Field Crops Research 102 (2007) 6063 www.elsevier.

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Diallel analysis of aatoxin accumulation in maize

Hilarie D. Gardner, W. Paul Williams *, Gary L. Windham
USDA-ARS Corn Host Plant Resistance Research Unit, Box 9555, Mississippi State, MS 39762, United States Received 2 February 2007; received in revised form 2 February 2007; accepted 17 February 2007

Abstract Since its discovery in numerous feedstuffs, aatoxin, a carcinogenic compound produced by the fungus Aspergillus avus Link ex Fries, has caused much concern among consumers and producers alike. This toxin poses a serious economic threat to maize (Zea mays L.) producers of the southeastern and midwestern regions of the United States. Efforts to identify maize germplasm that is resistant to aatoxin accumulation and to investigate the genetic basis of this resistance have been undertaken at numerous research institutions. The objectives of this study were to (1) evaluate aatoxin accumulation in grain harvested from maize inbred lines and a diallel cross among these lines, (2) determine the importance of general and specic combining abilities in inheritance of resistance to aatoxin accumulation, and (3) estimate general and specic combining ability effects associated with resistance to aatoxin accumulation in the inbred lines and crosses among them. Eight inbred lines and a diallel cross of the maize lines were inoculated with an A. avus spore suspension 1214 d after silk emergence. Following harvest, aatoxin content was determined from samples of grain. Statistical analyses performed using SAS general linear models (GLM) and DIALLEL-SAS indicated that general and specic combining ability were signicant sources of variation in the inheritance of resistance to aatoxin accumulation. The inbred line Mp313E, which was developed and released as a source of resistance to aatoxin contamination, had signicantly lower aatoxin accumulation than other lines. Mo18W exhibited excellent general combining ability for reduced aatoxin accumulation when crossed with the other lines. Both Mp313E and Mo18W could be useful in breeding programs to develop aatoxin-resistant maize hybrids. Mp339, SC212M, and Ab24E demonstrated aatoxin susceptibility as both inbreds and in single crosses. # 2007 Elsevier B.V. All rights reserved.
Keywords: Aatoxin; Diallel; Maize

1. Introduction Aatoxin, produced by the fungus Aspergillus avus Link ex Fries has been implicated in numerous human and animal diseases (Bennett and Klich, 2003; Gourama and Bullerman, 1995). Because inadvertent inhalation or ingestion of this toxin can be hazardous to health, contamination of maize (Zea mays L.) grain with aatoxin has lately become one of the primary concerns of maize producers in the southeastern and midwestern United States. Because aatoxin is a known carcinogen, stringent regulations have been put into effect by the U.S. Food and Drug Administration limiting the amount of aatoxin allowable in grain intended for human and animal consumption. Currently, grain having a concentration of
This paper is a joint contribution of USDA-ARS and the Mississippi Agricultural and Forestry Experiment Station and is published as journal no. J-10812 of the Miss. Agric. For. Exp. Stn. * Corresponding author. Tel.: +1 662 325 2735; fax: +1 662 325 8441. E-mail address: wpwilliams@msa-msstate.ars.usda.gov (W.P. Williams).

aatoxin B1 exceeding the threshold of 20 ng g1 is banned from interstate trade (NGFA, 1992). These restrictions have caused substantial economic losses among maize growers. Prompted by the growing concern for public safety and the nancial loss to producers, maize researchers have conducted careful studies of A. avus and the biosynthetic pathway of aatoxin. These efforts have provided the fundamental knowledge currently used to implement cultural control practices intended to minimize aatoxin contamination in pre- and post-harvest grain. Despite these added controls, higher temperatures, drought stress, insect damage, and other environmental conditions conducive to A. avus infection of maize and subsequent aatoxin production continue to promote contamination of maize grain. To reduce the incidence of aatoxin contamination of maize grain, plant breeders and geneticists are currently searching for aatoxin-resistant maize germplasm to expedite the incorporation of resistance traits into commercially grown hybrid maize. The objectives of this investigation were (1) to evaluate aatoxin accumulation in grain harvested from eight inbred

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lines and a diallel cross among these lines, (2) to determine the relative importance of general and specic combining ability in inheritance of resistance to aatoxin accumulation, and (3) to estimate general combining ability (GCA) effects associated with each line and specic combining ability (SCA) effects associated with each hybrid combination. The information gained from these analyses will allow for selection of inbred lines that demonstrate potential as sources of aatoxin-resistant germplasm for inclusion in a breeding program. 2. Materials and methods Eight maize inbred lines were planted at the R.R. Foil Plant Science Farm, Mississippi State, Mississippi on 17 April 2003 and 20 April 2004 for evaluation of aatoxin accumulation in the grain. Two of the lines, Mp313E and Mp420, were developed and released as sources of resistance to aatoxin contamination (Scott and Zummo, 1990, 1992). The other lines, Ab24E, Mo18W, Mp339, SC229, SC212M, and Tx601, had exhibited differing levels of susceptibility to aatoxin contamination in eld tests (Windham and Williams, 2002; Williams et al., 2003). A diallel cross of 28 F 1 hybrids produced from crossing the eight inbred lines was planted in an adjacent eld on 21 April 2003 and 29 April 2004. Because of the differences in growth, the parental inbred lines and F1 hybrids were planted in separate experiments. Inbred parents or F1 hybrids were assigned to single-row plots that were 5.1 m long, spaced 0.96 m apart, and thinned to 15 plants. Within each experiment, plots were arranged in a randomized complete block design with four replications. Standard production practices were followed and supplemental irrigation was used during the growing season to reduce drought stress and to improve seed production. The primary ear of each plant was articially inoculated with A. avus spores 1214 d after silk emergence using the side-needle technique described by Zummo and Scott (1989). A. avus isolate NRRL 3357 was used as inoculum. At maturity, ears were hand-harvested and shelled, and the grain was ground using a Romer mill (Union, MO). The concentration of aatoxin was determined using Vicams AaTest1 (Watertown, MA), which can detect aatoxin levels as low as 1 ng g1. Data were analyzed using SAS (SAS Institute, Cary, NC). All data were transformed as ln(y + 1), where y is equal to the concentration in ng g1 of aatoxin in a sample. This transformation was performed to provide a more normally distributed set of data for statistical analysis. The data on aatoxin contamination of the parental inbreds was analyzed using the SAS general linear models (GLM) procedure. The general linear model for an experimental plot of an inbred line was as follows: xi jk u vi b j bvi j ei jk ; where, xijk is the observed aatoxin level for the ith parent, jth replication, and kth environment, u is population mean, vi the effect of the ith parental line, bj the jth block effect, (bv)ij the

interaction between the ith genotype and the jth block, and eijk is the residual effect of the ijkth individual. It was assumed that the eijk were normally and independently distributed with mean = 0 and variance = s 2 . e Analysis of aatoxin accumulation in the seed harvested from the F1 hybrids was conducted using the DIALLEL-SAS procedure developed by Zhang and Kang (1997, 2003), according to Grifngs (1956) Method 4, Model 1, which includes the F1 progeny, but neither the parents nor the reciprocals. The DIALLEL-SAS program evaluated the data so as to account for environmental effects, as well as effects due to genotype, block, and the interactions between various effects. Means were compared using a least signicant difference (LSD) at P = 0.05 level of signicance. The general linear model for the open-pollinated diallel plots of F1 hybrids was as follows: xi jkl u vi j bk bvi jk ei jkl ; where, xijkl is the observed aatoxin level for the ith and jth parents, kth replication, and lth environment, u the population mean and vi j is the genotype effect, such as is equal to gi + gj + sij, where gi is the general combining ability (GCA) of the ith parent, gj the GCA of the jth parent, and sij the specic combining ability (SCA) of the ijth hybrid, bk the kth block effect, (bv)ijk the interaction between the ijth genotype within the kth block, and eijkl is the residual effect of the ijklth individual. It was assumed that the eijkl were normally and independently distributed with mean = 0 and variance = s 2 . e The signicance of the GCA of the inbred lines and SCA of the hybrid lines was determined by a t test, and the GCA and SCA values were estimated.

3. Results Analyses of the experimental data obtained from the inbred lines indicated that aatoxin concentration in the grain ranged from 6 to 1912 ng g1 in 2003, 83 to 2063 ng g1 in 2004, and 23 to 1986 ng g1 over both years (Table 1). The resistant line Mp313E had signicantly (P < 0.05) lower aatoxin accumulation (23 ng g1) than the other inbred lines, and inbred lines Tx601 and Mo18W exhibited the next lowest levels of aatoxin accumulation. On the other hand, inbred lines SC212M, Mp339, and Ab24E had signicantly (P < 0.05) higher concentrations of aatoxin in the grain, 1986, 1282, and 944 ng g1. Aatoxin accumulation in grain produced by the single cross hybrids was substantially lower than that for the inbred parents. Mean aatoxin levels ranged from a high of 364 ng g1 for SC229 Ab24E to a low of 34 ng g1 for Mp313E Mo18W (Table 2). Analysis of variance of aatoxin accumulation in the grain from single-cross hybrids revealed signicant (P < 0.01) differences among hybrids (Table 3). The results of this analysis also indicate a signicant (P < 0.05) interaction between years and hybrids. Both GCA and SCA were signicant (P < 0.01) sources of variation. The GCA year interaction was not

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H.D. Gardner et al. / Field Crops Research 102 (2007) 6063 Table 3 DIALLEL-SAS analysis of variance of aatoxin contamination of grain harvested from single crosses grown in a diallel in 2003 and 2004 at Mississippi State, MS Sourcea Years Replications (years) Hybrid GCA SCA Years hybrids GCA year SCA year Error
a

Table 1 Aatoxin contamination of inbred maize lines grown in 2003 and 2004 at Mississippi State, MS, and estimates of GCA effects associated with each line from diallel analysis Inbred line SC212M Mp339 Ab24E Mp420 SC229 Mo18W Tx601 Mp313E LSD(0.05)
a b * **

Logarithmic meana [ln(y + 1)] 7.59 7.15 6.85 5.92 5.86 5.34 4.61 3.18 1.09

Geometric meanb (ng g1) 1986 1282 944 391 350 207 99 23

GCA effect [ln(y + 1)] 0.34** 0.39** 0.39** 0.01 0.03 0.68** 0.29* 0.17

d.f. 1 6 27 7 20 27 7 20 161

MS 22.90** 0.28 3.07** 6.86** 1.75** 1.46* 0.88 1.65* 0.87

Values were transformed [ln(y + 1)] before analysis. Geometric mean, antilogarithm of logarithmic mean. Signicantly different from 0 at P < 0.05 level of probability. Signicantly different from 0 at P < 0.01 level of probability.

Test contained 224 observations, including one missing value. Means were transformed [ln(y + 1)] before analysis. * Signicant at P < 0.05 level of probability. ** Signicant at P < 0.01 level of probability.

signicant, but the SCA year interaction was signicant (P < 0.05). Estimates of GCA effects are provided in Table 1. Of the eight parental inbred lines, Mo18W had the greatest effect on
Table 2 Aatoxin contamination in a diallel cross among maize inbred lines grown in 2003 and 2004 at Mississippi State, MS, and estimates of SCA effects associated with each line Hybrid SC229 Ab24E Mp420 Mp339 SC212M Ab24E Mp339 SC212M Tx601 SC212M Mp313E Mp339 Mp339 SC229 Mp313E Mp420 Mp313E SC212M Mp420 Ab24E Tx601 Ab24E Mp420 SC212M Mp313E Ab24E Mp420 SC229 Mp339 Ab24E Mp313E SC229 Tx601 Mp339 Tx601 Mo18W Mp339 Mo18W SC229 Mo18W SC212M Mo18W SC229 SC212M Ab24E Mo18W Tx601 SC229 Mp420 Tx601 Mp313E Tx601 Mp420 Mo18W Mp313E Mo18W LSD(0.05)
a b * **

Logarithmic meana [ln(y + 1)] 5.90 5.72 5.70 5.64 5.46 5.22 5.18 5.10 5.06 5.04 5.03 5.03 5.01 4.98 4.94 4.71 4.69 4.64 4.49 4.40 4.36 4.34 4.28 4.19 3.93 3.86 3.71 3.56 0.92

Geometric meanb (ng g1) 364 305 298 281 235 185 177 163 157 153 152 151 149 145 139 110 109 103 88 80 77 75 71 65 50 47 40 34

SCA effect [ln(y + 1)] 0.82** 0.17 0.26 0.69* 0.03 0.21 0.82** 0.48 0.10 0.20 0.12 0.14 0.00 0.10 0.17 0.06 0.60* 0.63 0.38 0.01 0.09 0.39 0.22 0.14 0.55 0.47 0.34 0.57*

aatoxin accumulation in the grain of the hybrids, producing a signicant (P < 0.01) decrease in the amount of aatoxin detected. Parental line Tx601 contributed signicantly (P < 0.05) to reduced aatoxin accumulation. Although Mp313E exhibited the lowest level of aatoxin contamination as an inbred line, the GCA effect of this line was not signicant. Estimates of GCA effects associated with Mp339, SC212M, and Ab24E, were positive and signicant (P < 0.01). The crosses among these lines were among those exhibiting the highest levels of aatoxin contamination (Table 2). When evaluated as inbred lines (Table 1), these lines exhibited the greatest susceptibility to aatoxin accumulation in the grain. Specic combining ability (SCA) estimates were signicant and negative for only three hybrids: Mp339 SC229, Tx601 Mo18W, and Mp313E Mo18W (Table 2). These hybrids exhibited signicantly lower levels of aatoxin accumulation than would have been expected from their general combining abilities. Estimates of SCA were signicant and positive for only two hybrids: SC229 Ab24E and Tx601 Mp339. These SCA estimates indicated a tendency of these single-crosses to have signicantly higher levels of aatoxin contamination than expected based on estimates of GCA effects for these lines. 4. Discussion Diallel analysis revealed signicant differences in aatoxin contamination due to a GCA (Table 3). Although Mp313E was identied in the inbred evaluation as having the highest degree of resistance to aatoxin contamination (Table 1), its GCA effect was not signicant. When Mp313E was crossed with other lines that exhibited resistance, or at least reduced susceptibility, as inbreds (Tx601 and Mo18W), the hybrids exhibited excellent resistance (Table 2). When crossed with the more susceptible inbred lines such as Mp339 and SC212M, however, the resulting hybrids tended to have high levels of aatoxin. Mp313E was the rst maize inbred line developed and released as a source of resistance to A. avus infection and

Values were transformed [ln(y + 1)] before analysis. Geometric mean, antilogarithm of logarithmic mean. Signicantly different from 0 at P < 0.05 level of probability. Signicantly different from 0 at P < 0.01 level of probability.

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aatoxin contamination (Scott and Zummo, 1990). Mp420 was also developed and released as a source of resistance (Scott and Zummo, 1992). In this investigation, Mp420 ranked fourth among the eight inbreds in aatoxin contamination and its GCA effect was not signicantly different from 0 (Table 1). On the other hand, the GCA estimate for Mo18W revealed a tendency of this line to demonstrate greater aatoxin resistance in hybrids than it exhibited as an inbred line per se (Table 1). Of the 12 hybrids with the lowest levels of aatoxin, Mo18W was a parent of seven (Table 2). Because Mo18W generally combined well with other lines included in these experiments to yield grain with relatively low levels of aatoxin, it might be useful as a source of additional genes for aatoxin resistance that have yet to be identied. Whereas Mo18W and Mp313E demonstrated aatoxin resistance as either an inbred line or in hybrids, Mp339, SC212M, and Ab24E appeared to be consistently associated with aatoxin susceptibility, both as inbreds and in hybrids. It should be noted, however, that the GCA estimates presented in this paper are specic to this group of inbreds and introduction of other lines or exclusion of some of these lines could produce markedly different results. 5. Conclusion Mp313E demonstrated signicant aatoxin resistance when evaluated as an inbred line and was more reliable than other inbred lines for the years that this experiment was conducted. Mp339, SC212M, and Ab24E had consistently higher concentrations of aatoxin and, therefore, appeared to be reliable lines for use as susceptible checks. Analysis of the diallel cross indicated that GCA, SCA, and SCA year interactions were signicant sources of variation in the inheritance of resistance to aatoxin accumulation. The negative GCA effect estimated for Mo18W in the diallel experiment suggested that hybrids containing this line would, on average, have lower aatoxin concentrations than hybrids of the other inbred lines studied. Mo18W, which performed better in crosses than as an inbred line, combined particularly well with resistant lines Mp313E and Mp420. Because of its

resistant nature when used in hybrids, Mo18W may be a good candidate for QTL mapping and in identifying new genes associated with aatoxin resistance in maize. Acknowledgements The authors express their appreciation to Clarence E. Watson and Jixiang Wu who provided advice and statistical support for the experiments and to Ladonna Owens for her technical assistance. Mention of trade names or commercial products in this publication is solely for the purpose of providing specic information and does not imply recommendation or endorsement by the USDA. References
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