Many species in the genus Fusarium have the capacity to

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1 Published September, 01 RESEARCH Diallel Analysis of Resistance to Fusarium Ear Rot and Fumonisin Contamination in Maize Hsiao-Yi Hung and James B. Holland* ABSTRACT The fungus Fusarium verticillioides infects maize (Zea mays L.) ears and kernels, resulting in Fusarium ear rot disease, reduced grain yields, and contamination of grain with the mycotoxin fumonisin. Hybrid maize breeding programs involve selection for both inbred and hybrid performance; the emphasis placed on inbred versus hybrid selection depends on heritability of and the genetic correlation between resistance in the different generations. The objectives of this study were to assess the importance of general combing ability (GCA) and specific combining ability (SCA) for resistance to Fusarium ear rot and fumonisin contamination and to estimate the correlation between resistance measured in inbred and hybrid generations. We evaluated a diallel mating of 18 inbred lines from different heterotic groups with different levels of resistance. Hybrids had 7% less ear rot and 30% less fumonisin content than their inbred parents, demonstrating the importance of hybrid vigor to disease resistance. Both GCA and SCA were significant for yield and disease resistance, and inbred performance per se and corresponding GCA in hybrids were significantly correlated (r 0.78). Genetic variation for resistance was greater in inbred lines than among hybrids, however. These results suggest that the most efficient way to improve Fusarium ear rot and fumonisin contamination resistances in hybrids is to evaluate and select among inbred lines before using resources to create and evaluate hybrids. H.-Y. Hung and J.B. Holland, Dep. of Crop Science, North Carolina State Univ. Campus Box 760, Raleigh, NC 7695; J.B. Holland, U.S. Department of Agriculture Agricultural Research Service (USDA- ARS). Received 6 Mar. 01. *Corresponding author (james_holland@ ncsu.edu). Abbreviations: GCA, general combining ability; LNFUM, natural log transformation of fumonisin content; LNROT, natural log transformation of Fusarium ear rot percentage; SCA, specific combining ability. Many species in the genus Fusarium have the capacity to cause ear rots in maize and also contaminate grain with mycotoxins, which could damage the health of animals or humans (White, 1999). Ear rots in maize are most often the result of colonization by Fusarium verticillioides (Sacc.) Nirenberg (synonym F. moniliforme Sheldon) (teleomorph: Gibberella moniliformis) and Fusarium graminearum Schwabe [teleomorph Gibberella zeae (Schw.) Petch]. Fusarium verticillioides causes Fusarium ear rot while the disease caused by F. graminearum is called Gibberella ear rot (Bush et al., 004). Fusarium ear rot is more prevalent in warm dry conditions and associated with insect damage, so it is more common in the southern United States and lowland tropics (Miller, 1994). Ascospores or conidia of F. verticillioides can land on the silk and enter the cob through the silk channel and thus infect the kernel (Koehler, 194). Alternatively, F. verticillioides can systemically infect maize (Miller, 1994). Infections that begin in vegetative plant organs can spread through the plant and also affect the kernels (Bush et al., 004). Resistance to Fusarium ear rot found in maize is quantitative (King and Scott, 1981; Nankam and Pataky, 1996; Clements et al., 004), and no complete resistance has been discovered. Robertson Published in Crop Sci. 5: (01). doi: /cropsci Crop Science Society of America 5585 Guilford Rd., Madison, WI USA All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher. CROP SCIENCE, VOL. 5, SEPTEMBER OCTOBER

2 et al. (006) estimated an entry mean-basis heritability of at least 0.47 for ear rot and 0.75 for fumonisin content in two maize populations and estimated the genotypic correlations between ear rot and fumonisin concentration to be at least The high genetic correlations indicate that selection against ear rot should result in reduced susceptibility to fumonisin contamination. Direct selection against fumonisin content should be more efficient than indirect selection against ear rot for reducing susceptibility to fumonisin contamination, but fumonisin assays are much more time and money consuming than ear rot measurements, which can be done by visually rating. Therefore, selection against ear rot should frequently identify low fumonisin lines efficiently and is in most cases the economically more efficient selection criterion. To date, almost all research on the inheritance of resistance to Fusarium ear rot and fumonisin contamination (heritability and correlation estimates, response to selection, and quantitative trait locus mapping) has used partly or highly inbred lines (Robertson et al., 006; Robertson- Hoyt et al., 006, 007; Afolabi et al., 007; Eller et al., 008, 010). The usefulness of inbred line per se resistance data for predicting topcross hybrid resistance remains unclear. Eller et al. (008) compared the 10 lines with highest and 10 lines with lowest grain fumonisin contents from the BC 1 F 1: families of a cross between FR1064, a popular commercial line highly susceptible to Fusarium ear rot, to GE440, an inbred with a relatively high level of Fusarium ear rot resistance. The two groups of topcrosses did not differ significantly for ear rot resistance. Possible reasons for this result included (i) the topcross evaluation environments were not conducive to Fusarium infection, (ii) the tester, the F 1 of two related lines FR615 FR697, might contribute resistance, masking the variation among the lines being tested, (iii) hybrid vigor alone could provide a high level of resistance, and (iv) resistance in inbreds is not related to resistance in topcrosses. In a subsequent study, Eller et al. (010) evaluated the topcrosses of BC 4 derived lines from the same population and found that a selected set of 19 BC 4 F 1:3 lines had greater resistance to ear rot and fumonisin content than their recurrent parent FR1064. These results suggested that, when sufficient variation for Fusarium ear rot is expressed in topcrosses, inbred and topcross resistance to Fusarium ear rot are correlated. The confl icting results from these two studies suggested that a more detailed investigation of the correlation between inbred and hybrid resistance to Fusarium ear rot was warranted. One objective of this study was to compare resistance of a diverse sample of inbred lines inbred lines and their F 1 hybrids to understand the correlation between inbred and hybrid resistances to Fusarium ear rot. Second, the diallel analysis of crosses evaluated in this study permits testing the relative importance of general combining ability (GCA) and specific combining ability (SCA) for Fusarium ear rot resistance. Third, since the diallel involved crosses between pairs of related lines as well as unrelated lines, the resulting substantial variation for hybrid vigor among the crosses permits a test of the relationship between hybrid vigor and disease resistance. Finally, this experiment can help identify lines with good resistance for use as breeding parents and also to identify lines with less resistance, which may serve as more appropriate testers for future evaluations of topcrosses to develop elite inbred lines for Fusarium resistance. MATERIAL AND METHODS Population Development and Phenotype Evaluation The 18 parental maize inbred lines evaluated were chosen to represent a range of inbred per se resistance to Fusarium ear rot (based on previous screening experiments, Eller et al., 008, or based on nursery observations, M.M. Goodman, personal communication, 008) from among the Stiff Stalk, non-stiff Stalk, Iodent, tropical, and mixed heterotic groups (Table 1; Nelson et al., 008). Here, the two lines making up the mixed group were 50% tropical and 50% Stiff Stalk derived from the USDA Germplasm Enhancement of Maize program (Pollak, 003). The 18 inbred lines were crossed in all possible (153) combinations in 008 summer and 008/009 winter nurseries. Reciprocal F 1 seeds were bulked for testing, except NC444 was used only as a male parent because it carries a gametophyte factor for cross-sterility (Nelson, 1994). Inbred parents were evaluated using a randomized complete block design with three replications at each of four North Carolina locations (Clayton, Lewiston, Jackson Springs, and Kinston) in summer 009. Hybrids (153 experimental F 1 s plus three commercial check cultivars, Dekalb brand DK697 and Pioneer brand 31G66 and 31G98) were evaluated in separate disease and yield evaluation trials planted at the same four environments in two adjacent blocks at each location, using 1 13 lattice designs with two complete replications at each location. Inbred and hybrid disease trials were composed of single-row plots whereas the hybrid yield trials were composed of two-row plots. In all four locations, plots were approximately 3.66 m in length with a 1. m alley at the end of each plot. Interrow spacing was 0.91 m in Lewiston and 0.97 m for the other three. Plots were overplanted and thinned to approximately 40 plants per two-row plot for yield trials. Inbred and hybrid disease trials plots were planted at a rate of 7 kernels per one-row plot without thinning. Disease evaluation plots were artificially inoculated following as described by Robertson et al. (006) and Eller et al. (010). Six North Carolina isolates of F. verticillioides (i6, i7, i9, n16, n17, and n) identified as high-fumonisin producing in culture by Eller (009) were cultured separately on potato dextrose agar (Fisher Scientific). We used a mixture of isolates to represent the potential genetic variation within the local F. verticillioides population. Conidia were collected by washing the cultures with distilled water, loosening conidia by brushing with a paint brush, straining the suspension through cheese cloth, and diluting the conidia suspension of the six isolates to approximately 10 6 ml 1 in water. Two inoculations were conducted 7 d apart to reduce escapes and simulate common CROP SCIENCE, VOL. 5, SEPTEMBER OCTOBER 01

3 Table 1. Maize inbred lines used to form a diallel for analysis of Fusarium ear rot resistance, their heterotic groups, and their origin. Lines Heterotic groups Origin Pedigree B73 Stiff Stalk Iowa State Iowa Stiff Stalk Synthetic FAPW Stiff Stalk Dekalb ex-pvp B14AHt B37Ht FR1064 Stiff Stalk IFSI B73 type PHB47 Stiff Stalk Pioneer ex-pvp (B37*3) SD105 FR615 Non-Stiff Stalk IFSI C103 type FR697 Non-Stiff Stalk IFSI C103 type GE440 Non-Stiff Stalk Georgia Hasting s Prolifi c MBNA Non-Stiff Stalk Dekalb ex-pvp Mo17Ht MDA-8 PHT77 Non-Stiff Stalk Pioneer ex-pvp PH814 PH995 PHZ51 Non-Stiff Stalk Pioneer ex-pvp PH814 PH848 UR13085:N Non-Stiff Stalk Ames (Proprietary non-stiff Stalk*) Cateto Sulino (Uruguay) UR13085:N Non-Stiff Stalk Ames (Proprietary non-stiff Stalk*) Cateto Sulino (Uruguay) PHG9 Iodent Pioneer ex-pvp (PH07*) PH806) GEMS-000 Mixed Ohio State Florida Synthetic proprietary SS GEMS-0003 Mixed North Carolina State BR5051 (Dente Amarelo, Brazil) proprietary SS NC300 Tropical North Carolina State (PH X306B H5) PH X105A NC300/CML88-B-4 Tropical Texas A&M NC300 CML88 NC444 Tropical North Carolina State NC58 NC96* ex-pvp, expired Plant Variety Protection. IFSI, Illinois Foundation Seeds Inc. SS, Stiff Stalk. methods of natural infection. A silk channel inoculation 10 to 14 d post-midsilk was followed by a direct ear inoculation 7 d later. The primary ear of the first 1 to 15 plants in each row was injected with 5 ml of 10 6 conidial suspension using a 5-mL Allflex draw-off injection syringe (Allflex Inc.) fitted with a 16-gauge needle with a filed-down point. One drop of undiluted Tween-0 was added to each liter of inoculum suspension to break the surface tension of the suspension. The first 10 ears from each plot were harvested, rated for Fusarium ear rot by estimating the percentage of the infected kernels, and shelled. A sample of bulked grain from each plot was ground on a Romer II Series Mill (Romer Labs). A 0-g sample of ground grain from each plot was analyzed for fumonisin content using a quantitative enzyme-linked immunosorbent assay kit (EZ- Quant Fumonisin B1,, 3 from Diagnostix) (Eller et al., 008). For yield trials, stand counts were taken following thinning to target planting densities early in the season. Plots were mechanically harvested to measure grain yield and grain moisture for each plot. Statistical Analysis Each experiment (inbred line evaluation, hybrid disease evaluation, and hybrid yield trial) was analyzed separately. An initial analysis of Fusarium ear rot and fumonisin revealed that absolute residual values were correlated with predicted values; natural-log transformations were used to remove the dependency of residual values on predicted values (Rawlings et al., 1998). Yield was adjusted to 155 g kg 1 moisture. The linear model for the combined analysis of hybrids across environments for all three primary traits of interest (grain yield and natural log transformations of Fusarium ear rot percentage [LNROT] and fumonisin content [LNFUM]) were Y ijkl = μ + genotype i + loc j + (genotype loc ij ) + rep(loc) jk + block(rep loc) jkl + ε ijkl, in which μ is the overall mean, genotype i is the effect of the ith genotype, loc j is the effect of the jth location, genotype loc ij is the interaction effect of the ith genotype by the jth location, rep(loc) jk is the effect of the kth replication within the jth location, block(rep loc) jkl is the effect of the lth incomplete block within the kth replication within the jth location, and ε ijkl is the residual variance. The univariate and bivariate models were modified for inbred data by dropping the incomplete block term. Univariate analyses were conducted with Proc Mixed of SAS version 9. (SAS Institute, 009). A model with all terms considered random was also fit for the purpose of estimating entry mean heritabilities for each trait. Genotypic correlations between Fusarium ear rot and fumonisin were estimated from bivariate analyses of the combined data across environments using ASReml version 3 (Gilmour et al., 009; Holland, 006). In addition to the combined analysis across environments described above, least square means for hybrids at each individual location were estimated by separate analyses of each environment using SAS Proc Mixed (SAS Institute, 009). The environment-specific hybrid least square means (excluding check hybrids) were then analyzed with a model modified from Hallauer and Miranda (1988), following Griffi ng s (1956) analysis method 4 for half-diallel designs, Y lfm = μ + loc l + GCA f + GCA m + SCA fm + ε ifm, in which μ is the overall mean, loc l is the effect of the lth location, GCA f is the general combining ability for the fth female parent, GCA m is the general combining ability for the mth male parent, SCA fm is the specific combining ability of the cross between the fth female and the mth male, and ε lfm is the residual effect, which includes GCA and SCA environment interactions. The diallel analysis was done using ASReml version 3 (Gilmour et al., 009) to obtain F-tests for significance of GCA and SCA effects; GCA and SCA effect estimates were checked by spreadsheet computations. CROP SCIENCE, VOL. 5, SEPTEMBER OCTOBER

4 Table. Least square means and specific combining ability (SCA) effects of five highest and lowest ranking experimental hybrids and three check hybrids for grain yield and natural log transformation of (and back transformed to original scales) Fusarium ear rot percentage (LNROT) and fumonisin content (LNFUM). Best and worst hybrids for LNROT LNROT Fusarium ear rot Best performing hybrids ln(%) % GEMS0003 NC GEMS0003 GE GE440 GEMS NC444 UR13085:N GEMS000 NC Worst performing hybrids FAPW FR FR615 MBNA FR1064 FR B73 FR B73 FR Checks DK Pioneer 31G Pioneer 31G LSD (0.05) Best and worst hybrids for LNFUM LNFUM Fumonisin content Best performing hybrids ln(μg g 1 ) μg g 1 GEMS000 NC GE440 NC300/CML88-B GEMS0003 NC GEMS0003 GE GEMS000 NC Worst performing hybrids B73 FR B73 MBNA FR1064 FR FR697 MBNA B73 FR Checks DK Pioneer 31G Pioneer 31G LSD (0.05) Best and worst hybrids for grain yield Grain yield Best performing hybrids Mg ha 1 FR615 NC GEMS0003 NC B73 NC300/CML88-B NC300/CML88-B-4 PHZ FR1064 FR Worst performing hybrids FR615 FR NC300 NC300/CML88-B UR13085:N UR13085:N FR697 MBNA B73 FR Checks DK Pioneer 31G Pioneer 31G LSD (0.05) The average GCA and SCA for each heterotic group and the average SCA of crosses among three heterotic groups (including Iodent line PHG9 in the non-stiff Stalk category) were estimated to compare the differences among groups. The importance of GCA and SCA was assessed using the k / k + k, modified from Baker (1978), in equation GCA ( GCA SCA) which k GCA is the quadratic form (analogous to a variance component but referring to a fixed effect) derived from the mean square of GCA effect and k SCA is the quadratic form of SCA effects since the total genetic variation among single-cross progeny is equal to twice the GCA component plus the SCA component. The closer this ratio is to unity, the greater the predictability of a specific hybrid s performance based on GCA alone. Heterosis of Fusarium ear rot and fumonisin contamination were calculated using the equation heterosis (H) = ( ) F1 P1+ P /, in which F 1 is the average of F 1 progeny performance and P 1 and P are the average performance of parents of that specific cross. Correlations of GCA, SCA, heterosis, and least square mean values between the two disease traits and yield were calculated using Proc Corr in SAS (SAS Institute, 009). Analysis of covariance was used to compare the combined analysis for disease traits in hybrids with and without yield as covariate in ASReml version 3 (Gilmour et al., 009). RESULTS Genotype main effects and genotype environment interaction were significant (p < 0.001) for Fusarium ear rot and fumonisin contamination in the inbred and hybrid experiments (Supplemental Tables S1 and S). For grain yield, genotype main effects but not genotype environment interaction were significant (Supplemental Table S1). The pairwise correlations of hybrid entry means at each location were all significant (p < ) and of moderate to large magnitude (0.40 < r < 0.94; Supplemental Table S3). We checked the individual location performance of the five hybrids with lowest mean ear rot or fumonisin contamination across locations (Table ); the overall best hybrids had lower disease values than the experiment mean at each location and almost always were more than one standard deviation better than the location mean (Supplemental Fig. S1 and S). Entry mean heritabilities were moderate to high across traits and experiments (0.44 to 0.81; Supplemental Tables S1 and S). We focus on analysis of genotypic main effects because they were highly significant for disease traits in the inbred and hybrid experiments and for yield in the hybrid experiment and because the best performing hybrids exhibited good environmental stability. The correlation between entry means for Fusarium ear rot and fumonisin content was 0.87 in the hybrid trials and 0.93 for inbred trials. This result validates previous reports of high genetic correlations between these two aspects of disease resistance by Robertson et al. (006). A high genetic correlation between ear rot and fumonisin content suggests that indirect selection for reduced ear rot should be economically efficient for reducing fumonisin contamination susceptibility because scoring ear rot is much CROP SCIENCE, VOL. 5, SEPTEMBER OCTOBER 01

5 Table 3. Snedecor F statistic and associated p-values of general and specific combining ability (GCA and SCA) for natural log transformation of Fusarium ear rot percentage (LNROT), fumonisin content (LNFUM), and untransformed grain yield. The ability to predict hybrid performance based on parental GCA values is measured by ratio of k GCA to (k GCA + k SCA ). LNROT LNFUM Yield F value p-value F value p-value F value p-value GCA 5.7 < < <0.001 SCA 1.8 < <0.001 k / k + k ( ) GCA GCA SCA faster and less expensive than laboratory assays of fumonisin content (Robertson et al., 006; Eller et al., 008). Based on the diallel analysis of hybrids, both GCA and SCA were significant (p < 0.05) for Fusarium ear rot, fumonisin content, and grain yield (Table ). Baker (1978) noted that the proportion of variation among single cross hybrids of inbred parents is the sum of SCA variance and twice the GCA variance and suggested that the proportion of this total that is due to GCA variation, kgca /( kgca + ksca ), indicates the predictability of hybrid performance based on GCA alone. If the ratio is close to one, this suggests that SCA is not important and that hybrid performance can be accurately predicted based on the average of parental GCA values. We observed moderate (0.43 to 0.50; Table 3) ratios for Fusarium ear rot and fumonisin content, indicating that GCA is a useful guide to hybrid performance in general, so that evaluations based on a single representative tester should be sufficient for initial hybrid selections. Specific combining ability is important enough, however, that the initial screenings to identify better inbred parents should be followed by evaluations with multiple testers, as there is an important component of hybrid resistance that cannot be predicted from data on a different tester. In contrast, the predictive ratio for grain yield was close to zero (0.05; Table 3), indicating that SCA is very important for grain yield and that testing inbred parents in combination with multiple testers will be required to identify superior hybrids (Hallauer and Miranda, 1988). The inclusion of hybrids from within as well as among heterotic groups virtually guarantees that SCA will be important for yield; the importance of SCA should decrease if one considers only hybrids from crosses between a pair of previously defined heterotic groups. Based on GCA effects, the best inbred parents for producing hybrids with resistance to Fusarium ear rot and fumonisin content were GE440, GEMS-000, and NC444 (Table 4) whereas B73 and FR1064 (which are closely related lines) had the highest GCA values (indicating highest susceptibility) for both ear rot and fumonisin content (Table 4). Among heterotic groups, tropical lines had the lowest mean GCA values for ear rot percentage and fumonisin, indicating that tropical lines on average provided the best resistance to hybrids. A similar pattern of greatest resistance found in tropical lines was observed in the inbred per se experiment (Table 4). The correlations between inbred line per se values and their corresponding GCA values were 0.79 (p = ) for ear rot and 0.78 (p = ) for fumonisin content (Table 4). These high correlations indicate that inbred line per se evaluations are sufficiently reliable indicators of hybrid resistance to be useful initial screens in breeding programs. The five hybrids with lowest SCA for ear rot percentage (indicating better than predicted resistance) were GEMS-0003 NC444, FR615 PHG9, FR697 PHG9, NC444 UR13085:N015-01, and NC300 PHZ51. Four of these five hybrids represent crosses between distinct heterotic groups. Hybrids with lowest fumonisin content SCA effects are GEMS000 NC444, GEMS0003 NC444, B73 FR615, GE440 NC300/ CML88-B-4, and MBNA PHG9, all of which were crosses between heterotic groups. The highest yielding hybrids were also from crosses between heterotic groups: FR1064 GE440, FR1064 FR615, FR1064 MBNA, FR615 NC300, and FR697 UR13085:N (Table 4). Average SCA of crosses between heterotic groups show that, as expected, hybrids created from crosses between heterotic groups had positive yield and negative natural-log transformed ear rot SCA values on average whereas crosses within heterotic groups had negative yield and positive natural-log transformed ear rot SCA values (Table 5). Fumonisin content did not follow the same pattern exactly, as crosses between Stiff Stalk and tropical groups had a positive mean SCA (lower than expected hybrid resistance), indicating less hybrid resistance than predicted based on GCA. Commercial checks Dekalb brand DK697 and Pioneer brand hybrids 31G66 and 31G98 yielded better than most other hybrids in this study, but several experimental hybrids had significantly lower Fusarium ear rot or fumonisin content than all commercial hybrids (Table ). Correlations between GCA effects for grain yield and LNROT or LNFUM were not significant, indicating that additive genetic effects for grain yield and disease resistance are largely independent. Least square means of yield and LNFUM were significantly correlated (r = 0.8, p < 0.01; Fig. 1) in hybrids, however. Also, SCA effects for grain yield and LNROT or LNFUM were significantly correlated (r = 0.38, p < 0.01 in both cases; Fig. ). These results indicate that the nonadditive genetic components of CROP SCIENCE, VOL. 5, SEPTEMBER OCTOBER

6 Table 4. General combing ability (GCA) effect estimates of hybrids from a diallel crossing design and least square means of 18 maize inbred lines per se for grain yield and percent Fusarium ear rot (ROT) and concentration of fumonisin in grain (FUM) measured on their original scales and on a natural log transformed scale (natural log transformation of Fusarium ear rot percentage [LNROT] and natural log transformation of fumonisin content [LNFUM]). GCA Inbred GCA Inbred GCA Lines Heterotic groups LNROT ROT LNROT ROT LNFUM FUM LNFUM FUM Yield ln(%) % ln(%) % ln(μg g 1 ) μg g 1 ln(μg g 1 ) μg g 1 Mg ha 1 B73 SS FAPW SS FR1064 SS PHB47 SS FR615 NSS FR697 NSS GE440 NSS MBNA NSS PHT77 NSS PHZ51 NSS UR13085:N NSS UR13085:N NSS PHG9 Iodent GEMS0003 Mixed GEMS000 Mixed NC300 Tropical NC300/CML88-B-4 Tropical NC444 Tropical LSD (0.05) SE of GCA Average within group SS 0. a a a NSS 0.05 ab ab a Tropical 0.15 b b a Mixed 0.4 c b a Correlation (between GCA and inbred) p-value SS, Stiff Stalk; NSS, non-stiff Stalk. PHG9 (Iodent) included in temperate NSS group. LSD is at 0.05 signifi cance level. Letter code indicates the signifi cant difference between groups based on their LSD. Table 5. Average specific combining ability (SCA) effects for grain yield and percent Fusarium ear rot (ROT) and concentration of fumonisin in grain (FUM) on original measurement scales and for grain yield and natural log transformed scales (natural log transformation of Fusarium ear rot percentage [LNROT] and natural log transformation of fumonisin content [LNFUM]) for hybrids derived from crosses within and between heterotic groups. LNROT ROT LNFUM FUM Yield ln(%) % ln(μg g 1 ) μg g 1 Mg ha 1 NSS NSS NSS SS NSS Tropical SS SS SS Tropical Tropical Tropical SS, Stiff Stalk; NSS, non-stiff Stalk. yield are related to those of disease resistance in a manner favorable to breeding objectives. Average midparent heterosis for Fusarium ear rot was 7% and for fumonisin contamination was 30%. Heterosis for disease resistance can occur because of dominance for resistance as observed by Clements et al. (004) and because of an indirect effect of general hybrid vigor. The relationship between grain yield potential and disease resistance was tested by correlation and covariance analyses. Correlations between heterosis for LNROT, LNFUM, and grain yield were not significant. Analysis of covariance of hybrid disease traits including hybrid yield as a covariate revealed that grain yield was not a significant covariate for either trait, absorbing 4% or less of the mean square due to genotype. These results indicate that hybrid vigor plays a general role in increasing resistance in hybrids and may explain a large amount of the difference in disease between inbreds and hybrids but only accounts for a small proportion of the disease resistance variation among hybrids CROP SCIENCE, VOL. 5, SEPTEMBER OCTOBER 01

7 Figure 1. Scatterplots of general combing ability (GCA) effects or hybrid least square means between grain yield and natural log transformations of Fusarium ear rot percentage or fumonisin content. DISCUSSION Inbred line per se estimates of Fusarium ear rot and fumonisin content had moderate to high correlations with their corresponding hybrid GCA values. This suggests that inbred line evaluations provide breeders a good reference for selecting parents or picking a suitable inbred line tester. The correlations between inbred and hybrid Fusarium ear rot or fumonisin contamination reported here are greater than those reported by Bolduan et al. (010) for Gibberella ear rot and deoxynivalenol contamination in European maize germplasm but similar to those reported by Löffler et al. (011) for Gibberella and Fusarium ear rots as well as deoxynivalenol and fumonisin contaminations. This is helpful, because it permits breeders to reduce population sizes by selection in inbred generations before having to generate topcross seed for hybrid evaluations. The greater susceptibility of inbreds than hybrids to Fusarium ear rot permits greater variation for resistance to be expressed in inbred generations, which could make selection in inbred generations likely more efficient than direct selection in topcrosses. In contrast, however, Löffler et al. (011) reported that indirect selection among inbreds for ear rot resistance in hybrids was predicted to be effective but less so than direct selection among topcrosses. The most efficient use of inbred and topcross disease resistance screening likely depends on practical issues such as seed availability or expense of creating and evaluating topcrosses and also may vary for different germplasm groups. General combing ability made a significant and important contribution to hybrid variation for both Fusarium ear rot and fumonisin contamination. Specific combining ability accounted for half or more of the hybrid variation for these traits, suggesting that multiple testers may be needed to effectively screen for this disease resistance. The diallel mating included crosses both within and between heterotic groups, however, which tends to inflate the importance of SCA, particularly since we observed a general relationship between hybrid vigor and Fusarium ear rot and fumonisin contamination resistance. Therefore, in applied breeding programs where topcrosses are formed only with crosses between previously defined heterotic groups, it is likely that use of a single tester will be most efficient for initial topcross screening. These results indicate that selection for resistance to Fusarium ear rot and fumonisin content can be effectively and easily integrated into current hybrid maize breeding methods, similar to the conclusion of Bolduan et al. (010) for Gibberella ear rot resistance breeding. All the parents of the five most disease-resistant crosses had good inbred per se resistance and also favorable GCA resistance values. This suggests that, although resistance to Fusarium ear rot and fumonisin contamination tends to be at least partly dominant (Clements et al., 004), optimal resistance in hybrids will require crossing inbred lines that both carry resistance, in agreement with Löffler et al. (011). Therefore, selection for resistance should not be confined to a single heterotic group but should CROP SCIENCE, VOL. 5, SEPTEMBER OCTOBER

8 Figure. Scatterplots of specifi c combining ability (SCA) or heterosis estimates for grain yield and natural log transformation of Fusarium ear rot percentage (LNROT) and fumonisin content (LNFUM). Upper and lower diagonal scatterplots show same results, but with x and y axes transposed. Correlation coeffi cients are shown in lower diagonal scatterplots only when signifi cant (p < 0.01). *Signifi cant at the 0.05 probability level. be performed in parallel in all heterotic groups. We did not observe any cross between two susceptible lines that resulted in a hybrid with good resistance. We observed that heterosis is critical for Fusarium resistance. In contrast, Löffler et al. (011) observed that hybrids often had more disease than their parental inbreds, perhaps because they used quite susceptible tester lines and higher inoculum pressure for hybrids than inbreds. Bolduan et al. (010) demonstrated that expression of Gibberella ear rot resistance in hybrids depends on the tester used. In our study, GCA was most important for determining hybrid resistance, but SCA was also important for disease resistance. The existing breeding and selection strategies corn breeders have been using to develop good inbred lines are expected to generate good hybrids by following existing heterotic patterns. Specific combining ability is important for both disease resistance and yield; therefore, evaluation of topcrosses with multiple testers is recommended as a final stage in the breeding process. Supplemental Information Available Supplemental material is available at org/publications/cs. Supplemental Figure S1. Box plots showing distributions of percent ear rot for all entries in the hybrid experiment at each location. Supplemental Figure S. Box plots showing distributions of fumonisin content for all entries in the hybrid experiment at each location. Supplemental Table S1. Results of mixed models analyses of grain yield and natural log transformed Fusarium ear rot and fumonisin contents of F 1 hybrids tested at four North Carolina locations in 009. Supplemental Table S. Results of mixed models analyses of natural log transformed Fusarium ear rot and fumonisin contents of parental inbreds tested at four North Carolina locations in CROP SCIENCE, VOL. 5, SEPTEMBER OCTOBER 01

9 Supplemental Table S3. Correlation coefficients between entry least square means at each pair of locations for Fusarium ear rot or fumonisin contamination. Acknowledgments This research was supported by USDA-ARS, North Carolina State University, and Corn Growers Association of North Carolina. References Afolabi, C.G., P.S. Ojiambo, E.J.A. Ekpo, A. Menkir, and R. Bandyopadhyay Evaluation of maize inbred lines for resistance to Fusarium ear rot and fumonisin accumulation in grain in tropical Africa. Plant Dis. 91: doi: / PDIS Baker, R.J Issues in diallel analysis. Crop Sci. 18: doi:10.135/cropsci x x Bolduan, C., T. Miedaner, H.F. Utz, B.S. Dhillon, and A.E. Melchinger Genetic variation in testcrosses and relationship between line per se and testcross performance for resistance to Gibberella ear rot. Crop Sci. 50: doi:10.135/cropsci Bush, B.J., M.L. Carson, M.A. Cubeta, W.M. Hagler, and G.A. Payne Infection and fumonisin production by Fusarium verticillioides in developing maize kernels. Phytopathology 94: doi: /phyto Clements, M.J., C.M. Maragos, J.K. Pataky, and D.G. White Sources of resistance to fumonisin accumulation in grain and Fusarium ear and kernel rot of corn. Phytopathology 94: doi: /phyto Eller, M.S Improving resistance to Fusarium ear rot and fumonisin contamination and increasing yield with exotic maize germplasm. Ph.D. thesis, North Carolina State University, Raleigh, NC. handle/ /3519 (accessed 5 May 01). Eller, M.S., G.A. Payne, and J.B. Holland Selection for reduced Fusarium ear rot and fumonisin content in advanced backcross maize lines and their topcross hybrids. Crop Sci. 50: doi:10.135/cropsci Eller, M.S., L.A. Robertson-Hoyt, G.A. Payne, and J.B. Holland Grain yield and Fusarium ear rot of maize hybrids developed from lines with varying levels of resistance. Maydica 53: Gilmour, A.R., B.J. Gogel, B.R. Cullis, and R. Thompson ASReml user guide release 3.0. VSN International. (accessed 5 May 01). Griffi ng, B A generalised treatment of the use of diallel crosses in quantitative inheritance. Heredity 10: doi: /hdy Hallauer, A.R., and J.B. Miranda Quantitative genetics in maize breeding. nd ed. Iowa State Univ. Press, Ames, IA. Holland, J.B Estimating genotypic correlations and their standard errors using multivariate restricted maximum likelihood estimation in SAS Proc MIXED. Crop Sci. 46: King, S.B., and G.E. Scott Genotypic differences in maize to kernel infection by Fusarium moniliforme. Phytopathology 71: doi: /phyto Koehler, B Natural mode of entrance of fungi into corn ears and some symptoms that indicate infection. J. Agric. Res. 64: Löffler, M., B. Kessel, M. Ouzunova, and T. Miedaner Covariation between line and testcross performance for reduced mycotoxin concentrations in European maize after silk channel inoculation of two Fusarium species. Theor. Appl. Genet. 1: doi: /s y Miller, J.D Epidemiology of Fusarium ear diseases of cereals. In: J.D. Miller and H.L. Trenholm, editors, Mycotoxins in grain: Compounds other than afl atoxin. Eagan Press, St. Paul, MN. p Nankam, C., and J.K. Pataky Resistance to kernel infection by Fusarium moniliforme in the sweet corn inbred IL15b. Plant Dis. 80: doi: /pd Nelson, O.E The gametophyte factors of maize. In: M. Freeling and V. Walbot, editors, The maize handbook. Springer-Verlag, New York, NY. p Nelson, P.T., N.D. Coles, J.B. Holland, D.M. Bubeck, S. Smith, and M. Goodman Molecular characterization of maize inbreds with expired U.S. plant variety protection. Crop Sci. 48: doi:10.135/cropsci Pollak, L The history and success of the public-private project on germplasm enhancement of maize (GEM). Adv. Agron. 78: doi: /s (0) Rawlings, J.O., S.G. Pantula, and D.A. Dickey Applied regression analysis: A research tool. nd ed. Springer, New York, NY. Robertson, L.A., C.E. Kleinschmidt, D.G. White, G.A. Payne, C.M. Maragos, and J.B. Holland Heritabilities and correlations of Fusarium ear rot resistance and fumonisin contamination resistance in two maize populations. Crop Sci. 46: doi:10.135/cropsci Robertson-Hoyt, L.A., M.P. Jines, P.J. Balint-Kurti, C.E. Kleinschmidt, D.G. White, G.A. Payne, C.M. Maragos, T.L. Molnar, and J.B. Holland QTL mapping for Fusarium ear rot and fumonisin contamination resistance in two maize populations. Crop Sci. 46: doi:10.135/ cropsci Robertson-Hoyt, L.A., C.E. Kleinschmidt, D.G. White, G.A. Payne, C.M. Maragos, and J.B. Holland Relationships of resistance to Fusarium ear rot and fumonisin contamination with agronomic performance of maize. Crop Sci. 47: doi:10.135/cropsci SAS Institute SAS onlinedoc 9.. SAS Institute, Inc. Cary, NC. White, D Fusarium kernel or ear rot. In: D. White, editor, Compendium of corn diseases. American Phytopathological Society, St. Paul, MN. CROP SCIENCE, VOL. 5, SEPTEMBER OCTOBER