Browning-associated mechanisms of resistance to insects in corn callus tissue

Callus of the fungus- and insect-resistant corn inbred, Mp313E, of differing ages and degrees of brownness was tested for effects on neonate larvae of the corn earworm,Helicoverpa zea. Older, browner, 8-week-old callus caused significantly higher mortality ofH. zea after two days compared to larvae fed 5-week-old callus (brown or not) or pinto bean diet. Larvae fed on the 5-week-old callus were significantly smaller after nine days compared to those fed on nonbrown callus of the same age. Ferulic acid,p-coumaric acid, and sinapic acid, corn allelochemicals associated with cell walls, were oxidized (produced a brown product) up to 10-fold more rapidly by peroxidase preparations of the brown callus compared to nonbrown callus. Peroxidase isozymes from both types of brown callus separated by conventional gel electrophoresis and isoelectric focusing showed more intense anodic/acidic bands compared to peroxidase isozymes separated from the nonbrown callus. Some additional peroxidase isozymes were also present in the brown callus and were able to oxidize ferulic acid. Conventional extraction techniques for phenolic acids indicated free ferulic acid levels were similar for same-age callus, but somewhat less for older callus; no freep-coumaric acid was detected. For esterified and insoluble-bound phenolic content, the level ofp-coumaric acid was higher than that of ferulic acid and was somewhat greater in insoluble-bound fractions for the old callus compared to the younger callus. Color ratings of aqueous extracts and phenolic acid extract residues indicated much brown material remained, which was proportional in color to the original material. The differential insect response to, and peroxidase activity of, brown vs. nonbrown callus were similar to results previously noted for brown pericarps of corn compared to nonbrown pericarps. This information suggests the peroxidases are contributing to the browning through oxidation of phenolic acids and thereby enhancing resistance to insects.     

Factors contributing to resistance of exotic maize populations to maize weevil,Sitophilus zeamais

Factors contributing to resistance of maize to infestation by the corn weevilSitophilus zeamais were investigated in four populations of indigenous and improved maize from Belize. Resistance was related to the antifeedant properties of grain as well as sugar content and mechanical hardness. Grain extracts of all populations of maize significantly reduced insect feeding on treated artificial diets when compared to control diets. Consumption of treated diets was negatively correlated with phenolic content of the grain extract. An analysis by GC-MS indicated that ferulic acid andp-coumaric acid were the principal phenolics present in the extracts, and insect feeding was strongly deterred when pure substances were added to insect diets. Fluorescence associated with ferulic acid and related compounds in grain sections was located in the pericarp and aleurone layer and was especially intense in the most resistant grain variety.Contribution No. 838.     

Identification of food volatiles attractive to dusky sap beetle,Carpophilus lugubris (Coleoptera: Nitidulidae)

The chemical mediation of host-finding was investigated for the dusky sap beetle,Carpophilus lugubris Murray. GC-MS analysis of the headspace volatiles above whole-wheat bread dough inoculated with baker's yeast, a substrate previously determined to be an effective attractant, revealed seven major components in the following order of decreasing concentration: ethanol, acetaldehyde, 2-methylpropanol, 3-methylbutanol, propanol, 2-methylbutanol, and ethyl acetate. Solutions of these seven compounds blended so as to mimic the odor of whole-wheat bread dough elicited upwind orientation from 1.8 m in a wind tunnel byC. lugubris at a level comparable to that elicited by the bread dough. A series of bioassays investigating the role of individual components from the synthetic blend determined that all seven compounds contributed to behavioral activity; however, the simplest blend evoking attraction comparable to bread dough included acetaldehyde, ethyl acetate, 2-methylpropanol, and 3-methylbutanol. Of these compounds, acetaldehyde was essential, ethyl acetate was interchangeable with ethanol or partially replaceable with propanol, and 2-methylpropanol and 3-methylbutanol were partially replaceable with a combination of the other alcohols. Headspace volatiles above aseptic or fungus-inoculated tomato, banana, sweet corn, and strawberry were also qualitatively and quantitatively analyzed. This study suggests thatC. lugubris locates its food sources by response to variable blends of common volatile constituents of plants and fungi.     

Microbial decomposition of ferulic acid in soil

The suppression of plant growth by different phenolic acids is well known. This work was designed to determine if ferulic acid, a known phenolic inhibitor of plant growth, accumulates in the soil and if soil microorganisms could be isolated that metabolize it. Over 99% of the extractable ferulic acid was lost from decaying hackberry leaves in 300 days. During this time the amount in the top 15 cm of soil remained fairly constant at about 30 ppm, except for the March sample which was significantly higher than the rest. Addition of ferulic acid to soil caused an increase in CO₂ evolution and in numbers of a select group of microorganisms.Rhodotorula rubra andCepnalosporium curtipes, which actively metabolize ferulic acid, were isolated, but the metabolic pathways employed appear to be different from the reported one. The reported pathway for ferulic acid breakdown is ferulic acid to vanillic acid to protocatechuic acid to -keto-adipic acid.Rhodotorula Rubra was found to convert ferulic acid to vanillic acid, but no evidence was found for utilization of the rest of the pathway.Cephalosporium curtipes appears to use a different pathway or to metabolize intermediate compounds rapidly without accumulating them, because no phenolic compounds were found during the breakdown of ferulic acid. The presence in the soil of microorganisms that metabolize ferulic acid and other phenolic acids is ecologically significant because such organisms prevent long-term accumulations of these substances, which are toxic to many other microorganisms and higher plants.     

Aggregation pheromone ofCarpophilus antiquus (Coleoptera: Nitidulidae) and kairomonal use ofC. lugubris pheromone byC. antiquus

Males ofCarpophilus antiquus Melsheimer (Coleoptera: Nitidulidae) emit an aggregation pheromone that was found to be a novel hydrocarbon, (3E,5E,7E,9E)-6,8-diethyl-4-methyl-3,5,7,9-dodecatetraene. A synthetic scheme and spectra (mass and proton NMR) are given for the compound. Beetles produced the pheromone when feeding on a variety of media, including the brewer's yeast-based artificial diet, fermenting whole-wheat bread dough, corn, and prunes; live baker's yeast was generally added to the food media. Males held individually produced, on average, 25 × more pheromone per beetle than males held in groups of 10 or more. Pheromone was not produced until males were at least 5 days old but was still detected from the oldest beetles tested (47 days). In field tests, the pheromone was attractive to both sexes ofC. antiquus, and it was synergized by food volatiles: A combination of pheromone and fermenting whole wheat dough attracted 2.5× more beetles than pheromone alone, but dough by itself was not significantly more attractive than the control. Semiochemical interactions were studied amongC. antiquus and two other sympatric species for which pheromones are known,C. lugubris Murray andC. freemani Dobson.C. antiquus responded readily to the pheromone ofC. lugubris, but all other interspecific responses to the pheromones were weak. In a sample of naturally infested corn ears, the presence ofC. antiquus was strongly associated with the presence ofC. lugubris, as would be expected if the pheromone ofC. lugubris serves as a kairomone forC. antiquus.     

Correlation of phenolic acid content of maize to resistance toSitophilus zeamais,the maize weevil,in CIMMYT'S collections

The (E)-ferulic acid content of the grain of nine populations of land races of maize derived from CIMMYT's collections was found to be negatively correlated to susceptibility characteristics towards the maize weevilSitophilus zeamais. Correlation coefficients for six susceptibility parameters and (E)-ferulic acid content were significant and ranged from –0.58 to –0.79. A multiple regression analysis by the SAS forward procedure using the primary seed characteristics associated with susceptibility indicated that the ferulic acid content was the only significant factor in explaining variation in at least two susceptibility parameters: the Dobie index and adult preference. In 15 CIMMYT pools, correlations between four susceptibility parameters and (E)-ferulic acid content were also significant (–0.76 to –0.81). The results suggest that phenolic acid content is a leading indicator of grain resistance or susceptibility to insects and may represent a newly identified mechanism of resistance.     

Allelopathy in agroecosystems: Wheat phytotoxicity and its possible roles in crop rotation

The germination rates of cotton and wheat seeds were significantly affected by various extracts of wheat mulch and soils collected from the wheat field. This toxicity was even more pronounced against seedling growth. Five allelochemics: ferulic,p-coumaric,p-OH benzoic, syringic, and vanillic acids, were identified from the wheat mulch and its associated soil. Quantitatively, ferulic acid was found at higher concentrations thanp-coumaric acid in the soil. Various concentrations of ferulic andp-coumaric acids were toxic to the growth of radish in a bioassay. The functional aspects of allelochemic transfer from decaying residue to soil and the subsequent microbial degradation within agroecosystems are discussed, particularly as they relate to wheat crop rotation, with wheat and cotton, in Pakistan.     

Simultaneous effects of ferulic andp-coumaric acids on cucumber leaf expansion in split-root experiments

Experiments were conducted to determine how plant responses to mixtures of allelochemicals may change as the proportion of roots in contact with allelochemicals is modified. Thirteen-day-old cucumber seedlings were treated with ferulic and/orp-coumaric acid in a split-root nutrient culture system. Leaf areas were determined just prior to treatment and at harvest, 24 hr after treatment. Ferulic acid was more inhibitory to cucumber leaf expansion thanp-coumaric acid. The effects of ferulic andp-coumaric acids on leaf expansion were additive. For individual acids, mixtures of ferulic andp-coumaric acids in the same container and combinations of ferulic andp-coumaric acids in separate containers, the inhibition of leaf expansion was directly related to the product of the concentration of the acid(s) and the proportion of roots treated with the acid(s). Pretreatment with 0.2 or 0.4 mM ferulic acid and subsequent treatment with 0.5 mM ferulic acid failed to show evidence of acclimation by cucumber seedlings.The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of products named, nor criticism of similar ones not mentioned.     

Effects of ferulic acid onGlomus fasciculatum and associated effects on phosphorus uptake and growth of asparagus (Asparagus officinalis L.)

The effect of ferulic acid, an allelochemical produced by asparagus, on hyphal elongation and colonization of asparagus byG. fasciculatum was studied. Spore germination in vitro was not affected, but hyphal elongation decreased significantly with increasing ferulic acid concentration. In the greenhouse, mycorrhizal colonization of roots and growth of mycorrhizal asparagus decreased significantly with increasing ferulic acid concentration, while growth of nonmycorrhizal plants was not affected by ferulic acid. Although plant tissue phosphorus levels were not affected by ferulic acid or mycorrhizal status, ferulic acid inhibition of hyphal elongation in vitro and fungal root colonization in vivo suggests that production of ferulic acid by asparagus reduces the symbiotic effectiveness of the fungus and subsequently reduces plant growth.Michigan Agricultural Experiment Station Journal Article Number 13065.     

Fatty acid composition of two new pepper varieties (Capsicum annuum L. cv. Jaranda and Jariza). Effect of drying process and nutritional aspects

Composition in fatty acids of the pericarp and seeds of two new pepper fruits (Capsicum annuum L. cv. Jaranda and Jariza) and the effects of different processing stages on the fatty acid composition of these tissues and of paprika are shown. In the pericarp the polyunsaturated fatty acids (PUFA), linoleic and linolenic, both in the same proportion, are the major acids; in the seed, linoleic is in a very high concentration as compared to in the pericarp. In the pericarp, a storage zone of carotenoid pigments, linolenic acid does not participate in the carotenoid esterification process. From the different lipid patterns, nutritional aspects are deduced. In the drying step the concentrations of constituent fatty acids are constant in the seed, while in the pericarp there is a net increase in the total content of fatty acids.     

Physiological, Nutritional, and Biochemical Bases of Corn Resistance to Foliage-Feeding Fall Armyworm

Three corn (Zea mays) germplasm lines [i.e., Ab24E (susceptible control), Mp708 (resistant control), and a locally selected partial inbred line FAW7050 (resistant)] were examined for Spodoptera frugiperda (J.E. Smith; Lepidoptera: Noctuidae) resistance. Nutritional [i.e., total protein content, amino acids, glucose, total nonstructural carbohydrates (TNC), protein to TNC (P/C) ratios] and biochemical (i.e., peroxidase and lipoxygenase 3) properties in the seedlings of these corn lines were examined to categorize resistance mechanisms to S. frugiperda. Physiological changes in photosynthetic rates also were examined in an attempt to explain nutritional and biochemical dynamics among corn germplasm lines and between insect-infested and noninfested corn plants within a germplasm line. Results indicated that S. frugiperda larvae survived better and developed faster in susceptible Ab24E than in resistant FAW7050 or Mp708. The three germplasm lines differed in resistance mechanisms to S. frugiperda, and the observed patterns of resistance were probably collective results of the P/C ratio and defensive proteins. That is, the susceptibility of Ab24E to S. frugiperda was due to a high P/C ratio and a low level of induced defensive compounds in response to insect herbivory, while the resistance of FAW7050 resulted from elevated defensive proteins following insect herbivory, low P/C ratio, and elevated defensive proteins in Mp708 contributed to its resistance to S. frugiperda. The elevated protein amounts in resistant Mp708 and FAW7050 following S. frugiperda injury were likely due to greater conversion of photosynthates to defensive proteins following the greater photosynthetic rates in these entries. Greater photosynthetic capacity in Mp708 and FAW7050 also led to higher amino acid and glucose contents in these two lines. Neither amino acid nor lipoxygenase 3 played a critical role in corn resistance to S. frugiperda. However, high inducibility of peroxidase may be an indicator of S. frugiperda susceptibility as observed elsewhere.     

Effects of phenolic acids on germination and early growth of herbaceous woodland plants

Four herbaceous plant species from woodland (clearings),Deschampsia flexuosa, Scrophularia nodosa, Senecio sylvaticus, andChamaenerion angustifolium, were tested for their sensitivity to phenolic acids. Seven commonly occurring phenolic compounds were used in a germination experiment in concentrations ranging from 0.01 to 10 mM, i.e., salicylic,p-hydroxybenzoic, syringic, caffeic, vanillic,p-coumaric, and ferulic acids. Germination was delayed rather than inhibited. Radicle elongation was strongly affected; at lower concentrations stimulatory effects were observed, whereas at high concentrations radicle elongation was severely reduced. Salicylic acid was the most effective phenolic compound, whereas caffeic acid caused no effects. Early growth was studied in more detail in a second experiment withDeschampsia flexuosa andSenecio sylvaticus and the phenolic acids, ferulic and p-coumaric acid. Primary root length, number and length of secondary roots, and dry weight were stimulated at 0.01 mM but were inhibited at 10 mM of both compounds. The results are discussed in view of the allelopathic relations between trees and herbaceous understory vegetation.     

Effects of Microbial Utilization of Phenolic Acids and their Phenolic Acid Breakdown Products on Allelopathic Interactions

Reversible sorption of phenolic acids by soils may provide some protection to phenolic acids from microbial degradation. In the absence of microbes, reversible sorption 35 days after addition of 0.5–3 mol/g of ferulic acid or p-coumaric acid was 8–14% in Cecil A_p horizon and 31–38% in Cecil B_t, horizon soil materials. The reversibly sorbed/solution ratios (r/s) for ferulic acid or p-coumaric acid ranged from 0.12 to 0.25 in A_p and 0.65 to 0.85 in B_t horizon soil materials. When microbes were introduced, the r/s ratio for both the A_p and B_t horizon soil materials increased over time up to 5 and 2, respectively, thereby indicating a more rapid utilization of solution phenolic acids over reversibly sorbed phenolic acids. The increase in r/s ratio and the overall microbial utilization of ferulic acid and/or p-coumaric acid were much more rapid in A_p than in B_t horizon soil materials. Reversible sorption, however, provided protection of phenolic acids from microbial utilization for only very short periods of time. Differential soil fixation, microbial production of benzoic acids (e.g., vanillic acid and p-hydroxybenzoic acid) from cinnamic acids (e.g., ferulic acid and p-coumaric acid, respectively), and the subsequent differential utilization of cinnamic and benzoic acids by soil microbes indicated that these processes can substantially influence the magnitude and duration of the phytoxicity of individual phenolic acids.     

Effects of ferulic andp-coumaric acids in nutrient culture of cucumber leaf expansion as influenced by pH

Cucumber seedlings were grown in 5 mM MES [2-(N-morpholino)ethanesulfonic acid] -buffered nutrient solutions adjusted to a pH of 5.5, 6.25, or 7.0. Nutrient solutions were changed on alternate days. Seedlings were treated for a two-day period with various concentrations (0–1 mM) of ferulic acid,p-coumaric acid, or mixtures of these phenolic acids when 16 days old. Leaf growth, dry weight, and water utilization of the seedlings; pH of the solutions; and disappearance of the phenolic acids from nutrient solutions were monitored. Leaf area expansion of cucumber seedlings was inhibited by both ferulic andp-coumaric acid, and the magnitude of these inhibitions was influenced by concentration and pH. Inhibition of leaf area expansion was greater at pH 5.5 and nominal at pH 7.O. Ferulic acid was more inhibitory thanp-coumaric acid. The effect of pH on growth was best described by data for mean relative rates of leaf expansion. For example, the mean relative rates of leaf expansion by both acids at 0.5 mM for the 16- to 18-day growth period (treatment period) were reduced by 45, 31, and 8% for the pH 5.5, 6.25, and 7.0 treatments, respectively. The dry weight of seedlings at harvest (day 22) was significantly reduced for seedlings grown in the pH 5.5 and 6.25 treatments, but not for the pH 7.0 treatment. There was, however, one exception; the dry weight of seedlings treated withp-coumaric acid solutions adjusted to a pH of 5.5 was not significantly reduced. Water utilization by the seedlings was reduced by both ferulic andp-coumaric acid. Again, the impact of ferulic acid was greater thanp-coumaric acid. The effect of ferulic acid on water utlization decreased with increasing pH of the nutrient solution. The pH effects were not so consistent forp-coumaric acid. The effects of equimolar mixtures of the two phenolic acids were additive for all variables measured. There was a linear correlation between mean relative rates of leaf expansion and water utilization.Paper No. 9693 of the Journal Series of the North Carolina Agricultural Research Sevice, Raleigh, North Carolina. Mention of a trademark or a propriety product does not constitute a guarantee or warranty of the product by the Agricultural Research Service and does not imply its approval to the exclusion of other products that may be suitable.     

Phenylacetic acid as a phytotoxic compound of corn pollen

Phenylacetic acid (PAA), one of the phytotoxic compounds in corn (Zea mays) pollen, was identified by GC-MS and by direct comparison with a pure commercial sample of PAA. Bioassays were carried out by testing whole pollen, methylene chloride extract of the pollen, and pure PAA on germination and radical growth ofAmaranthus leucocarpus andEchinochloa crusgalli. The effect of corn pollen was compared with that ofZea mexicana (Teosinte), one of the wild relatives of cultivated maize.     

Ultrasonic extraction of ferulic acid from Ligusticum chuanxiong

The extraction of ferulic acid, a pharmacologically active ingredient from the root of Ligusticum chuanxiong, with ultrasonic extraction was investigated. Percolation and supercritical fluid extraction (SFE) were employed to make comparisons with ultrasonic extraction. Three variables, which included the concentration of solvent, the ratio of solvent volume/sample (mL/g), and extraction time, were found to have a great influence on ultrasonic extraction. The optimum extraction was with pure ethanol with a solvent volume/sample ratio 8:1 (mL/g) and extraction time of 30 min. Under the optimum extraction conditions, the extraction yield could reach 8.8% which was higher than that using SFE with ethanol as co-solvent and a content of ferulic acid of 0.7%; both the yield and the content were higher than those obtained by percolation. Ultrasonic extraction significantly shortened the time required for the extraction process. Overall, ultrasonic extraction was shown to be highly efficient in the extraction of ferulic acid from Ligusticum chuanxiong.     

Possible ecological significance of within-fruit and seed furocoumarin distribution in twoPsoralea species

Furocoumarin distribution patterns in the fruits and seeds ofPsoralea macrostachya andP. onobrychis were investigated. Both species contain the linear furocoumarin psoralen and its angular isomer, angelicin. In the monospermous indehiscent fruit ofP. macrostachya, furocoumarins occur in the pericarp and all seed parts. InP. onobrychis, the pericarp of which is easily detached at ripeness, no furocoumarins were found in the pericarp tissues and only traces occur in the embryo axis; cotyledons are the preferential accumulation site. The within-fruit and -seed furocoumarin variations associated with the developmental stages of fruit were followed inP. onobrychis, in view of changes in the defensive value of the pericarp before and after ripening. Rapid furocoumarin biosynthesis after fertilization was observed in both pericarp and seed; ripening is associated with furocoumarin decrease in the seed and complete disappearance in the pericarp tissues. Such findings are consistent with the chemical defense role of these substances. The cooccurrence of linear and angular isomers seems to be a chemical marker of the genusPsoralea: the biosynthetic pathway leading to the angular isomer as an evolutionary response to selective pressure from herbivore insects is suggested.     

Comparison of volatiles from beetle-transmittedCeratocystis fagacearum and four non-insect-dependent fungi

Ceratocystis fagacearum (Bretz) Hunt is the causative agent of oak wilt disease, which is transmitted primarily by nitidulid beetles. This fungus was compared with four non-insect-dependent fungi for their volatile profiles using gas chromatography and mass spectrometry (GC-MS) and for their attractiveness to nitidulids using a wind-tunnel bioassay. The four additional fungi includedXerula radicata Sing,Pluteus atricapillus Kumm,Tyromyces chioneus Karst, andBotrytis cinerea. Nitidulids have been reported in association with each of these fungi, but unlikeC. fagacearum, they are dispersed primarily by wind or rain. Significant attraction of three nitidulid species,Carpophilus hemipterus (Linne),C. lugubris Murray, andStelidota geminata (Say) was elicited byC. fagacearum and to a lesser extent byX. radicata, but not by the others. A comparison of headspace volatile profiles showed that the odor ofC. fagacearum was the strongest, both with regard to the number of components and in their rates of production. Chemical characterization of the headspace profile ofC. fagacearum revealed 16 components: one aldehyde, one ketone, five alcohols, and nine esters. These components were all common fruit-odor constituents and many of them were previously shown to be attractive to nitidulid beetles. The results of this study suggest that, by mimicking food odors,C. fagacearum odor is an adaptation for attracting nitidulid and possibly other insect vectors.     

Inducible Production of Phenolic Acids in Wheat and Antibiotic Resistance to Sitodiplosis mosellana

Larvae of the wheat midge, Sitodiplosis mosellana (Géhin) feed on the surface of wheat seeds for about 10 days beginning when pollination occurs. A few wheats have a high level of antibiotic resistance to the larvae, which suppresses their growth and development. Nearly all larvae develop successfully on susceptible wheats. Analysis by HPLC of seed extracts produced by alkaline hydrolysis revealed rapid changes in the levels of p-coumaric and ferulic acids during early seed development. Seed infested by wheat midge larvae showed induced changes in the dynamics of these phenolic acids. The most resistant wheats had a higher constitutive level and a more rapid induction of ferulic acid than susceptible wheats. Levels of ferulic acid exceeding 0.35 g/g fresh weight were associated with a high mortality of newly hatched larvae. In one wheat line, resistance also was associated with induced production of p-coumaric acid. The induction of ferulic acid was similar in wheat from the laboratory and field, except in one resistant wheat that produced higher levels in the field. In ripe seeds, resistant and susceptible wheats had similar levels of phenolic acids.