxin production as high as or higher than the best lines from the United States. These results prompted the initiation of a breeding project to combine resistance factors from the IITA lines with resistance factors from the US inbred lines. Several crosses and backcross populations were made from selected resistant or potentially resistant inbred lines from the US and IITA. Sixty-five S5 lines were developed from the backcross populations and 144 S5 lines were derived from the F1 crosses. Kernels from these lines were screened in a laboratory assay. Significant differences in aflatoxin accumulation were detected amongst the lines within each group. Several S5 lines in which aflatoxin contamination was significantly less than in either parent were selected for resistance-confirmation tests. We found pairs of S5 lines with 88-97% common genetic backgrounds that differed significantly in aflatoxin accumulation. These pairs of lines are being used for proteomic analyses to identify the proteins and the corresponding genes that limit aflatoxin accumulation. We also found significant differences in fumonisin accumulation amongst 58 elite maize inbred lines in which variation in aflatoxin accumulation was found. Both the new inbreds and the elite lines can be exploited as new genetic sources in breeding programs in which the objective is to develop maize cultivars/hybrids that accumulate lesser amounts of mycotoxins.

Introduction

Maize is a major staple food crop widely grown from the wet climate of the coast to the dry Sudanese savannas of West and Central Africa. The most common ear and kernel rot causing fungal species in maize in this area include Aspergillus, Botryodiplodia, Diplodia and Fusarium (Fajemisin et al., 1985). Some of these fungi are widespread in different tropical maize growing environments and can cause considerable economic losses by reducing grain quality in maize (Miller, 1996). When two of these fungal species, Aspergillus flavus and Fusarium verticillioides, infect maize plants, the developing kernels can be contaminated with aflatoxins and fumonisins, respectively, that render the harvested grain unfit for use as human food or animal feed.

Contamination with aflatoxins and fumonisins occur in warm, humid, tropical and subtropical maize-growing environments that are conducive to growth and development of the two fungi (Widstrom, 1996; Kpodo and Bankole, Chapter 9; Siame and Nawa; Chapter 10). Maize contaminated with mycotoxins is a serious problem in Sub-Saharan Africa because most of the maize grain in many countries is used for human consumption and the capacity to monitor the mycotoxin levels in the grain is limited (Widstrom, 1996). The widespread exposure to aflatoxin in Africa has been implicated in the increased incidence of acute toxicosis, liver cancer, and morbidity in children suffering from kwashiorkor (Miller, 1996; Widstrom, 1996; Gong et al., Chapter 6). The consumption of fumonisin-contaminated maize foods also has been associated with outbreaks of esophageal cancer (Rheeder et al., 1992; Thiel et al., 1992) and neural tube birth defects in humans (Stack, 1998). Some reports suggest that various processing methods including roasting, boiling, frying, baking and fermenting, may not effectively eliminate either aflatoxin (Widstrom, 1996) or fumonisin (Hendrich et al., 1993; Voss et al., 1996; Fandohan et al., Chapter 26) from contaminated maize food products.

Several approaches have been proposed for reducing aflatoxin (Widstrom, 1996) and fumonisin (Norred et al., 1991; Hendrich et al., 1993; Voss et al., 1996; Katta et al, 1997) levels in unprocessed grain and processed maize-based food products. One promising strategy is to grow maize cultivars that are resistant to Aspergillus and Fusarium ear rot and accumulate less mycotoxin in the grain (Widstrom, 1996; Kleinschmidt et al., 2005). Moderate to high levels of resistance to A. flavus (Gorman and Kang, 1991; Brown et al., 1999, 2001) and F. verticillioides (Widstrom, 1996; Clements et al, 2004) are known in maize. The use of these types of resistance in combination with appropriate cultural practices can reduce the total amount of mycotoxin accumulation. Preharvest host resistance to A. flavus and F. verticillioides is a simple and economical technology that leaves no harmful residue in food or the environment, and can be applied over a broad range of environmental and socioeconomic conditions. Although several maize genotypes that accumulate only low levels of aflatoxin and fumonisin have been identified, most of these lines lack desirable agronomic backgrounds, with adaptation problems and the relatively high levels of toxin that can still accumulate sufficing to prevent commercial deployment (Gorman and Kang, 1991; Brown et al, 1999; Clements et al., 2004; Brooks et al., 2005; Kleinschmidt et al., 2005). Thus, there is a need to develop maize germplasm with desirable agronomic traits and reduced levels of mycotoxin contamination. This chapter evaluates the breeding strategies currently used at IITA to develop germplasm resistant to infection by A. flavus and F. verticillioides, which includes: (i) field selection for reduced ear rot infection, (ii) breeding for reduced aflatoxin accumulation, and (iii) screening elite germplasm for resistance to specific fungi.

Field selection for reduced ear rot infection

Selecting germplasm resistant to ear rots has a crucial role in IITA's strategy for developing maize germplasm targeted to the forest zones and mid-altitude regions of Central and West Africa. Every year, early generation and advanced breeding lines developed from diverse sources of germplasm, as well as varieties and hybrids, are screened in "hot spot" locations representative of the production zones where ear rot occurs regularly at high levels. Promis ing materials are selected following visual assessment for reduced levels of ear rot. Environmental factors affect naturally occurring ear rot severity, so the breeding materials are evaluated repeatedly at the "hot spot" locations and at different stages of development to identify materials with reasonable levels of resistance to kernel and ear rot infection. The breeding materials also have been evaluated for tight husk cover, which is the first line of defense against infection by fungi causing ear rot (Widstrom, 1996). The detection of high concentrations of mycotoxins in maize genotypes exhibiting minimal ear rot infection symptoms (Clements et al., 2004; Kleinschmidt et al., 2005) emphasizes the need to select directly for maize germplasm with reduced mycotoxin accumulation.

Breeding for reduced aflatoxin accumulation

Screening for reduced aflatoxin accumulation in parental materials

Effective, reliable, rapid screening techniques are indispensable for breeding for lower levels of aflatoxin accumulation in maize (Gorman and Kang, 1991; Brown et al., 1999). Brown et al. (1995) developed a rapid laboratory-based kernel-screening assay that creates high uniform levels of infection and aflatoxin production and enables the differentiation of maize genotypes that accumulate low and high levels of aflatoxins. This assay provides a consistent ranking of maize genotypes in different tests and the results are correlated with results obtained in field trials (Brown et al., 1995).

Seventy-six inbred lines from IITA with moderate to high levels of field resistance to ear rots in the forest zone and the mid-altitudes were evaluated at the Southern Regional Research Center (SRRC)-USDA-ARS laboratory with the kernel screening assay (Brown et al., 2001). Eighteen of these inbred lines had aflatoxin levels that were as low as or lower than those of the best lines from the United States. Further studies with some lines found that the protein profiles of the IITA lines were different from those developed in the United States, suggesting that the IITA lines and those from the United States carry different al-leles for the reduction of aflatoxin accumulation. By assessing fungal growth on selected lines, a unique line with low aflatoxin accumulation, but a high level of fungal growth was identified (Brown et al., 2001). This result suggests that toxin accumulation may be inhibited directly in addition to being related to the amount of fungal infection present. The lowest toxin accumulating lines from IITA were crossed with similar genotypes from the United States in a collaborative breeding project (Brown et al., 2003). This strategy increases the probability of developing inbred lines with good agronomic traits that accumulates less toxin than do the currently available commercial lines.

Genetics of resistance to aflatoxin accumulation

The mode of inheritance of resistance to Aspergillus ear rot and reduced aflatoxin accumulation in maize grain is not settled. In some studies resistance to ear rot and lower aflatoxin accumulation levels are quantitatively inherited (Walker and White, 2001), with additive gene effects playing a major role the inheritance of resistance (Norred et al., 1991; Miller, 1996; Naidoo et al., 2002). In other studies, dominance has a greater effect on the reduction of aflatoxin accumulation than does additive gene action (Campbell et al., 1997; Campbell and White, 1995; Maupin et al., 2003; Busboom and White, 2004). Broad-sense herita-

Table 1. Mean and range of values (ng/g) for each group of inbred lines evaluated with the kernel screening assay in 2003 and 2004.

Group Number Range of lines

Mean ± Standard error

Lines derived from

I 14

II 6

III 10

IV 13

V 15

VI 12

VII 8 Lines derived from

I 12

II 10

III 11

IV 9

VI 9

VII 9

VIII 7

IX 11 Lines derived from

II 11

III 11

IV 11

V 12

backcrosses 6 - 6,000 0 - 5,200 360 - 5,100 75 - 10,200 40 - 2,100 150 - 7,900 140 - 4,800 tropical x temperate 890 - 15,000 740 - 14,000 3,000 - 9,100 0 - 4,400 80 - 3,700 500 - 9,300 390 - 3,600 250 - 5,600 230 - 4,600 tropical x temperate 990 - 14,000 130 - 7,400 460 - 6,600 650 - 9,500 700 - 12,000

6,500 6,500 6,000 1,900 2,200 5,300 1,500 3,300 1,300 crosses -7,900 ± 2,100 ± 2,500 ± 5,000 ± 4,200 ±

420 670 530 740 170 660 740 Set-1 1,300 1,400 530 450 380 880 310 740 430 Set-2 1,500 790 660 830 1,300

bility estimates for both ear rot resistance and lowered aflatoxin levels are moderate to high (Norred et al., 1991; Maupin et al., 2003), suggesting that selection for resistance should be feasible. Significant progress has been made in identifying sources of resistance and understanding their genetic basis, but neither the germplasm nor the genetic information has been used to breed commercially useful maize that accumulates less aflatoxin. The trait's complex of inheritance, the erratic nature of field infection by A. flavus, and the year-to-year variability in aflatoxin levels have limited transfer of these traits to elite maize inbred lines since selections made under field conditions often could not be relied on (Gorman and Kang, 1991; Brooks et al., 2005). The development of new efficient tools for screening maize genotypes in both field and laboratory settings increased the number of breeding strategies available for developing resistant maize germplasm that accumulates less toxin (Brown et al., 2003).

Creating populations and developing lines

Both pedigree and backcross breeding methods have been used to develop maize lines with new combinations of agronomic traits and resistance to diseases. Five elite tropical inbred lines from IITA

Table 2. Mean aflatoxin values (ng/g) for a group of inbred lines derived from backcrosses evaluated with the kernel screening assay.

Line

Pedigree

Aflatoxin

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