Evaluation of maize inbred lines and topcross progeny for resistance to pre-harvest aflatoxin contamination

Jak C.Fountain,Ham K.Aas,Brian T.Sully,Hong Li,Rort D.L,Rort C.Kmrait,Baozhu Guo*

aUSDA-ARS Crop Protection and Management Research Unit,Tifton,GA,USA

bDepartment of Plant Pathology,University of Georgia,Tifton,GA,USA

cUSDA-ARS Biological Control of Pests Research Unit,Stoneville,MS,USA

dUSDA-ARS National Horticultural Research Laboratory,Fort Pierce,FL,USA

eShanxi Academy of Agricultural Sciences,Millet Research Institute,Changzhi,Shanxi,China

fDepartment of Crop and Soil Sciences,University of Georgia,Tifton,GA,USA

Keywords:Aspergillus flavus Pre-harvest Aflatoxin Maize Topcross

ABSTRACT Pre-harvest aflatoxin contamination occurs in maize following kernel colonization by Aspergillus flavus.Aflatoxin contamination resistance is a highly desired trait in maize breeding programs.The identification of novel sources of resistance to pre-harvest aflatoxin contamination is a major focus in germplasm screening efforts.Here,we performed a field evaluation of 64 inbred lines over two years for pre-harvest aflatoxin contamination.Topcrosses were also performed with two testers,B73 and Mo17,to generate 128 F1hybrids which were also evaluated over two years.Hybrid performance was used to calculate both general combining ability(GCA)of the inbreds,and observed heterosis for aflatoxin resistance.Over both years of the study,aflatoxin concentrations ranged from 80± 47 to 17,617± 8816 μg kg-1for inbreds,and from 58 ± 39 to 2771 ± 780 μg kg-1for hybrids with significant variation between years and lines.The inbred linesCML52,CML69,CML247,GT-603,GEMS-0005,Hi63,Hp301,and M37 Wshowed＜1000 μg kg-1 of aflatoxin accumulation in both years of the study and less than the resistant check,Mp313E,in at least one season.Among these,CML52,GT-603, and Hi63 also showed significant GCA with the testers in hybrid progeny.CML52,GT-603,and M37 W also showed heterotic effects of-13.64%,-12.47%,and-24.50%,respectively,with B73 resulting in reduced aflatoxin contamination.GT-603 also showed a similar heterotic effect for aflatoxin contamination,-13.11%,with Mo17 indicating that this line may serve as a versatile source of aflatoxin contamination resistance in breeding programs.

1.Introduction

Aspergillus flavus Link is a facultative pathogen of maize(Zea mays L.)and produces potent mycotoxins collectively known as aflatoxins[1].These mycotoxins are highly carcinogenic and are also acutely toxic in sufficient quantities resulting in increased likelihood of developing hepatitis,cirrhosis,hepatocellular carcinoma,and birth defects[2,3].Due to the hazards associated with acute and chronic aflatoxin exposure,the US Food and Drug Administration(FDA)currently regulates aflatoxin content in maize products for human consumption at 20 μg kg-1[4].It is estimated that aflatoxin contamination of maize results in losses of over$225 million per year in the United States alone[5],but models estimate losses of$1.68 billion in years where conditions are highly conducive for aflatoxin contamination[6].Given the potential losses,both human and economic,the development of commercial varieties with aflatoxin contamination resistance is a major focus of breeding efforts.

Numerous breeding approaches have been applied since the mid-1970s following a severe outbreak of aflatoxin contamination thatreached theMidwestern US.This underlined the importance of pre-harvest aflatoxin contamination resistance,and stimulated research in a new direction considering this was previously considered primarily a postharvest storage issue[7-9].Traditional breeding approaches have focused on the identification of resistant germplasm using field evaluations and laboratory kernel assays in combination [10,11].These efforts have resulted in the identification of several aflatoxin resistant inbred lines including Mp420 and Mp313E[12,13],African and tropical germplasm such as TZAR101-TZAR106 developed by the International Institute of Tropical Agriculture(IITA)in collaboration with other institutions[14],and southern-adapted inbred lines such as GT-601,GT-602,GT-603,Mp715,Mp717,Mp718,and Mp719[15-19].

Recent advances in DNA marker technologies and genotyping capabilities have also allowed for the application of molecular breeding techniques to this issue.Several quantitative trait loci(QTL)for aflatoxin resistance have been identified using both traditional bi-parental mapping populations and using diverse germplasm collections with a genome-wide association study(GWAS)method.For example,Dhakal et al.[20]utilized an F2:3mapping population derived from Mp715 and B73 to identify seven QTL for aflatoxin contamination resistance explaining＜10%phenotypic variance(PVE).Recent genome wide-association study(GWAS)has also been used to identify QTL.For example,Farfan et al.[21]used 346 inbred lines testcrossed to Tx714 for GWAS resulting in six minor QTL with PVE of approximately 5%,and Zhuang et al.[22]used a combination of GWAS using 437diverse lines and a recombinant inbred line(RIL)population with 228 individuals derived from RA×M53 to identify QTL and markers with 6.7%to 26.8%PVE.

These molecular studies also detected significant genotype×environment interactions,which,along with the relatively low PVEs obtained for detected QTL,are major confounding factors for aflatoxin resistance breeding.To account for this and improve cultivar selection,multi-location germplasm selection trials such as the Southeast Regional Aflatoxin Test(SERAT)have been performed to examine germplasm performance across multiple environments[23].Another confounding factor is the poor and/or inconsistent agronomic performance of inbred lines observed due to inbreeding depression.In order to compensate for this,F1topcross hybrids can be used to screen inbreds as potential sources of aflatoxin contamination resistance.This has been employed both in standard testcross designs,and in diallel designs to examine both general and specific combining ability for aflatoxin resistance in addition to other relevant agronomic traits[24-27].

Given the potential for more consistent agronomic performance in hybrids,and the possibility of effects from heterosis and/or hybrid performance on aflatoxin resistance,the objective of this study was to evaluate breeding germplasm available in our breeding program in Tifton,Georgia for aflatoxin contamination resistance in both inbreds and in F1topcross hybrids.Both GCA and heterotic effects on aflatoxin contamination observed in the generated hybrids were also investigated.By identifying novel sources of resistance,these lines can be used in breeding commercial varieties with enhanced aflatoxin resistance.

2.Materials and methods

2.1.Plant materials

Sixty-four maize inbred lines were selected from among diverse germplasm available in our breeding program in Tifton,Georgia,and were cultivated as described by Guo et al.[28].Briefly,these inbred lines were grown at the USDA-ARS Crop Protection and Management Research Unit(CPMRU)Belflower Research Farm,Tifton,GA,in 2014 and 2015.The inbreds were planted in a randomized complete block design with three replicates.Each inbred was planted in two row plots 3.0 m in length with 0.6 m row spacing,1.0 m alleys between plots,and seed spacing of 15.2 cm.Irrigation was applied as needed and recommended management practices were employed for all plots.F1topcross hybrids were generated at the Belflower Farm,USDA-CPMRU,and the University of Georgia Gibbs Farm,Tifton,GA in 2014 and 2015.Hybrids were generated using the 64inbred lines planted in crossing blocks in isolation from other maize plots and were open pollinated following inbred line emasculation prior with a tester line,B73 or Mo17.Each tester was used at a different location to avoid cross-contamination.The testers were also included with the other 64 inbreds for inoculation in 2014 and 2015.The 128 F1hybrids generated(64 inbreds×B73 and 64 inbreds×Mo17)were then grown in 2015 and 2016 at the Belflower Farm as previously described for the inbreds.Two commercial hybrids,Dekalb DKC69-43 and Dupont-Pioneer P-2023HR,were also included in 2016 as controls.

2.2.A.flavus inoculation

The A.flavus isolate NRRL3357was used for all field inoculations.The isolate was cultured on V8 agar(20%V8 juice,1%CaCO3,and 2%agar)at 37°C for 5-7 days.Conidia were then harvested into 0.01%(v/v)Tween 20 buffer and quantified using a hemocytometer.Inoculum concentration was then adjusted to 4.0×106conidia/mL and refrigerated at 4°C until use.Both the inbred and the hybrid plants were inoculated using a side-needle inoculation method as previously described[28,29].Briefly,at 14 days after 50%silk emergence(DAS)within each individual plot,an Idico treemarking gun outfitted with a 14-guage hypodermic needle was used to inoculate maize ears by inserting the needle into the husk and injecting 3.0 mL of inoculum.Only the uppermost ear on each plant was inoculated.In total,five plants per row in each two-row plot were inoculated,and bulk harvested at 45-55 days after inoculation(DAI).In 2014,inbreds were harvested at multiple times corresponding to a range of 45-50 DAI for each plot.In 2015 and 2016,harvest was conducted as a single event relative to the median inoculation date for each year and test.This was done since～80%of plots had inoculation dates within one week of the median corresponding to a single harvesting event in both years.Late-flowering plots with inoculation dates more than one week later than the median were harvested individually at 45-50 DAI.Ears from each bulk harvested row were dried and shelled,all kernels were ground together,and a 50.0 g subsample was taken for aflatoxin analyses.

2.3.Aflatoxin quantification

Aflatoxin quantification was performed as previously described[28].Ground subsamples from both the inbred lines and the hybrids were tested for total aflatoxin content using a Neogen Veratox for Aflatoxin ELISA kit(Neogen,Lansing,MI,USA)according to the manufacturer's instructions.Initially,20.0 g of ground kernel tissue was placed into an 8.0 oz.opaque container with a lid,and aflatoxin was extracted in 100 mL 70%(v/v)methanol with gentle shaking for 3 min.The mixture was then allowed to stand for several minutes before an aliquot of liquid extract was transferred to a 1.5 mL microcentrifuge tube and stored at 4°C until use in ELISA.Spectrometric analysis using a Veratox ELISA reader(Neogen)was then performed to quantify total aflatoxin content.

2.4.Statistical analysis

The normality of the obtained aflatoxin data was assessed based on skewness(Sk)and kurtosis(K)using PROC Univariate using SAS v9.2(SAS Institute,2003).Given that the original data were highly skewed and not normally distributed,the data were log2(y+1)transformed(Fig.S1).These transformed aflatoxin data for both inbred and hybrid samples were used for analysis of variance(ANOVA)based on the model yijk=μ+yeari+linej+(year×line)ij+eijk,where both year and line were considered fixed effects using PROC GLM followed by Tukey's post-hoc analysis for pairwise comparisons within inbreds and hybrids using SAS v9.2(SAS Institute,2003)and R(v.3.3.0).Data presented in the text and tables are means and standard deviations calculated from the original data prior to transformation.General combining ability(GCA)for each inbred was calculated using line×tester analysis using the Analysis of Genetic Designs with R for Windows(AGD-R,v.4.0)software package[30].For all statistical analyses,significance was defined based on α=0.05.Hybrid vigor(heterosis)was calculated as described by Fehr[31]using the equation heterosis(%)=[(F1-MP)/(MP)]×100 where F1is the average F1hybrid performance and MP is the average performance of the parent inbreds.

3.Results and discussion

Over the course of two years of replicated analyses,64 maize in bred lines and128 F1hybrids generated from to pcrosses of the inbreds with testers B73 and Mo17 were evaluated for aflatoxin contamination resistance.All of the field evaluated maize samples contained total aflatoxin in concentrations exceeding the FDA limit of 20.0 μg kg-1[4].For the inbreds,an overall average of 2275 ± 2323 μg kg-1ranging from 201± 186 to 5000± 4876 μg kg-1in 2014 and from 80± 47 to 17,617 ± 8816 μg kg-1in 2015 were measured.Averages of 1258 ± 1196 μg kg-1(coefficient of variation(CV)=0.9507)and 2941 ± 3784 μg kg-1(CV=1.2866)were observed for the inbreds in 2014 and 2015,respectively.For the hybrids,an overall average of 1045 ± 784 μg kg-1ranging from 58 ± 39 to 2387± 430 μg kg-1in 2015 and from 157± 135 to 2771±780 μg kg-1in 2016 were measured.Averages of 666 ±811 μg kg-1(CV=1.2177)and 1230 ± 855 μg kg-1(CV=0.6951)were observed for the hybrids in 2015 and 2016,respectively.Interestingly,2015 showed higher degrees of variation compared to the other years possibly indicating a greater seasonal effect in that year on aflatoxin accumulation.Bulk harvesting at a single time point may also result in elevated aflatoxin in inbreds with earlier anthesis or less in lines with later anthesis.While no apparent trend was observed in the data,this may contribute to higher aflatoxin levels and variation observed in inbreds compared to hybrids and warrants further study.Overall,the hybrid progeny showed lower and less variable total aflatoxin content than the inbreds.The high standard deviations observed were also likely due to the skewed distribution of the raw aflatoxin data across the experiment necessitating data transformation for further analyses.Therefore,aflatoxin content for both inbreds and hybrids was log2(y+1)transformed for downstream analyses(Fig.S1).

Analysis of variance(ANOVA)showed that there was a significant year effect and variety×year interaction for both inbred lines and hybrids(Table S1).This interaction is representative of genotype×environment interactions classically reported for aflatoxin contamination resistance in maize[32].The overall range of aflatoxin content observed in the hybrids,however,does suggest that the magnitude of this variation may indeed be reduced due to more consistent agronomic performance within and across environments compared to the inbred lines although variability between hybrids as indicated by CVs was similar to that seen in the inbreds in 2015.Given this significant interaction,inbred line and hybrid effects were analyzed separately by year,both of which resulted in significant effects on aflatoxin content in both inbred and hybrid samples in all years(Table S1).

Statistical examination for differences in aflatoxin accumulation among the inbreds showed very little mean separation in post-hoc analysis(Table 1).No significant differences were observed between the resistant control Mp313E and theother lines in 2015.This was the case at both α=0.05 and 0.10 which is reflective of the high degree of variability in aflatoxin contamination existing within each line,and significant plot to plot variability within the field environments.The only inbred line found to significantly differ from B73 and Mo17 in the post-hoc analysis in 2015,though not in 2014,was CML247 which had the lowest consistent level of aflatoxin across both years(Table 1).

Table 1-Mean aflatoxin contamination and general combining ability(GCA)for the inbred lines.

Table 1(continued)

Numerically,the inbred lines CML52,CML69,CML247,GT-603,GEMS-0005,Hi63,Hp301,and M37 W showed ＜1000 μg kg-1of aflatoxin accumulation in both years of the study(Table 1).This is consistent with previous reports on the observed resistance of these inbred lines in other environments.For example,Mideros et al.[33]found that CML52,CML247,and M37 W accumulated levels of aflatoxin and A.flavus mycelial growth comparable to Mp313E in Mississippi field conditions.CML69 was also found to be resistant to aflatoxin contamination in Mississippi and Texas field environments in previous evaluations[34].GT-603 was derived from the GT-MAS:gk population,and has been shown to exhibit resistance in Georgia and other Southern US environments[35].GEMS-0005 was developed as a part of the Germplasm Enhancement of Maize(GEM)cooperative effort to enhance maize resistance to disease,and hybrids of GEMS-0005 have been shown to exhibit resistance to aflatoxin contamination across multiple environments in the Southern US[23,27].Hi63 has no previously reported resistance to aflatoxin contamination,but has been reported to possess resistance to Puccinia polysora[36].Hp301 has been found to possess an active ZmLOX5 gene which has been linked to aflatoxin contamination resistance in previous QTL studies[37].This indicates that these inbred lines may serve as novel sources of resistance for use in breeding programs and warrant additional investigation.

This reduction in aflatoxin content in the hybrids is consistent with the observed GCA(Table 1)and heterotic(Table S2)effects observed for these lines.While no significant GCA effects were observed for the 2015 hybrid samples,in 2016,CML52,GT-603,and Hi63 showed significantly negative GCA effects(P＜0.05)toward reduced aflatoxin content in their hybrids with B73 and Mo17.Previous studies have also found that GCA effects for inbred lines in diallel crosses are negatively correlated with observed aflatoxin contamination in hybrids.For example,Williams and Windham[27]found that during examination of a diallel cross between 10 inbred lines with contrasting aflatoxin resistance GCA effects observed for susceptible lines(i.e.,B73,PHW79,T173,and Va35)tended to be significantly positive while for lines with less contamination(i.e.,Mp313E,Mp494,Mp717,and Mp719)GCA effects were significantly negative.The results in the present study also reflect this negative correlation,though few significant GCA effects were observed,indicating that inbred and hybrid performance may be correlated.While the number of testers used in this experiment do not allow for a complete evaluation of the potential GCA of these lines,the lines with significant GCA here may be useful in diallel crosses and broader topcrosses to investigate their GCA and specific combining ability(SCA)in future studies.

Testcross hybrids generated in also exhibited varying levels of resistance to aflatoxin contamination,though as with the inbreds little means separation was observed in the post-hoc analysis(Table 2).In particular,very few differences were observed in 2015 with only M37 W×B73 being significantly lower compared to the other hybrids at both α=0.05 and 0.10.This may be due either to both plot-to-plot variation as mentioned for the inbreds,or lack of significant GCA effects detected between the inbreds and the testers in 2015.In 2016,a greater number of significant differences could be observed thought the hybrid with the resistant check,Mp313E×B73,showed no significant differences with any other hybrid except GT-603×Mo17 which accumulated the least aflatoxin in the 2016 study(Table 2).Along with GT-603×Mo17,the hybrids CML52×B73,Hp301×Mo17,and M37 W×B73 also showed significantly less aflatoxin accumulation compared to the most heavily contaminated hybrids(Table 2).These additional hybrids were also not significantly different from Mp313E×B73.

Numerically,hybrids of several of the most resistant inbred lines including CML247,CML52,GT-603,and Hi63 showed among the lower levels of aflatoxin contamination observed when crossed with both B73 or Mo17(Table 2).CML52,GT-603,and Hi63 also had the only significant GCA effects detected in 2016(Table 1).M37 W×B73 was also the most consistently resistant F1hybrid identified in the study with an average of 200 ± 180 μg kg-1across both years of the study.In contrast to these lines,the inbred lines used as testers in the topcrosses,B73 and Mo17,showed higher levels of aflatoxin contamination with an average of 1824±1091 and 759±654,respectively(Table 1).These hybrids also exhibited lower aflatoxin contamination than observed in the two commercial hybrids included as checks in 2016,Dekalb DKC69-43 and Dupont-Pioneer P-2023HR,which accumulated an average of 764 ± 661 and 1404 ± 962 μg kg-1,respectively.

When examining mean parent heterosis(Table S2),CML52 and M37 W show heterotic effects across years on aflatoxin content of-24.50%and-13.64%when crossed with B73.Incontrast,if crossed with Mo17,these lines showed effects of 13.98%and-0.68%,respectively.This indicates that these lines show greater heterotic effects for aflatoxin contamination resistance with the stiff-stalk tester B73.For GT-603 and Hi63,negatively heterotic effects across years were observed with both testers with-12.47%and-8.84%,respectively,with B73 and-13.11 and-9.90%,respectively,with Mo17.This suggests that these lines may serve as versatile sources of resistance in breeding programs.In addition,the greatest heterotic effect was observed with Mo18W which showed a heterotic effect across years of-29.23%with B73 and-11.38%withMo17.These results are consistent with previous observations of heterosis having an effect on aflatoxin contamination through unique genetic combinations,or through heterotic increases in hybrid yield relative to the parental inbred lines[23,24,26,34].In this case,however,these effects are more likely due to the more consistent and superior agronomic performance of hybrids compared to inbred lines given that elevated heterotic effects were observed for crosses within the same heterotic groups in this study.Regardless,these inbred lines and hybrid combinations can be useful sources of aflatoxin resistance in breeding programs and potentially useful in commercial applications for the development of resistant germplasm.

Table 2-Mean aflatoxin contamination for F1topcross hybrids.

Table 2(continued)

4.Conclusions

Aflatoxin contamination of maize and other staple food crops is a serious threat to food safety and security,particularly in developing countries.Since the discovery of pre-harvest aflatoxin contamination,breeding efforts have been focused on the discovery of novel sources of resistance,and the investigation of the interactions of maize resistance and the environment toward the development of high yielding varieties with stable aflatoxin contamination resistance across multiple environments.We evaluated the resistance of 64 maize inbred lines from our breeding program,and 128 topcross hybrids with two testers,B73 and Mo17 across two years in a field environment.Eight inbred lines,i.e.,CML52,CML69,CML247,GT-603,GEMS-0005,Hi63,Hp301,and M37 W,were found to have aflatoxin contamination levels＜1000 μg kg-1which were less than or comparable to the resistant check,Mp313E.In particular,CML247 significantly accumulated less aflatoxin compared to the susceptible tester lines in both years of the study.Significant GCA effects were also detected for CML-52,GT-603,and Hi63.F1hybrids of these lines also possessed reduced aflatoxin contamination,and heterotic effects demonstrating their potential use in breeding programs for the development of resistant cultivars.

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2018.10.001.

Acknowledgments

We thank Billy Wilson and Hui Wang for technical assistance in the field.This work is partially supported by the U.S.Department of Agriculture,Agricultural Research Service(USDA-ARS),the Georgia Agricultural Commodity Commission for Corn,and AMCOE(Aflatoxin Mitigation Center of Excellence,Chesterfield,MO,USA).Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA.The USDA is an equal opportunity provider and employer.