Responses of Carpophilus hemipterus larvae and adults to selected secondary metabolites of maize

Selected secondary metabolites produced by maize (Zea mays L.) were tested for effects on larvae and adults of the dried-fruit beetle [Carpophilus hemipterus (L.)] in no-choice and choice assays. Feeding by adults and larvae was significantly reduced by ferulic acid and 6-methoxy-2-benzoxazolinone (MBOA) in no-choice assays. In choice assays, larvae and adults generally preferred diets with trans-cinnamic acid, quercetin, rutin, and thymol, but were repelled by diets with either ferulic acid or MBOA.

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Résumé Les réactions de larves et adultes du nitidulidé C. hemipterus (L.), vecteur de champignons produisant la mycotoxine, aux composés phénoliques caractéristiques, aux flavonoïdes et aux acides hydroxamiques, métabolites secondaires qui provoquent la résistance du maïs (Zea mays L.) ont été examinées au cours d'expériences avec et sans choix. L'alimentation des adultes et des larves est généralement réduite par les acides coumarique et férulique et par la 6-méthoxy-2-benzoxazolinone dans des expériences sans choix; les insectes évitent généralement les aliments qui contiennent ces produits. Quoi qu'il en soit, les larves préfèrent consommer d'autres aliments contenant les autres phénoliques ou flavonoïdes examinés. Les adultes sont plus inconstants dans leur choix alimentaires, mais préfèrent souvent des aliments contenant de la quercetine. Ainsi, des programmes de sélection orientés contre les principaux ravageurs comme Heliothis zea (Boddie) ou Ostrinia nubilalis (Hübner), impliquant la sélection de plantes à teneur élevée en acides phénolique ou hydroxamique, augmentant probablement aussi la résistance aux nitidulidés.

     

Biological activity of maize leaf extracts against the African armyworm,Spodoptera exempta

Maize (Zea mays L.) leaf tissue of cv Bastille and cv Michoacan 12 was extracted with n-hexane. The extracts were bioassayed against 5th instar African armyworm,Spodoptera exempta (Walker)(Lepidoptera: Noctuidae), by feeding the larvae on agar based media or sucrose impregnated glass fibre discs. The hexane extract of the ‘resistant’ cv Bastille exhibited feeding deterrency and toxicity which were not shown by the ‘susceptible’ cv Michoacan 12. The hexane extract of cv Bastille was adsorbed onto silica gel, the solution filtered off and the adsorbed component taken up into ethyl acetate. Bioassay of these fractions indicated that the toxic and deterrent action was retained in the ethyl acetate fraction. Preparative thin layer chromatography of the ethyl acetate fraction isolated two biologically active constituents. These were both growth inhibitors and lethal by ingestion to the 5th instar African armyworm. Implications for resistance in maize varieties to insect pests are discussed.     

The binding motifs for Ac transposase are absolutely required for excision of Ds1 in maize

A reverse genetic system for studying excision of the transposable elementDs1 in maize plants has been established previously. In this system, theDs1 element, as part of the genome of maize streak virus (MSV), is introduced into maize plants via agroinfection. In the presence of theAc element, excision ofDs1 from the MSV genome results in the appearance of viral symptoms on the maize plants. Here, we used this system to study DNA sequences requiredin cis for excision ofDs1. TheDs1 element contains theAc transposase binding motif AAACGG in only one of its subterminal regions (defined here as the 5′ subterminal region). We showed that mutation of these motifs abolished completely the excision capacity ofDs1. This is the first direct demonstration that the transposase binding motifs are essential for excision. Mutagenesis with oligonucleotide insertions in the other (3′) subterminal region resulted in elements with either a reduced or an increased excision efficiency, indicating that this subterminal region also has an important function.     

Mechanism of Ds1 excision from the genome of maize streak virus

Summary We have previously shown that the maize transposable element Ds1 introduced into maize plants by agroinfection can be excised from the genome of geminivirus maize streak virus (MSV). Excision depended strictly on the presence of an active Ac element in the plants. In this study, the excision products or footprints left in the MSV genome after Ds1 excision were extensively characterized and the effects of flanking sequences on Ds1 excision were analysed. Most types of footprints obtained were comparable to those described for Ds1 excision in the maize genome, and could be explained by the models proposed for excision of plant transposable elements. In two revertants, however, some terminal sequences of the Ds1 element were found to have been left behind at the excision site. The finding of this novel type of Ds1 footprint indicated that gene conversion events occurred during and/or after Ds1 excision from the MSV genome. A partial deletion of one copy of the 8 by duplications flanking the Ds1 element had no effect on the frequency or on the types of footprints of Ds1 excision from the MSV genome. Thus, the duplicated 8 by sequences flanking the transposable element are not involved in Ds1 excision. These results, as well as a statistical analysis of the modifications of the bases flanking the Ds1 element after excision, are discussed in terms of excision models.     

The Phylogenetics of Mycotoxin and Sclerotium Production in Aspergillus flavus and Aspergillus oryzae

Aspergillus flavus is a common filamentous fungus that produces aflatoxins and presents a major threat to agriculture and human health. Previous phylogenetic studies of A. flavus have shown that it consists of two subgroups, called groups I and II, and morphological studies indicated that it consists of two morphological groups based on sclerotium size, called “S” and “L.” The industrially important non-aflatoxin-producing fungus A. oryzae is nested within group I. Three different gene regions, including part of a gene involved in aflatoxin biosynthesis (omt12), were sequenced in 33 S and L strains of A. flavus collected from various regions around the world, along with three isolates of A. oryzae and two isolates of A. parasiticus that were used as outgroups. The production of B and G aflatoxins and cyclopiazonic acid was analyzed in the A. flavus isolates, and each isolate was identified as “S” or “L” based on sclerotium size. Phylogenetic analysis of all three genes confirmed the inference that group I and group II represent a deep divergence within A. flavus. Most group I strains produced B aflatoxins to some degree, and none produced G aflatoxins. Four of six group II strains produced both B and G aflatoxins. All group II isolates were of the “S” sclerotium phenotype, whereas group I strains consisted of both “S” and “L” isolates. Based on the omt12 gene region, phylogenetic structure in sclerotium phenotype and aflatoxin production was evident within group I. Some non-aflatoxin-producing isolates of group I had an omt12 allele that was identical to that found in isolates of A. oryzae.     

Inhibition of phosphoenolpyruvate carboxylase from maize by 2-phosphoglycollate

The phosphoenolpyruvate carboxylase from maize leaf was strongly inhibited by 2-phosphoglycollate. The pH of the reaction did not influence the extent of inhibition by 2-phosphoglycollate. The kinetic analysis of the inhibition data by Lineweaver-Burk method showed that 2-phosphoglycollate inhibition was competitive with respect to phosphoenolpyruvate. The secondary plot of the data showed nonlinearity indicating that there may be two 2-phosphoglycollate binding sites with Ki values of 0.4 mM and 0.16 mM. The biphasic nature of the inhibition was also evident when the data were plotted using the method of Dixon. 2-phosphoglycollate inhibition was uncompetitive with respect to Mg²⁺ suggestting that it binds only to enzyme-Mg²⁺ complex.     

Modification of maize phosphoenolpyruvate carboxylase by Woodward's reagentK

Maize leaf phosphoenolpyruvate carboxylase was completely and irreversibly inactivated by treatment with micromolar concentrations of Woodward's reagentK (WRK) for about 1 min. The inactivation followed pseudo-first-order reaction kinetics. The order of reaction with respect to WRK showed that the reagent causes formation of reversible enzyme inhibitor complex before resulting in irreversible inactivation. The loss of activity was correlated to the modification of a single carboxyl group per subunit, even though the reagent reacted with 2 carboxyl groups per protomer. Substrate PEP and PEP + Mg²⁺ offered substantial protection against inactivation by WRK. The modified enzyme showed a characteristic absorbance at 346 nm due to carboxyl group modification. The modified enzyme exhibited altered surface charge as seen from the elution profile on FPLC Mono Q anion exchange column. The modified enzyme was desensitized to positive and negative effectors like glucose-6-phosphate and malate. Pretreatment of PEP carboxylase with diethylpyrocarbonate prevented WRK incorporation into the enzyme, suggesting that both histidine and carboxyl groups may be closely physically related. The carboxyl groups might be involved in metal binding during catalysis by the enzyme.     

Polyamine metabolism in maize tumors induced by Ustilago maydis

Alterations occurring in polyamine metabolism of maize in tumors formed during the interaction with the biotrophic pathogenic fungus Ustilago maydis were analyzed. During the process, a striking increase in maize polyamine biosynthesis, mainly free and conjugated putrescine occurred in the tumors induced by the fungus, and in the neighbor plant tissues. This increase correlated with an activation mainly of Adc, Samdc1, Zmsamdc2 and Zmsamdc3, but not of Zmodc, Zmspds1 and Zmspds2 genes, and an elevation in arginine decarboxylase activity, confirming a predominant role of this enzyme in the process. Evidences for a possible contribution of spermidine and spermine degradation by polyamine oxidase activity, probably related to cell wall stiffening or lignification during tumor growth, were also obtained. It is suggested that polyamines, mainly putrescine, might play an active role in the pathosystem maize-U. maydis.     

Binding sites for maize nuclear proteins in the subterminal regions of the transposable elementActivator

Genetic data suggest that transposition of the maize elementActivator (Ac) is modulated by host factors. Using gel retardation and DNase I protection assays we identified maize proteins which bind to seven subterminal sites in both ends ofAc. Four DNase I-protected sites contain a GGTAAA sequence, the other three include either GATAAA or GTTAAA. The specificity of the maize protein binding toAc was verified by using a synthetic fragment containing four GGTAAA motifs as probe and competitor in gel retardation assays. All seven binding sites are located within regions requiredin cis for transposition. A maize protein binding site with the same sequence has previously been identified in the terminal inverted repeats of the maizeMutator element. Thus, the protein, that recognizes this sequence is a good candidate for a regulatory host factor forAc transposition.     

Newly activated germinal Uq elements in maize are clustered on one linkage group independently of the standard Uq element

Summary Allelism tests between the standard Uq element (Uq1) and five newly activated germinal Uq elements (Uq2, Uq3, UQ4, Uq5, and Uq6) demonstrate that these new Uq elements are independent of Uq1. Gametes that either contain one Uq or various combinations of two different and phenotypically distinguishable Uq elements, have been constructed either with or without the a-ruq reporter allele. Genetic analyses of the progenies of the gametes (using the standard a-ruq tested line as the other parent) have indicated that (i) each Uq element, when present alone, has the capacity to express full activity except when a secondary transposition or loss of activity has occurred; (ii) all five new Uq elements are independent of Uq1 with respect to transposition activity; and (iii) these newly originated Uqs are clustered on one linkage group. Uq2 is allelic to Uq4, and Uq3 is allelic to Uq5, whereas Uq6 is linked to both allelic pairs. A putative linkage map of these Uq elements is presented. In reciprocal crosses there is a striking difference in phenotypic segregation of Uq; when transmitted via the male parent Uq loses full expression capacity.     

Breeding for resistance to downy mildews and stalk rots in maize

Summary The present review includes information on distribution, symptoms, inoculation techniques, disease rating, sources of resistance, genetics of resistance, breeding approaches for resistance, and the present status of resistance breeding with respect to Sclerophthora and Peronosclerospora downy mildews and Erwinia, Cephalosporium and Fusarium stalk rots. Some suggestions highlighting research gaps pertinent to future breeding strategies are mentioned.Publication No. 2993, Experiment Station, GB Pant University of Agriculture and Technology, Pantnagar, India     

Agronomic evaluation of inbred lines derived from tissue cultures of maize

Summary Tissue culture-induced variation has been proposed as a novel source of variation for crop improvement. In maize (Zea mays L.), chromosome aberrations and qualitative genetic variants have been induced during in vitro culture. The proportion of regenerated plants carrying such variants has been shown to increase with culture age. The objective of this research was to evaluate the relationship between culture age and somaclonal variation for several agronomic traits. Six sib-pollinated ears of S₀ (F₂) plants in four OH43 ms/A188 populations each provided control seed and embryos for culture initiation. S₂ lines derived from control seed and from plants regenerated 4 and 8 months after culture initiation were grouped according to their source ear and grown in 6 separate trials. A total of 305 tissue culture-derived and 48 control lines were evaluated as lines per se and in a testcross at each of three locations. Tissue culturederived lines and their testcrosses generally had lower grain yield and moisture. Since grain yield and moisture were not positively correlated in any trial, the highest yielding lines could be selected without increasing grain moisture. Grain yield and plant height tended to decrease with culture age. Although tissue culture-derived lines were, on average, inferior, the highest yielding line per se in three of six trials and the top-ranked line in five of six trials for yield and moisture were derived from tissue culture. The results indicate that tissue culture may generate variation for agronomic traits. Some of the variation, particularly the trend towards earlier maturity, could be useful. However, this method may require screening large populations because of the tendency to generate a large proportion of inferior lines.Contribution from Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108. Minnesota Agric. Exp. Stn. Scientific Journal Series Paper No. 15,172     

Molecular analysis of the dhp1+ gene of Schizosaccharomyces pombe: an essential gene that has homology to the DST 2 and RAT 1 genes of Saccharomyces cerevisiae

We report here the first cloning of a chalcone flavonone isomerase gene (CHI) from maize. Northern blot experiments indicate that the maize CHI gene (ZmCHI1) is regulated in the pericarp by the P gene, a myb homologue. The ZmCHI1 gene encodes a 24.3 kDa product 55% and 58% identical to CHI-A and CHI-B from Petunia, respectively. This maize CHI gene has four exons and an intron-exon structure identical to the CHI-B gene of Petunia hybrida. RFLP mapping data indicate that some inbred lines contain two additional CHI-homologous sequences, suggesting an organization more complex than that found in Petunia or bean. The possibility that the additional CHI-homologous sequences are responsible for the lack of CHI mutants in maize will be discussed.     

Reversion of Texas male-sterile cytoplasm maize in culture to give fertile,T-toxin resistant plants

Summary Plants carrying Texas male-sterile (Tms) cytoplasm are normally sensitive to Drechslera maydis T-toxin. Tissue cultures were initiated from immature embryos of maize carrying Tms-cytoplasm, and plants were regenerated after selection for resistance to T-toxin. Fertile, T-toxin resistant plants were obtained from the unselected control cultures as well as from the selected material. In addition, one regenerant from an unselected culture was fertile and T-toxin sensitive. The progeny of the regenerants showed the phenotype of the female parent with respect to pollen-fertility, and T-toxin resistance. The data are consistent with the heritable changes observed being the result of the expression of an altered mitochondrial genome.     

Evaluation of three test statistics used to identify maize inbred lines with new favorable alleles not present in elite single cross

Summary The identification of inbred lines useful for improvement of an elite single cross hybrid is an essential part of a pedigree maize (Zea mays L.) breeding program. The objectives of this study were to identify lines that could be useful for improvement of hybrid B73 × Mo17 and to relate the values of estimators of new favorable alleles with test cross yields. Crosses of parents of hybrid B73 × Mo17 with 10 public lines from the United States (US), and 14 Maize Research Institute Zemun Polje proprietary lines (lines per se, and test crosses from 3 F₂ populations) were evaluated at 4 locations in Yugoslavia in 1986. Significant differences in grain yield were found among lines in minimally biased estimates of favorable alleles (G) present in a donor inbred but not present in a B73 × Mo17, in minimum upper bound (UBND) estimates and in predicted three-way performance (PTC). Twenty-one lines had a significant number of dominant favorable alleles for grain yield not present in B73 × Mo17. The highest values for all estimators of new favorable alleles were found for donor lines which belonged to different heterotic groups than the B73 and Mo17. For most of the inbreds, the (C + F) – (D + E) statistics agreed with predigree information. Simultaneous increases in grain yield and decreases in grain moisture content for B73 × Mo17 are possible with several donor inbred lines. All of the lines with a high number of new favorable alleles for grain yield not present in B73 × Mo17 had negative D (F)-G values for low plant height. Line N152 had the most new favorable alleles for grain yield not present in single cross B73 × Mo17. Population (N152 × Mo17) F₂ had the highest difference of observed test cross means from check mean, the most test crosses with significantly higher yields than the check, and the largest estimate of number of segregating loci.This project was partly supported by the United States Department of Agriculture and Republic Funds for Scientific Work of Serbia through funds available to the United States-Yugoslav Joint Board on Scientific and Technological Cooperation. Project No. JFP 662     

Improved Agrobacterium-mediated transformation of three maize inbred lines using MS salts

Transformation technology as a research or breeding tool to improve maize is routinely used in most industrial and some specialized public laboratories. However, transformation of many inbred lines remains a challenging task, especially when using Agrobacterium tumefaciens as the delivery method. Here we report success in generating transgenic plants and progeny from three maize inbred lines using an Agrobacterium-mediated standard binary vector system to target maize immature embryos. Eleven maize inbred lines were pre-screened for transformation frequency using N6 salts. A subset of three maize inbred lines was then systematically evaluated for frequency of post-infection embryogenic callus induction and transformation on four media regimes: N6 or MS salts in each of two distinct media backgrounds. Transgenic plants recovered from inbred lines B104, B114, and Ky21 were analyzed for transgene integration, expression, and transmission. Average transformation frequencies of 6.4% (for B104), 2.8% (for B114), and 8% (for Ky21) were achieved using MS salts. Availability of Agrobacterium-mediated maize inbred line transformation will improve future opportunities for maize genetic and functional genomic studies.     

Cleavage polyembryony in maize

Summary Two types of cleavage polyembryony are described in the inbred line VIR 17 of maize. Suspensorial embryony was observed to occur spontaneously. Typical cleavage of the zygotic proembryo occurred spontaneously, but could also be induced by treating the developing caryopses with 2,4-Dichlorophenoxyacetic acid (2,4-D) on the second day after pollination. 2,4-D was active as a decorelative factor also evoking the expression of totipotency in individual proembryonal cells.     

Uptake and distribution of cadmium in maize inbred lines

Genotypic variation in uptake and distribution of cadmium (Cd) was studied in 19 inbred lines of maize (Zea mays L.). The inbred lines were grown for 27 days on an in situ Cd-contaminated sandy soil or for 20 days on nutrient solution culture with 10 µg Cd L^-1. The Cd concentrations in the shoots showed large genotypic variation, ranging from 0.9 to 9.9 µg g^-1 dry wt. for the Cd-contaminated soil and from 2.5 to 56.9 µg g^-1 dry wt. for the nutrient solution culture. The inbred lines showed a similar ranking for the Cd concentrations in the shoots for both growth media (r²=0.89). Two main groups of inbreds were distinguished: a group with low shoot, but high root Cd concentrations (shoot: 7.4±5.3 µg g^-1 dry wt.; root: 206.0±71.2 µg g^-1 dry wt.; shoot Cd excluder) and a group with similar shoot and root Cd concentrations (shoot: 54.2±3.4 µg g^-1 dry wt.; root: 75.6±11.2 µg g^-1 dry wt.; non-shoot Cd excluder). The classification of the maize inbred lines and the near equal whole-plant Cd uptake between the two groups demonstrates that internal distribution rather than uptake is causing the genotypic differences in shoot Cd concentration of maize inbred lines. Zinc (Zn), a micronutrient chemically related to Cd, showed an almost similar distribution pattern for all maize inbred lines. The discrepancy in the internal distribution between Cd and Zn emphasizes the specificity of the Cd distribution in maize inbred lines.