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Field trials using Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) and Euxesta stigmatias Loew (Diptera: Ulidiidae) were conducted to evaluate resistance and potential damage interactions between these two primary corn, Zea mays L., pests against Lepidoptera-resistant corn varieties derived from both endogenous and exogenous sources. The endogenous source of resistance was maysin, a C-glycosyl flavone produced in high concentrations in varieties 'Zapalote Chico 2451' and 'Zapalote Chico sh2'. The exogenous resistance source was the Bacillus thuringiensis (Bt)11 gene that expresses Cry1A(b) insecticidal protein found in 'Attribute GSS-0966'. Damage by the two pests was compared among these resistant varieties and the susceptible 'Primetime'. Single-species tests determined that the Zapalote Chico varieties and GSS-0966 effectively reduced S. frugiperda larval damage compared with Primetime. E. stigmatias larval damage was less in the Zapalote Chico varieties than the other varieties in single-species tests. E. stigmatias damage was greater on S. frugiperda-infested versus S. frugiperda-excluded ears. Ears with S. frugiperda damage to husk, silk and kernels had greater E. stigmatias damage than ears with less S. frugiperda damage. Reversed phase high-performance liquid chromatography analysis of nonpollinated corn silk collected from field plots determined that isoorientin, maysin, and apimaysin plus 3'-methoxymaysin concentrations followed the order Zapalote Chico sh2 > Zapalote Chico 2451 > Attribute GSS-0966 = Primetime. Chlorogenic acid concentrations were greatest in Zapalote Chico 2451. The two high maysin Zapalote Chico varieties did as well against fall armyworm as the Bt-enhanced GSS-0966, and they outperformed GSS-0966 against E. stigmatias.

Maysin is a naturally occurring C-glycosyl flavone found in maize (Zea mays L.) silk tissue that confers resistance to corn earworm (Helicoverpa zea, Boddie). Recently, two new maize populations were derived for high silk maysin. The two populations were named the exotic populations of maize (EPM) and the southern inbreds of maize (SIM). Quantitative trait locus (QTL) analysis was employed to determine which loci were responsible for elevated maysin levels in inbred lines derived from the EPM and SIM populations. The candidate genes consistent with QTL position included the p (pericarp color), c2 (colorless2), whp1 (white pollen1) and in1 (intensifier1) loci. The role of these loci in controlling high maysin levels in silks was tested by expression analysis and use of the loci as genetic markers onto the QTL populations. These studies support p, c2 and whp1, but not in1, as loci controlling maysin. Through this study, we determined that the p locus regulates whp1 transcription and that increased maysin in these inbred lines was primarily due to alleles at both structural and regulatory loci promoting increased flux through the flavone pathway by increasing chalcone synthase activity.

In the United States, insecticide is used extensively in the production of sweet corn due to consumer demand for zero damage to ears and to a sweet corn genetic base with little or no resistance to ear-feeding insects. Growers in the southern United States depend on scheduled pesticide applications to control ear-feeding insects. In a study of quantitative genetic control over silk maysin, AM-maysin (apimaysin and methoxymaysin), and chlorogenic acid contents in an F2 population derived from GE37 (dent corn, P1A1) and 565 (sh2 sweet corn, p1a1), we demonstrate that the P1 allele from field corn, which was selected against in the development of sweet corn, has a strong epistatic interaction with the a1 allele in sh2 sweet corn. We detected that the p1 gene has significant effects (P < 0.0001) not only on silk maysin concentrations but also on AM-maysin, and chlorogenic acid concentrations. The a1 gene also has significant (P < 0.0005) effects on these silk antibiotic chemicals. Successful selection from the fourth and fifth selfed backcrosses for high-maysin individuals of sweet corn homozygous for the recessive a1 allele (tightly linked to sh2) and the dominant P1 allele has been demonstrated. These selected lines have much higher (2 to 3 times) concentrations of silk maysin and other chemicals (AM-maysin and chlorogenic acid) than the donor parent GE37 and could enhance sweet corn resistance to corn earworm and reduce the number of applications of insecticide required to produce sweet corn.

Several resistance sources and resistance mechanisms to aflatoxin formation and corn earworm (Helicoverpazea Boddie) damage to maize (Zeamays L.) have been identified. Based on this knowledge, experiments were initiated toward achievement of the following objectives: (1) to confirm earlier determinations on resistance traits of germplasm sources and to identify quantitative trait loci (QTL) associated with each of the traits, and (2) upon estimation of the degree of QTL effects on each trait, to generate a maize population, with chemical and physical resistance to Aspergillus spp. and ear-feeding insects, for inbred development. A 2-year field experiment to evaluate selected genotypes inoculated with A. flavus and infested with corn earworm revealed that significant variation exists among the genotypes for aflatoxin contamination and corn earworm damage. The protection of maize ears against aflatoxin contamination was primarily dependent on resistance to fungal infection and ear-feeding insects, and excellent husk coverage and tightness. A major QTL (p1) identified on chromosome 1S had effects of 54.0, 42.1, and 28.3% on the phenotypic variability for concentrations of silk maysin, 3′-methoxymaysin+apimaysin, and chlorogenic acid, respectively. Markers/QTLs for husk phenotypic traits and total aflatoxin concentrations have been determined, but more detailed mapping of these chromosomic regions will be necessary to locate precise markers/QTLs for husk traits and aflatoxin production. Realizing the complexity of the Aspergillus–aflatoxin-maize system and the factors affecting aflatoxin contamination, we are directing our program toward marker-assisted breeding to enhance or improve general genetic resistance to ear-feeding insects and invasion by Aspergillus spp., A. Butron thanks ‘Ministerio de Educacion y Ciencia’ of Spain that supported her while she was carrying out the present study. Research was supported by funds provided by USDA Agricultural Research Service and by the Georgia Agricultural Commodity Commission for Corn., MEC, USDA Agricultural Research Service, Georgia Agricultural Commodity Commission for Corn

Aflatoxin contamination of corn in the field is influenced by several factors. In the southern U.S., insect populations are usually large every year. Drought caused by warmer and drier than normal weather is conducive to A. flavus infection and aflatoxin contamination of corn, Zea mays L. When loose-husked hybrids are used in the southern U.S., they accentuate insect damage and aflatoxin contamination. The development and breeding of ‘‘southern-type’’ hybrids is essential for control of preharvest aflatoxin contamination. Molecular biotechnology may make an impact on tackling the complexity of preharvest aflatoxin contamination of corn. Integration of crop management tactics and genetic

Aspergillus flavus (Link:Fr.) infection and aflatoxin contamination of maize (Zea mays L.) grain are an extremely serious problem. Maize genotypes resistant to A. flavus attack are needed. Maize breeders and plant pathologists must identify resistance sources and incorporate resistance into adapted breeding material. Maize population GT-MAS:gk has been released for use as a resistance source. In this study, we surveyed the genetic variation in this population and made the breeders/plant pathologists aware of the heterogeneous nature in this maize population by using RAPD analysis and correlated the RAPD marker association with the resistance to A. flavus and aflatoxin production. Of 40 RAPD primers, only 15 gave sufficient numbers of reproducible and readily scored polymorphic bands suggesting that this population was highly homogeneous. However, genetic distances, ranging from 0.08 to 0.28 and averaging 0.17, suggest that there is variation within the population. Cluster analysis distinguished three major polymorphic groups. Laboratory bioassay revealed that group I contained the most resistant individuals, i.e., those with less aflatoxin production. Group II had the least resistance, and group III was intermediate. This study showed that the maize population GT-MAS:gk is heterogeneous and individuals are different in resistance to A. flavus and aflatoxin production. Resistance should be confirmed through progeny testing before further development. The RAPD marker OPX-04, which may be associated with the resistance trait, has been cloned and further characterization will be pursued.

Maysin, a C-glycosylflavone in maize silk, has insecticidal activity against corn earworm, Helicoverpa zea (Boddie), larvae. Sweet corn, Zea mays L., is a vulnerable crop to ear-feeding insects and requires pesticide protection from ear damage. This study was conducted to identify maize chromosome regions associated with silk maysin concentration and eventually to transfer and develop high silk maysin sweet corn lines with marker-assisted selection (MAS). Using an F2 population derived from SC102 (high maysin dent corn) and B31857 (low maysin sh2 sweet corn), we detected two major quantitative trait loci (QTL). It was estimated that 25.6% of the silk maysin variance was associated with segregation in the genomic region of npi286 (flanking to p1) on chromosome 1S. We also demonstrated that a1 on chromosome 3L had major contribution to silk maysin (accounted for 15.7% of the variance). Locus a1 has a recessive gene action for high maysin with the presence of functional p1 allele. Markers umc66a (near c2) and umc105a on chromosome 9S also were detected in this analysis with minor contribution. A multiple-locus model, which included npi286, a1, csu3 (Bin 1.05), umc245 (Bin 7.05), agrr21 (Bin 8.09), umc105a, and the epistatic interactions npi286 x a1, a1 x agrr21, csu3 x umc245, and umc105a x umc245, accounted for 76.3% of the total silk maysin variance. Tester crosses showed that at the a1 locus, SC102 has functional A1 alleles and B31857 has homozygous recessive a1 alleles. Individuals of (SC102 x B31857) x B31857 were examined with MAS and plants with p1 allele from SC102 and homozygous a1 alleles from B31857 had consistent high silk maysin. Marker-assisted selection seems to be a suitable method to transfer silk maysin to sweet corn lines to reduce pesticide application.

The presence of maysin, a flavone glycoside, and its analogues in the silks of corn is an important defence against invasion of the ear by corn earworm in the southeastern USA. Three dent maize inbreds with high silk-maysin concentrations were evaluated for chromosomal location of major genes controlling synthesis of three antibiotic chemicals by crossing them to a series of waxy-marked chromosome 9 reciprocal translocations. The data indicated that genes for maysin and its analogues are probably present on the short arms of chromosomes 1 and 10, and long arms of chromosomes 5 and 8 for inbred line GT114, the short arms of chromosome 1 and 6, and the long arms of chromosome 5 and 8 for inbred line GE37, and the short arms of chromosomes 1 and 10, and the long arm of chromosome 4 for inbred line SC102. These results are in general agreement with other translocation studies on corn earworm injury to sweet corn inbreds and gene and restriction fragment length polymorphism marker studies to locate quantitative trait loci (QTL) for maysin synthesis, with the exception that QTL on chromosome 9 have been found to be strongly associated with flavone synthesis. The most significant concordance between conventional and molecular techniques for locating chromosome regions influencing synthesis of antibiotic chemicals from silks of high silk antibiosis sources is found for the short arm of chromosome 1. This region is the most likely one on which to place emphasis during the initial stages of transferring high-maysin silks to elite inbred lines. A chromosome 1 marker plus at least one more from any chosen high-maysin inbred source should be sufficient to transfer high maysin silks to other lines. Other markers may be needed for transfer of specific traits when recovering recurrent parent genome types in a backcrossing procedure.

J. Econ. Entomol. 92(3): 746-753 (1999) Sweet corn is one of the highest ranking vegetable crops in the United States in terms of both market value and total acreage: it also ranks among the top vegetable crops in terms of pesticide usage. Ear damage caused by insects is the most significant concern to producers. An estimated 25-40 insecticide applications may be made in a single season to control ear-feeding insects. Plant resistance is an alternative strategy to alleviate environmental concern when pesticides are used. In this study we investigated the feasibility of transferring antibiosis to corn earworm. Helicoverpa zea (Boddie), from dent corn with high silk maysin to sweet corn. Relationships among silk maysin concentration, antibiosis to corn earworm, and silk browning in their F 2 segregating populations were determined. Lyophilized high maysin silks bioassays produced low larval weights, thus providing evidence that damage to sweet corn by corn earworm could be reduced by increasing silk maysin concentration. Simple correlation coefficients indicated a highly significant negative association between silk maysin concentrations and 8-d corn earworm larval weights (r -0.80, P< 0.0001). Silk browning was also closely associated with silk maysin concentrations and antibiosis. Diets containing mid-dark silks reduced larval weights by 68% compared with larval weights (588 mg) on the bean diet control. Maysin analyses and dry-silk bioassays also revealed that if maysin is present in F 2 plants from sweet corn kernels, it is always in a higher concentration than in plants from dent kernels, These results suggest that sweet corn parents in these populations may have a recessive gene, which may be closely linked to sh2 locus enhancing/regulating maysin synthesis when crossed to a maysin producing dent parent.

C-glycosyl flavones in maize silks confer resistance (i.e., antibiosis) to corn earworm (Helicoverpa zea [Boddie]) larvae and are distinguished by their B-ring substitutions, with maysin and apimaysin being the di- and monohydroxy B-ring forms, respectively. Herein, we examine the genetic mechanisms underlying the synthesis of maysin and apimaysin and the corresponding effects on corn earworm larval growth. Using an F2 population, we found a quantitative trait locus (QTL), rem1, which accounted for 55.3% of the phenotypic variance for maysin, and a QTL, pr1, which explained 64.7% of the phenotypic variance for apimaysin. The maysin QTL did not affect apimaysin synthesis, and the apimaysin QTL did not affect maysin synthesis, suggesting that the synthesis of these closely related compounds occurs independently. The two QTLs, rem1 and pr1, were involved in a significant epistatic interaction for total flavones, suggesting that a ceiling exists governing the total possible amount of C-glycosyl flavone. The maysin and apimaysin QTLs were significant QTLs for corn earworm antibiosis, accounting for 14.1% (rem1) and 14.7% (pr1) of the phenotypic variation. An additional QTL, represented by umc85 on the short arm of chromosome 6, affected antibiosis (R2 = 15.2%), but did not affect the synthesis of the C-glycosyl flavones.

The interpretation of quantitative trait locus (QTL) studies is limited by the lack of information on metabolic pathways leading to most economic traits. Inferences about the roles of the underlying genes with a pathway or the nature of their interaction with other loci are generally not possible. An exception is resistance to the corn earworm Helicoverpa zea (Boddie) in maize ( Zea mays L.) because of maysin, a C-glycosyl flavone synthesized in silks via a branch of the well characterized flavonoid pathway. Our results using flavone synthesis as a model QTL system indicate: ( i ) the importance of regulatory loci as QTLs, ( ii ) the importance of interconnecting biochemical pathways on product levels, ( iii ) evidence for “channeling” of intermediates, allowing independent synthesis of related compounds, ( iv ) the utility of QTL analysis in clarifying the role of specific genes in a biochemical pathway, and ( v ) identification of a previously unknown locus on chromosome 9S affecting flavone level. A greater understanding of the genetic basis of maysin synthesis and associated corn earworm resistance should lead to improved breeding strategies. More broadly, the insights gained in relating a defined genetic and biochemical pathway affecting a quantitative trait should enhance interpretation of the biological basis of variation for other quantitative traits.

Growth of the corn earworm (Helicoverpa zea Boddie), a major pest of maize (Zea mays L.), is inhibited by high concentrations of maysin, a C-glycosyl flavone, in maize silks. To advance understanding of the genetic control of silk maysin concentration and antibiosis to corn earworm, we conducted a quantitative trait locus (QTL) study in a population derived from a cross of high-maysin (GE37) and low-maysin (FF8) lines. Both lines have phenotypically identical, functional alleles at the p1 locus, a regulator of the biochemical pathway leading to maysin synthesis. We evaluated 250 (GE37 x FFB)F 2:3 families for (i) restriction fragment length polymorphisms at 121 loci, (ii) silk maysin concentrations in two locations (in Georgia and Missouri), and (iii) weight of corn earworm larvae grown on artificial diet containing oven-dried silks. For maysin concentration in both locations, composite interval mapping revealed major QTLs in the asg20-whpl interval of chromosome 2 and near the wxl locus on chromosome 9; smaller effects were detected on chromosomes 1, 6, and 8. For larval weight, the largest QTL was detected in the asg20-whpl interval, and major effects were also associated with chromosomes 1 and 6. However, we found no effect on larval weight at wxl. Significant (P < 0.001) epistasis was observed for all traits. Our results suggest that whp1 on chromosome 2, a gene that encodes chalcone synthase and sm1, a silk-specific gene on chromosome 6, are candidate genes affecting maysin concentration and corn earworm larval weight in this population.

The isolation and identification of maysin and two of its analogues, 3'-methoxymaysin and apimaysin (3'M+A),in the silks of corn, Zea mays L., has given plant breeders a new tool for reducing damage by larvae of the corn earworm, Helicoverpa zea (Boddie). This study was initiated to examine the inheritance of these compounds so that a logical course for developing germplasm with high concentrations can be determined. Incorporation of the compounds into corn hybrids for the southern USA can then proceed. Twelve inbred lines were crossed in all combinations and evaluated for maysin and the sum of 3'-methoxymaysin plus apimaysin concentrations in their silks. Analysis of data from 2 yr by Griffing's Method 3 Model I revealed that reciprocal and maternal effects were not significantly different from zero. Subsequent pooling of reciprocal crosses in a Gardner-Eberhart Analysis II gave significant main effects (P < 0.01) and years x entries interactions (P < 0.05). Further partitioning of the variation established additive effects as the predominant source of genetic variation among the 66 crosses evaluated. Selection for increased maysin and (3'-methoxymaysin plus apimaysin) concentrations in corn silks should be successful by means of a simple backcrossing procedure or routine recurrent selection within a population. One or two inbreds, among the 12 tested, appear to be good candidates for establishing a selection program because they have both good general and specific combining ability. Maysin concentration and the sum of its analogues 3'-methoxymaysin and apimaysin are closely correlated in corn silks (r = 0.81, P < 0.01).

Mechanisms of resistance to infection by the fungus Aspergillus flavus and accumulation of aflatoxin were studied in kernels of resistant (GT-MAS:gk, Mp420) and susceptible (Pioneer 3154, Deltapine G-4666) corn genotypes. Proteins from kernel extracts of corn genotypes were analyzed by several methods of polyacrylamide gel electrophoresis. Consistent differences in protein profiles were detected among genotypes. Several proteins were unique to or present in greater concentration in resistant genotypes, whereas others were present only in susceptible genotypes. Extracts of resistant kernels showed markedly greater antifungal activity against A. flavus than did susceptible kernel extracts. Results from the present study suggest a role for kernel proteins in resistance to A. flavus infection and aflatoxin contamination in corn genotypes GT-MAS:gk and Mp420.

Guo, B. Z., Chen, Z.-Y., Brown, R. L., Lax, A. R., Cleveland, T. E., Russin, J. S., Mehta, A. D., Selitrennikoff, C. P., and Widstrom, N. W. 1997. Germination induces accumulation of specific proteins and antif ungal activities in corn kernels. Phytopathology 87:1174-1178. This study examined protein induction and accumulation during imbibition and germination of corn kernels, as well as antifungal activ ities of extracts from germinating kernels against Aspergillus flavus and Fusarium moniliforme. Genotypes studied included GT-MAS:gk and Mp420, which are resistant to A. flavus infection and aflatoxin accumulation, and Pioneer 3154 and Deltapine G-4666, which are susceptible to A. flavus infection and aflatoxin accumulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved five protein bands that were present at higher concentrations in germinated kernels than in nongerminated kernels. Western blot analyses revealed that one of these proteins reacted with the 22-kDa zeamatin antiserum, and a zeamatin-like protein accumulated to a higher concentration in germinated kernels. Two protein bands from dry kernels that reacted with ribosome-inactivating protein (RIP) antiserum were identified as the 32-kDa proRIP-like form and an 18-kDa peptide of the two peptides that form active RIP. However, in germinated kernels, two protein bands that reacted with RIP antiserum were identified as two RIP-like peptides with a molecular mass of ≈18 and 9 kDa. Purified RIP and zeamatin from corn inhibited growth of A. flavus. Bioassays of germinated kernel extracts from all four genotypes exhibited antifungal activity against A. flavus and F. moniliforme, with extracts from the susceptible genotypes showing greater inhibition zones. This study provides evidence of protein induction in corn kernels during imbibition or the early stages of germination, and the induced proteins may be related to our previous findings of germination-associated resistance in the corn kernel, especially in the susceptible kernels.

A better understanding of the genetic basis of antibiosis to the corn earworm, Helicoverpa zea (Boddie), in maize, Zea nays L., silks will help breeders develop hybrids with improved resistance to this pest. We identified maize chromosome regions associated with antibiosis to the corn earworm, and compared the results with those previously obtained for concentration of silk maysin, a C-glycosyl flavone that inhibits larval growth. We evaluated antibiosis with a dried-silk bioassay of 76 (GT114 X GT119)F 2:3 lines, derived by self-pollinating F2 plants from the high and low tails of the distribution for silk maysin concentration. Variation in 8-d larval weights resulting from silks of the F 2:3 lines was compared with variation in restriction fragment length polymorphism genotypes at 39 flavonoid pathway loci or linked markers of corresponding F 2 plants. The multiple-locus model that best explained variation of larval weights included a major effect at the p1 region of chromosome 1 and smaller effects in the umc166b region (chromosome 1) and the sh1 region (chromosome 9). The model for maysin concentration of the corresponding F2 plants was nearly identical, with the exception that the sh1 locus was replaced by the nearby u1tlciosa locus. Our results provide evidence that in this population silk maysin concentration and corn earworm antibiosis are under similar genetic control and that the p1 locus plays the major role in determining variation in both traits. Because we evaluated the tails of the distribution and probed only at flavonoid pathway loci, all chromosome regions associated with corn earworm resistance in this population may not have been detected.

Russin, J. S., Guo, B. Z., Tubajika, K. M., Brown, R. L., Cleveland, T. E., and Widstrom, N. W. 1997. Comparison of kernel wax from corn genotypes resistant or susceptible to Aspergillus flavus. Phytopathology 87: 529-533.Kernels of corn genotype GT-MAS: gk are resistant to Aspergillus flavus. Earlier studies showed that this resistance is due in part to kernel pericarp wax. Experiments were conducted to compare wax from GTMAS: gk kernels with that from kernels of several susceptible commercial hybrids. GT-MAS: gk had more pericarp wax than did the susceptible hybrids. Scanning electron microscopy revealed that GT-MAS: gk kernels appeared rough and showed abundant wax deposits on kernel surfaces. Susceptible kernels appeared much more smooth and lacked the abundant surface deposits observed in GT-MAS: gk. In vitro bioassays showed that kernel wax from GT-MAS: gk reduced A. flavus colony diameter by 35%. Colony diameters on a medium amended with wax from susceptible kernels did not differ from those of controls. Thin-layer chromatography and analyses of chromatograms using NIH Image software showed a distinctive composition for GT-MAS: gk kernel wax. Chromatograms of wax from GT-MAS: gk contained a peak unique to this genotype, but also lacked a peak common to all susceptible hybrids. This is the first report of specific kernel factors involved in resistance to A. flavus in corn.

A maize inbred extracted from an Iowa synthetic (BSBB) produces silks with an odor that is easily detectable by most humans. The inbred did not demonstrate greater field resistance to ear-feeding by insects than sister lines from the same synthetic. The odorous trait was determined to be inherited as a recessive trait but could not be positively associated with a single gene when the odorous line was crossed with unrelated normal lines. Failure to produce a single-gene Mendelian ratio for F2 and backcross generations was attributed to errors in classification for a subjectively evaluated trait and the seeming incomplete penetrance of the trait, possibly due to the presence of modifying genes. Eleven compounds that differed from those obtained from nonodorous sister lines were separated and identified by headspace trapping and analysis by gas chromatographic and mass spectrometric techniques. A five-member panel clearly identified the odorous compound as indole.

Interpretation of quantitative trait locus (QTL) studies of agronomic traits is limited by lack of knowledge of biochemical pathways leading to trait expression. To more fully elucidate the biological significance of detected QTL, we chose a trait that is the product of a well-characterized pathway, namely the concentration of maysin, a C-glycosyl flavone, in silks of maize, Zea mays L. Maysin is a host-plant resistance factor against the corn earworm, Helicoverpa zea (Boddie). We determined silk maysin concentrations and restriction fragment length polymorphism genotypes at flavonoid pathway loci or linked markers for 285 F2 plants derived from the cross of lines GT114 and GT119. Single-factor analysis of variance indicated that the p1 region on chromosome 1 accounted for 58.0% of the phenotypic variance and showed additive gene action. The p1 locus is a transcription activator for portions of the flavonoid pathway. A second QTL, represented by marker umc 105a near the brown pericarp1 locus on chromosome 9, accounted for 10.8% of the variance. Gene action of this region was dominant for low maysin, but was only expressed in the presence of a functional p1 allele. The model explaining the greatest proportion of phenotypic variance (75.9%) included p1, umc105a, umc166b (chromosome 1), r1 (chromosome 10), and two epistatic interaction terms, p1 x umc105a and p1 x r1. Our results provide evidence that regulatory loci have a central role and that there is a complex interplay among different branches of the flavonoid pathway in the expression of this trait.

Silks of 50 field corn, Zea mays L., inbreds were evaluated in 4 separate bioassays (fresh silks, 2- and 4-g oven-dried silks/100 inl diet, and silk methanol extract deposited on celufil) for their effects on the growth of corn earworm, Helicoverna zea (Boddie) larvae. The silks also were assayed for maysin, isomaysin, and apimaysin plus 3'-methoxymaysin content. Significant differences in larval growth were found among silks from inbreds within each bioassay. The dried silk bioassay was easier to mix and dispense and permitted the use of silks of germplasm with varying maturity. A highly significant negative relationship was found between weight of larvae and maysin content from fresh silk, and maysin and isomaysin content from oven-dried silk. The correlation between larval weight and apimaysin plus 3'-methoxymaysin was not significant. The correlation between larval weight and total flavones from oven dried silks seas enhanced (-0.667 for maysin alone to -0.979 for total flavones) compared with that between larval weight and maysin alone.

A small percentage (< 5%) of dent corn inbreds was sensitive to the herbicide nicosulfuron. Segregation within a backcross to inbred Ab18 demonstrated simple inheritance as a nuclear gene for corn sensitivity to this herbicide. Greenhouse tests indicated that cytoplasmic or maternal effects were not important in conditioning sensitivity. Tolerance in some crosses began to break down at application rates approaching twice that registered. Sensitivity was controlled by a single autosomal recessive gene in the inbred Ab18. Further testing will be required to determine whether this gene is common to other sensitive inbreds. Thorough screening continues to be necessary to identify sensitive elite inbreds in public and private breeding programs.

Reduced derivatives of maysin [2-O-α-L-rhamnosyl-6-C-(6-deoxy-xylo-hexos-4-ulosyl)luteolin] and 3'-methoxymaysin have been isolated and identified from several corn (Zea mays L.) inbreds. These include 2-O-α-L-rhamnosyl-6-C-quinovosylluteolin (equatorial 4-OH-maysin, eq-4-OH-maysin), 2-O-α-L-rhamnosyl-6-C-fucosylluteolin (axial 4-OH-maysin, ax-4-OH-maysin), and 2-O-α-L-rhamnosyl-6-C-fucosyl-3'-methoxyluteolin (ax-4-OH-3'-methoxymaysin). In addition to maysin and 3'-methoxymaysin, inbred Tx501 contains minor amounts of ax-4-OH-maysin and ax-4-OH-3'-methoxymaysin. Corn lines A103, GE275, ESDJ1, and CML131 contained relatively large levels of both ax-4-OH-maysin and eq-4-OH-maysin. Synthetic 4-OH-maysin (obtained by NaBH 4 reduction of maysin) was tested for growth inhibition of corn earworm (Helicoverpa zea Boddie) larvae in a laboratory bioassay and found to be almost as active as maysin, suggesting that incorporation by breeders of these compounds into silks could enhance the resistance of corn to corn earworm larvae.

Thirteen maize hybrids and one maize population, MAS:gk, were screened for susceptibility to aflatoxin production by Aspergillus flavus . Marked differences in aflatoxin B1 production were detected among the maize genotypes tested. Most commercial hybrids consistently supported high levels of aflatoxin accumulation. Aflatoxin levels did not differ between intact and wounded kernels of these genotypes. However, different results were obtained from 4 of the 13 hybrids and the maize population MAS:gk. Levels of aflatoxin accumulation in intact kernels of these genotypes were lower than in the previous susceptible group of genotypes. In addition, aflatoxin levels were higher in wounded than in intact kernels. MAS:gk not only supported the lowest levels of aflatoxin production in intact kernels, but aflatoxin levels in endosperm-wounded kernels also were significantly lower in MAS:gk than in wounded kernels of all tested hybrids. Treatment with KOH to remove cutin from intact kernels prior to inoculation with A. flavus effected substantial increases in aflatoxin accumulation in MAS:gk, but only marginal increases in the susceptible hybrid Pioneer 3154. Removing wax from the surface of MAS:gk kernels greatly increased the susceptibility of this genotype to aflatoxin accumulation. When wax removal was combined with treatment with potassium hydroxide (KOH) or purified cutinase, aflatoxin levels in kernels were equal to those in wounded control kernels in both genotypes. These results indicated that wax and cutin layers of maize kernel pericarps may play a role in resistance to aflatoxin accumulation in MAS:gk and some other genotypes. However, results suggest further that resistance in MAS:gk also may be due to other preformed compounds as well.

Several phenols and related compounds have been shown to have detrimental effects on insects while others have antibiotic activity against fungi which attack higher plants. Insects have also been implicated as contributors to preharvest contamination of corn [maize], Zea mays L, by aflatoxin. The objectives, therefore, were to determine (a) if commercial corn hybrids vary in their silk maysin content and aflatoxin contamination of the grain, and (b) if grain aflatoxin contamination is correlated with maysin and related compounds. During 4 years of testing, 16 corn hybrids varied significantly for silk maysin content and grain aflatoxin contamination. Based on correlations, grain aflatoxin content of the hybrids tested was not significantly associated with maysin, chlorogenic acid, and 3′-methoxymaysin contents. It was concluded that other untested phenolic compounds in the category of compounds analyzed in the present study could be involved in resistance to aflatoxin formation, and that other classes of compounds should also now be assessed to locate major chemical resistance components.

A two-year factorial experiment was utilized to test plants field-inoculated singly and in combination withAspergillus flavus andFusarium moniliforme. Pinbar inoculations were made through the husks with conidial suspensions, and 10-ear maize samples were harvested at 60 days post-silking for aflatoxin determinations. When ears were inoculated with both fungi simultaneously,F. moniliforme reduced aflatoxin formation byA. flavus isolate NRRL 3357 by approximately two-thirds.F. moniliforme had no significant effect on naturally occurring aflatoxin contamination byA. flavus. This may be due to the timing of infection by both fungi in the field. In nature,A. flavus andF. moniliforme respond differently to the environment, offering one explanation of whyF. moniliforme did not measurably affect the other fungus.

Genetic information is needed on maysin, a compound in maize, Zea mays L., silks that is antibiotic to corn earworm larvae, Helicoverpa (formerly Heliothis) zea (Boddie), to assist in the process of breeding for resistance to this pest. This study was initiated to elucidate the inheritance of maysin content in the silks of a cross between two maize inbreds (GT114 and GT119) having resistance to the corn earworm. Reversed phase high performance liquid chromatography (HPLC) procedures were used to quantify the maysin concentration of silks from individual plants in the parent, F1, F2, and first backcross generations of the cross GT114 × GT119. A model assuming dominance at a single locus for low maysin content which is expressed only when a dominant modifier is present was proposed to explain the segregation ratios obtained in the F2 and first back-crosses of GT114 × GT119. This model also provided a fit for maysin data previously generated from a cross between inbreds F6 and F44. Observations of selfed families in the backcross of GT114 × GT119 to the low maysin parent confirmed the two-locus model (one factor with dominance for low maysin expressed only when a modifier gene is also present) as a plausible explanation for inheritance of silk maysin in the cross. The process of selection for high maysin will be complicated somewhat by the presence of a gene which modifies the expression of dominance unless a method of easily identifying the gene, independent of maysin content, can be developed.

Kernels from two maize populations, MAS:gk and MAS:pw,nf, showed significant postharvest resistance to aflatoxin contamination by Aspergillus flavus but showed no significant inter-population variation for this resistance. Growth of A. flavus on both populations was significantly less than on susceptible control lines. Kernels from the resistant populations retained resistance when wounded through the pericarp prior to inoculation with A. flavus , despite the fact that the exposed endosperm supported good fungal growth. Kernels from these populations also retained resistance when they were acetone washed before inoculation. Resistance to aflatoxin contamination was lost in kernels that were autoclaved, crushed, or embryo wounded. All assays were incubated under conditions favorable to kernel germination. Results suggest that postharvest resistance to aflatoxin contamination in these two populations is related to metabolic activities of the living com embryo.

Information on heterosis is critical to assimilation of desirable traits into usable maize, Zea mays L., germplasm. Our objective was to identify maize ppopulations with heterosis for desirable traits. Twelve southern-adapted maize populations with resistance to damage by ear and leaf-feeding insects and all 66 possible population crosses were evaluated in a randomized complete-block design in three environments. Populations effects, heterosis, and specific combining ability (SCA) effects were estimated for seven plant traits [...]

Maysin [2"-O-alpha-L-rhamnosyl-6-C-(6-deoxy-xylo-hexos-4-ulosyl) luteolin], the resistance factor of Zapalote Chico corn (Zea mays L.) silks to the corn earworm (Helicoverpa zea Boddie), was determined by HPLC in the silks of over 600 inbreds, populations, plant introductions (PI), and various unassigned collections. Maysin levels ranged from 0% to 0.9% fresh weight with approximately 19% of both the inbreds and populations containing maysin levels above 0.2%, a level considered to be necessary for resistance, as based on the laboratory bioassay determined level (for larval) of maysin toxicity. Over 25 lines yielded higher silk maysin than found in Zapalote Chico (0.347%). High levels of 2"-O-alpha-L-rhamnosyl-6-C-(6-deoxy-xylo-hexos-4-ulosyl) apigenin (apimysin) were found in inbred NC7, while Tx501 contained appreciable amounts of 2"-O-alpha-L-rhamnosyl-6-C (6-deoxy-xylo-hexos-4-ulosyl)-3'-methoxyluteolin (3'-methoxymaysin). Spectral data (13C NMR and FAB-MS) are given for the characterization of maysin, apimaysin, and 3'-methoxymaysin.

A thorough knowledge of the inheritance of maysin, a flavone glycoside with antibiosis to corn earworm, Helicoverpa zea Boddie, in the silks of maize will assist breeders in choosing the most efficient method of incorporating this trait into elite inbreds. Two breeding populations, one having exotic origin (EPM), the other from southern inbred lines (SIM) were subjected to six cycles of recurrent selection for increased silk maysin. Ten per cent of the individuals evaluated in populations were selected for recombination as S 1 progenies in each cycle. Progress was evaluated as C0 to C6 population cycles in a randomized complete-block experiment with five replications in 1997 and 1998. Maysin fresh weights in silks of the C0 populations were 0.54% for EPM and 0.36% for SIM. Responses of 0.19% per cycle in EPM and 0.22% per cycle in SIM resulted in an EPM (C6) population with 1.76% maysin and an SIM (C6) population with 1.69% maysin. Silk maysin concentrations above 0.2% begin to substantially reduce larval growth and prevent completion of the life cycle when husk coverage is sufficient to force the insect to feed on silks while entering the ear. Chlorogenic acid and two analogues of maysin, apimaysin and 3'-methoxymaysin, were found in such minor quantities in silks that they could not be credited with any impact on antibiotic activity against the insect. Selection has effectively increased silk maysin concentration in both EPM and SIM. Trait responses for maysin are highly heritable and will allow the plant breeder to introgress resistance to the corn earworm into elite material easily.

Plant resistance to insects, specifically antibiotic resistance, offers biologically, economically, and environmentally sound alternatives to pesticides for controlling the corn earworm, Helicoverpa zea (Boddie), and fall armyworm, Spodoptera frugiperda (J. E. Smith), in corn, Zea mays L. Our study was done to determine the relationship between maysin concentration in corn silks and growth of corn earwonn and fall armyworm larvae. Silks, or methanol extracts from the silks of a large number of corn entries mixed in meridic diets, were evaluated by feeding them to neonate larvae and measuring subsequent larval growth at 8 or 9 d. Silks from several corn entries showed high antibiosis and a high maysin content. A range of highly significant negative relationships between weight of larvae and maysin concentration was observed.

Aflatoxin contamination in preharvest maize, Zea mays L., grain is a serious problem in the United States, especially in the southeastern production areas. Development of genotypes with resistance to ear-infesting insects, resistance to Aspergillus spp., and resistance to production of aflatoxin are maize-breeding objectives. Two cultivars, Mo20W × Teosinte and ‘lbadan B’, have been identified as having resistance to earinfesting insects when compared with the widely grown check hybrid B73 × Mo17. Although it was not determined if there was resistance to growth of Aspergillus flavus (Link ex Fries) or aflatoxin production per se, it was confirmed that these two cultivars produced less aflatoxin in preharvest grain than B73 × Mo17. Also, Mo20W × Teosinte tended to have less aflatoxin than ‘lbadan B’.

Populations and inbreds of maize, Zea mays L., with effective levels of resistance to damage by fall armyworm, Spodoptera frugiperda (J.E. Smith), are needed to transfer resistance to commercial hybrids for the U.S. Southeast. Recurrent S 1 selection for resistance to leaf feeding by larvae of fall armyworm was conducted with an exotic maize synthetic, FAW-CC (.)

The corn earworm, Helicoverpa zea (Boddie), is the most important pest of corn, Zea mays L., in the southeastern United States. This study was conducted to determine whether tolerance or antibiosis mechanisms (or both) of resistance function in 18 populations of corn that are being developed for resistance to ear and leaf damage in the field to the corn earworm and fall armyworm, Spodoptera frugiperda (J. E. Smith). The populations and resistant and susceptible checks were evaluated in the field for corn earworm injury and husk tightness and in the laboratory for feeding responses of neonate corn earworm larvae on fresh silks or oven-dried silks mixed in a meridic diet. Several populations approached the resistance level of the resistant check, ‘Zapalote Chico’, to ear damage in the field, but none equaled ‘Zapalote Chico’ in husk tightness. In 1987, the growth reduction of corn earworm larvae that were fed silk-diets of five populations was similar to the growth reduction of larvae that were fed a silk diet of ‘Zapalote Chico’. In 1988, only one population showed a growth reduction similar to that of ‘Zapalote Chico’, However, none of the populations approached the level of resistance exhibited by ‘Zapalote Chico’ when other antibiotic measurements such as development time and weight of pupae were considered. Based on data from both the field and laboratory, the resistance of recently released populations, ‘DDSA(C5)’ and ‘DDSB(C5)’, is primarily tolerance. Tolerance to ear damage in the field is also present in the 18 populations evaluated, although a few populations also possess a minor level of antibiosis.

Six recently released dent inbreds of corn, Zea mays L., with resistance to ear damage by corn earworm, Helicoverpa zea (Boddie), larvae were evaluated in the field and laboratory to determine mechanisms ofresistance. ‘GT117’ and ‘GT118’ were tolerant to corn earworm based on ear damage in the field and negligible biological effects on larvae that were fed fresh silks, silk diets, or both, in the laboratory. ‘GT114’ was susceptible to corn earworm in the field but highly antibiotic when larvae were fed silks in the laboratory. Development time to pupation for larvae that were fed silk diets of ‘GT114’ was more than twice that required for larvae that were fed check diets. Weight of pupae for larvae fed silk diets of ‘GT114’ was significantly less than for larvae that fed on any of the other entries, including the resistant check ‘Zapalote Chico’. ‘CT115’ possessed resistance to ear damage in the field as well as antibiosis in the silks in laboratory bioassays. ‘GT114’ will require additional breeding for husk characters to make the antibiotic silks effective against ear damage by larvae of the corn earworm in the field.

Aflatoxin B1 is a potent hepatacarcinogen that occurs in corn worldwide. The aflatoxin‐producing fungus Aspergillus flavus can grow and produce aflatoxin on corn preharvest and in storage. Within the U.S., aflatoxin contamination of preharvest corn has been reported in 23 states, and contamination is chronic in the southeastern U.S. where hot, drought conditions often favor the fungus and the production of aflatoxin. Management practices have been developed to reduce aflatoxin contamination, but in years when environmental conditions are extremely favorable for the fungus, no control strategy is effective. Resistance to aflatoxin accumulation appears to be heritable, but no commercial hybrids are available with adequate resistance. This review covers the epidemiology of A. flavus in preharvest corn from the infection process to the factors that influence aflatoxin formation. Also discussed are the problems associated with the development of resistant varieties and new strategies that are being de...

Improvement of resistance to insects while maintaining other desirable agronomic traits in maize (Zea mays L) populations has been an elusive goal for breeders. This study was conducted to evaluate crosses among 11 insect-resistant breeding populations to identify paired populations for selection or extraction of inbreds. A complete diallel was evaluated for lodging, plant height, ear height, yield, ear damage by insects, and maturity in a complex-block experiment with five replicates at two locations(.)

Corn (Zea mays L.) genotypes with resistance to Aspergillus flavus Link ex Fr. are needed to reduce aflatoxin contamination of grain. The primary objective of this study was to determine if the pinbar inoculation technique was effective in separating hybrids for resistance and susceptibility to kernel infection by A. flavus and aflatoxin contamination of the grain at a number of locations, but other inoculation techniques were evaluated at individual locations (...)

(...) Our primary objective was to assess the effect of planting date on aflatoxin concentrations in grain from knife-wound inoculated ears during a five-year study at Tifton, GA. We also evaluated the relationship between aflatoxin concentrations in maize grain and several environmental variables, especially those involving temperature and moisture. Five plantings of Funk's G-4507 were made in 1982, six in 1983 and 1984, and 10 in 1986 and 1987. Average planting dates varied from about 1 March to 1 August. Eight to 12 wound-inoculated, 10-ear samples were assayed per planting date. (...)

Two maize (Zea mays L.) breeding populations with very high concentrations of maysin, a silk-expressed flavone glycoside, were tested for their ability to resist ear damage by the corn earworm, Helicoverpa zea Boddie, under field conditions. Tests were conducted in 2000 and 2001 at multiple locations in Georgia. The high maysin populations, EPM6 and SIM6, as well as resistant and susceptible checks, were scored for silk-maysin content, H. zea damage, and husk characters. In 2000, there was a negative correlation between husk tightness and earworm damage at three of five locations, while there was no significant correlation between damage and maysin content at any location. In 2001, EPM6 and SIM6 had approximately ten times the maysin content of the low-maysin control genotypes; nevertheless, earworm damage to EPM6 and SIM6 was either greater than or not significantly different from the low-maysin genotypes at all locations. The resistant control genotype, Zapalote Chico, had significantly less earworm damage than EPM6 and SIM6 for both years at all locations. The results of this study highlight the importance of identifying and quantifying husk and ear traits that are essential to H. zea resistance in maize.

Aflatoxin, produced by Aspergillus flavus, is one of the most toxic and carcinogenic substances known and contaminates many agricultural commodities such as corn, peanuts, cottonseed, and tree nuts. The challenge to breeders/plant pathologists is to identify lines that have resistance to aflatoxin production. Maize population GT-MAS:gk has been identified and released as a germplasm with resistance to aflatoxin contamination. In the present study, we assessed genetic divergence in the GTMAS:gk population using restriction fragment length polymorphism (RFLP) DNA markers to survey 11 selfed inbred lines and conducted field evaluations for the dissimilarities in aflatoxin production among these inbred lines in comparison with a sister population, GT-MAS:pw,nf. The 11 selfed inbred lines were assayed for DNA polymorphism using 113 RFLP markers in 10 linkage groups covering 1,518.2 centimorgans (cM; unit of gene or chromosome size). Considerable variation among the inbreds was detected with RFLP markers, of which 42 probe-enzyme combinations gave 102 polymorphic bands. Cluster analysis based on genetic similarities revealed associations and variations among the tested lines. Three polymorphic groups were distinguished by cluster analysis. Two years of field evaluation data showed that aflatoxin concentrations among the lines were significantly different in both years (P < 0.001). Maturity data were also different. Thus, this study demonstrates that the maize population GT-MAS:gk is heterogeneous and that individuals may be different in resistance to A. flavus infection and aflatoxin production. Therefore, the most resistant lines should be inbred to increase homogeneity, and resistance should be confirmed through progeny testing.

A thin-layer chromatographic method has been developed for the analysis of maysin, a flavonoid C-glycoside, and its analogues, which have been implicated as resistance factors for the corn earworm (Helicoverpa zea, formerly Heliothis zea) and the fall armyworm (Spodoptera frugiperda). Boric acid present on the TLC plate enhances the fluorescence of luteolins, including maysin, and increases sensitivity to these compounds by a factor of 10 or more. Another resistance factor, found in smaller amounts, chlorogenic acid, interferes with maysin unless the developing solvent is made basic with dilute ammonium hydroxide or the TLC plate is treated with boric acid. Leaves, the major food source of the fall armyworm on corn, were not as readily analyzed because of interference due to streaking of the TLC plate.

(.)Spectrophotometrically analyzed maysin concentrations of individual silks were similar, but variances differed from those analyzed by HPLC. Spectrophotometric values were scattered for SC235×F44 and its related populations in 1983, but 1988 HPLC determinations produced interpretable data. Data from F6×F44 populations in 1983 suggested that F6 has a gene for reducing maysin content, but 1988 HPLC distributions indicated that the gene in F6 is dominant for low maysin content(.)

This study examined the distribution of two antifungal proteins, ribosome-inactivating protein (RIP) and zeamatin, in maize kernel tissues. Proteins were extracted from endosperm (including aleurone layer) and embryo tissues of imbibed maize kernels. Western blot analyses revealed that RIP-like protein was present at higher levels in endosperm than in embryo tissues, whereas zeamatin-like protein was more concentrated in embryo tissues than in endosperm tissues. However, there were three protein bands in the endosperm and two bands in the embryo that reacted to anti-RIP antibody in Western blot analyses. Tissue prints were conducted to localize the antifungal proteins. Imbibed kernels were cut longitudinally and transversely and blotted onto nitrocellulose membranes. Using antibodies against maize RIP and zeamatin, RIP was found primarily in the aleurone layer of the endosperm and glandular layer of scutellum, whereas zeamatin was located mainly in the kernel embryo. These results provide insight into the potential functions of these antifungal proteins, especially since the presence of RIP and zeamatin within maize kernels uniquely protects kernels from pathogens.

Kernels of corn population GT-MAS:gk, resistant to aflatoxin B1 production by Aspergillus flavus, and susceptible Pioneer hybrid 3154 were tested for aflatoxin when incubated under different relative humidities (RH). High aflatoxin levels were not detected in either genotype at RH < 91%. Resistance in GT-MAS:gk was consistent across all RH levels (91 to 100%) at which significant aflatoxin accumulation was detected. Aflatoxin levels in GT-MAS:gk averaged about 98% less than those in susceptible Pioneer 3154, which suggests that storage of this or other genotypes with similar resistance mechanisms may be possible under moisture conditions less exacting than are required with susceptible hybrids. Results for fungus growth and sporulation ratings on kernel surfaces were similar to those for aflatoxin levels. When kernels of both genotypes were preincubated 3 days at 100% RH prior to inoculation with A. flavus, germination percentages increased to very high levels compared to those of kernels that were not preincubated. In preincubated kernels aflatoxin levels remained consistently low in GT-MAS:gk but decreased markedly (61%) in Pioneer 3154. When eight susceptible hybrids were evaluated for aflatoxin accumulation in preincubated kernels, seven of these supported significantly lower toxin levels than kernels not subjected to preincubation. Average reduction across hybrids was 83%, and reductions within hybrids ranged from 68 to 96%. Preincubated kernels of one susceptible hybrid (Deltapine G-4666) supported aflatoxin levels comparable to those in resistant GT-MAS: gk. Data suggest that an inhibitor of aflatoxin biosynthesis may be induced during kernel germination. Possible mechanisms for embryo effects on resistance to aflatoxin accumulation are discussed.