CHARACTERIZATION OF A SALIVARY LYSOZYME IN LARVAL <Emphasis Type="Italic">Helicoverpa zea</Emphasis>

The cDNA sequence of a salivary lysozyme in Helicoverpa zea (Lepidoptera: Noctuidae) was determined. The full-length cDNA is 1,032 bp, and it encodes a protein of 142 amino acids. This lysozyme has 90% identity with Heliothis virescens lysozyme and 76% identity with Manduca sexta lysozyme. There is a signal peptide of 20 amino acids at the N-terminus. The mature protein is about 14.4 kDa without the signal peptide. The pI value is greater than 9.5 as determined by isoelectric focusing. From genomic DNA, two introns and three exons were within the open reading frame (ORF). Southern blot analysis indicated that it is a single-copy gene. A time-course study revealed that the H. zea lysozyme gene was differentially expressed in the labial glands during the development of fifth-instar larvae, with the peak level of lysozyme mRNA being detected on day 1. Dot blot analysis showed different levels of H. zea lysozyme expression when the caterpillars fed on different plants. Further, the H. zea lysozyme could be detected with antibodies raised against the M. sexta lysozyme, and it was one of the most abundant secreted proteins in saliva collected directly from the caterpillars spinneret. The potential role of the lysozyme on host plants in mediating susceptibility to bacterial disease is discussed in the context of tritrophic interactions.     

Peripheral coding of sex pheromone and a behavioral antagonist in the Japanese beetle,Popillia japonica

Male antennae of the Japanese beetle, Popillia japonica, possess olfactory receptor neurons ORNs cocompartmentalized in the same sensilla placodea, one tuned to the sex pheromone, (R)-japonilure, and the other to the detection of a behavioral antagonist, (S)-japonilure. In-depth electrophysiological experiments revealed mutual inhibitory and synergistic effects in ORNs stimulated simultaneously with the two semiochemicals. The olfactory system of P. japonica exhibited a remarkable ability to discriminate completely coincident strands of pheromone and behavioral antagonist from strands of the two semiochemicals temporally isolated (by 1.5–3 msec). The mutual inhibition was reflected mainly by the delay of onset or total lack of spikes and by the significant increase in the rise time of potentials generated by blends of (R)- and (S)-japonilure. In contrast, synergist ORNs showed no neural activity (spikes) when stimulated with either the sex pheromone or the behavioral antagonist, but showed clear responses to blends of the two semiochemicals. Evidence for mixture-suppressed responses was observed not only in the Japanese beetle, but also in the Osaka beetle, Anomala osakana, and the Oriental beetle, Exomala orientalis, thus suggesting that it is a common feature in the sensory physiology of scarab beetles.     

Phenolic acids affect photosynthesis and protein synthesis by isolated leaf cells of velvet-leaf

The effects ofp-coumaric, ferulic, chlorogenic, and vanillic acids on photosynthesis and protein synthesis by isolated leaf cells of velvetleaf (Abutilon theophrasti Medik) were investigated. Photosynthesis and protein synthesis were measured in cell suspensions by the incorporation of¹⁴CO₂ and [¹⁴C]leucine, respectively. None of the tested phenolic acids except vanillic reduced photosynthesis by more than 50% at the highest concentration and 30 min of incubation. At 100M concentrations and 60-min incubation periods,p-coumaric, ferulic, chlorogenic, and vanillic acids inhibited photosynthesis by 33, 37, 57, and 65%, respectively. Ferulic acid was the most inhibitory to protein synthesis and reduced the incorporation of [¹⁴C]leucine by 50% at about 1.0M after 60 min of incubation. At the highest concentrations tested in this study, vanillic and ferulic acids were inhibitory to photosynthesis and protein synthesis, respectively, whereas chlorogenic andp-coumaric acids did not inhibit either physiological process. The maximum inhibition of protein synthesis by chlorogenic acid was 19% and by vanillic acid was 28% at 100M concentrations. Chlorogenic, vanillic, andp-cou-maric acids at 0.1M caused increased protein synthesis over the untreated control. Overall, photosynthesis was more sensitive than protein synthesis to the four phenolic acids tested.Florida Agricultural Experiment Station Journal Series No. 9228.     

Synthesis of Ethyl 5 Z ,9 Z ,12 Z -octadecatrienoate (ethyl pinolenate) and Methyl 12 Z ,15 Z -octadecadienoate

Pinolenic acid (5Z,9Z,12Z-octadecatrienoic acid, 1a), one of the most abundant trienoic fatty acids in nature, is very difficult to obtain in quantity in a pure state from the highly complex mixture of unsaturated tall oil fatty acids. For this reason its chemistry has been little studied when compared to linolenic or linoleic acids. A simple synthesis of esters of 1a and of 12Z,15Z-octadecadienoic acid 3 using the one pot double Wittig procedure is described here. The products of double Wittig reactions were purified by argentation chromatography, and their structural purity was established by ¹H-, ¹³C-NMR and 2D-NMR spectroscopies.

Allelopathic effect of alfalfa (Medicago sativa) on bladygrass (Imperata cylindrica)

Greenhouse and laboratory experiments were conducted at the Agricultural and Water Resources Research Center Station, Baghdad, in 1985 and 1986 to investigate the possible allelopathic potential of alfalfa (Medicago saliva L.) and its decomposed residues on bladygrass (Imperata cylin-drica L. Beauv.), a noxious weed in Iraq, and to isolate, characterize, and quantify possible allelopathic agents in alfalfa residues and root exudates. Results indicated that decomposed alfalfa roots and their associated soil produced a 51–56% reduction in bladygrass seed germination. Root and shoot length of bladygrass seedlings were reduced by an average of 88%. Decayed and undecayed mixtures of alfalfa roots and soil at 0.0151 (w/w) inhibited bladygrass seedlings reproduced from rhizomes by 30 and 42%. It was found that root exudates of alfalfa seedlings caused significant reduction in shoot and root dry weights of bladygrass seedlings when alfalfa and bladygrass were grown together in nutrient culture. Caffeic, chlorogenic, isochloro-genic,p-coumaric,p-OH-benzoic, and ferulic acids were detected in alfalfa root exudates and residues. The highest amount (126 fig phenolic acids/g soil) of these compounds was found in alfalfa root residues after six months of decomposition in soil.     

Acidic fog-induced changes in host-plant suitability

Phaseolus lunatus L. (Henderson Bush lima beans) were exposed to 2 hr acidic fogs with 2.51.0 (v/v) nitrogen-sulfur ratio typical of the west coast of the United States. Fogs with pH values of 2.0 (P < 0.01,t tests), 2.5 (P < 0.05), or 3.0 (P < 0.01) increased percent total nitrogen (dry weight) of foliage as compared to plants subjected to control fogs with a pH of 6.3–6.5. Fresh weight concentrations of soluble protein and certain free amino acid concentrations were increased by plant exposure to acidic fogs with a pH of 2.5 (t tests,P < 0.05). Concentrations of free amino acids considered essential for insect growth, as well as nonessential and total free amino acids were not significantly affected by any treatment (P > 0.05,t test). Water content (%) of foliage was not changed significantly (P > 0.05,t test) by exposure to any of the fogs.Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) larvae ate significantly more foliage and gained significantly more weight on plants treated with 3.0 pH fogs (P < 0.01,t test). Several potential explanations are offered for the lack of significant weight gain by larvae on plants in which soluble protein levels, free amino acid concentrations, or percent total nitrogen contents were enhanced by acidic fogs with a pH of 2.5 and 2.0. No larval feeding preference was detected for foliage exposed to acidic versus control fogs, and no significant differences were detected in percent survival ofT. ni eggs exposed to acidic or control fogs. Some implications of acidic fogs for population dynamics ofT. ni are discussed.     

Phytotoxicity of Vulpia Residues: II. Separation, Identification, and Quantitation of Allelochemicals from Vulpia myuros

Phytotoxicity-based extraction and fractionation were employed to separate and purify the allelochemicals from an aqueous extract of vulpia (Vulpia myuros) residues. Further analyses, identification, and quantitation of these allelochemicals were conducted by gas chromatography–mass spectrometry on the most toxic fraction. Twenty-one compounds were identified in the ether fraction from the vulpia residue aqueous extract. They were catechol, hydroquinone, 3,4-dimethoxyphenol, pyrogallol, coniferyl alcohol, and benzoic, succinic, hydrocinnamic, salicylic, protocatechuic, vanillic, gentisic, syringic, p-hydroxybenzoic, -hydroxybenzenepropanoic, p-hydroxyphenylacetic, p-hydroxybenzenepropanoic, hydroferulic, p-coumaric, hydrocaffeic, and ferulic acids. A chromatographic internal standard method with multiple-point calibration graphs was used to quantify the identified compounds. Quantities in the vulpia residues ranged from 1.36 to 81.0 g/g dry residue, in total accounting for 0.05% of the dry weight residue. The combined syringic and hydroferulic acids were present in the largest amount, 140.11 g/g residue. Vanillic, succinic, p-hydroxybenzenepropanoic, and salicylic acids were next in amount, ranging from 37.24 to 81.24 g/g residue. Catechol, hydrocinnamic acid, and hydroquinone were present in the smallest quantities, ranging from 1.36 to 1.82 g/g residue. The remainder of the compounds were intermediate in amount, ranging from 2.33 to 18.1 g/g residue.     

Selective and pH-Dependent Binding of a Moth Pheromone to a Pheromone-Binding Protein

Fluorescence and circular dichroism (CD) data suggest that the major pheromone-binding protein (PBP) from the wild silkmoth, Antheraea polyphemus, ApolPBP1, undergoes a pH-dependent conformational change similar to that previously observed for the PBP from the silkworm moth, Bombyx mori, BmorPBP. All three constituents of the sex pheromone, E6,Z11-16Ac, E6,Z11-16Ald, and E4,Z9-14Ac, bound to ApolPBP1 with apparent high affinity at high pH, but reduced binding at low pH when tested individually in a “cold binding assay.” In competitive assays, however, ApolPBP1 showed considerable preference for the major constituent of the sex pheromone, E6,Z11-16Ac. These data suggest that specificity of PBPs contributes at least in part to the remarkable selectivity of moth's olfactory system.     

Phytotoxic and antimicrobial activity of volatile constituents ofArtemisia princeps var.orientalis

The volatile constituents ofArtemisia princeps var.orientalis (wormwood) were investigated for phytotoxic and antimicrobial activities. The germination and radicle elongation of receptor plants were inhibited by volatile substances emitted from wormwood leaf and effects were concentration-dependent. Essential oil of the plant extracted by Karlsruker's apparatus suppressed seed germination and seedling elongation of the receptor plants at a threshold concentration of 4.8l/100 ml.Escherichia coli was not susceptible to the wormwood essential oil, but the growth ofBacillus subtilis, Aspergillus nidulans, Fusarium solani, andPleurotus ostreatus was inhibited severely.     

Assessment of allelopathic potential inArtemisia princeps var.Orientalis residues

Field and laboratory studies were conducted to examine the differential phytotoxicity of residues ofArtemisia princeps var.orientalis (wormwood) using various plants as test species. Seedling elongation and dry weights of receptor plants were inversely proportional to the concentration and incubation time of dry leaves ofA. princeps var.orientalis in vermiculite. In seedling growth tests with abandoned field soils (control) and soil underneath wormwood plants (test), the elongation, dry weight, and caloric content of seedlings grown in the soil from under wormwood plants were severely inhibited, thereby suggesting that certain growth inhibitors were released from wormwood and the inhibitor remained in the soil.     

Roles of amino acids,protein, and fiber in leaf-feeding resistance of corn to the fall armyworm

The free amino acids have been shown by isolational work and choice bioassays to be more important than all other factors evaluated in defining leaf-feeding resistance of corn (Zea mays L.) to fall armyworm (FAW) [(Spodoptera frugiperda J.E. Smith)] larvae. 6-MBOA (6-methoxybenzoxazolinone) and maysin, toxins present in corn, were shown not to be significant factors for leaf-feeding resistance to first-instar FAW larvae because of their low concentrations in the whorl. Amino acid analysis showed that while the ratios of the essential amino acids in susceptible (S) and resistant (R) lines were similar, there were differences in the nonessential amino acids, particularly aspartic acid, which was higher in R lines. Also, the ratio of essential amino acids to nonessential amino acids was important, being too low in expressed whorl leaf juice (obtained from V₈–V₁₀ growth stage plants) to support larval growth, although juice was stimulatory in choice tests. The total protein content of whorls in S lines was about 15% higher than in R lines, but the significance of this difference is uncertain, because nutritional tests showed that larval growth increased with total protein only up to 12% protein. Sugars were only slightly stimulatory. Thus, the amino acids along with higher hemicellulose content of R lines, established by us earlier, appear to explain much of the basis of resistance in corn to larval leaf-feeding of the FAW.     

Leaf Epicuticular Wax Chemicals of the Japanese Knotweed Fallopia japonica as Oviposition Stimulants for Ostrinia latipennis

Extraction, fractionation, and gas chromatography−mass spectrometry analyses guided by bioassays have shown that n-alkanes and free fatty acids in leaf epicuticular wax of the Japanese knotweed Fallopia (Reynoutria) japonica stimulate oviposition in the Far-Eastern knotweed borer, Ostrinia latipennis (Lepidoptera: Crambidae). n-Alkanes made up 48.1% of the total amount of epicuticular wax, and their carbon chain length was in the C₁₆−C₃₃ range, with n-nonacosane (n-C₂₉) most abundant, followed by n-C₂₇, n-C₂₅, and n-C₃₁. Free fatty acids with C₉−C₂₂ accounted for 22.3%, and hexadecanoic acid was predominant. A mixture of authentic n-alkanes and fatty acids of the composition found in the epicuticular wax, a mixture of n-alkanes, and a mixture of fatty acids significantly enhanced oviposition. Thus, it was demonstrated that both n-alkanes and free fatty acids in leaf epicuticular wax of F. japonica are naturally occurring oviposition stimulants for O. latipennis.     

Identification and directed biosynthesis of efrapeptins in the fungusTolypocladium geodes gams (Deuteromycotina: Hyphomycetes)

HPLC analysis of crude dichloromethane extracts of shaken liquid cultures of the hyphomycetous fungusTolypocladium geodes Gams revealed the presence of efrapeptins. These peptides, which have mitochondrial ATPase inhibitory activity as well as antifungal and insecticidal properties, are previously known only from the congeneric species,T. niveum Rostrup. The identity of efrapeptins was confirmed by fast atom bombardment mass spectrometry and by amino acid analysis. HPLC analyses of efrapeptins extracted from single isolates of bothT. geodes andT. niveum indicated that both species produced the same efrapeptins but the profile of relative abundance of the compounds produced was diagnostic for the isolates examined. Efrapeptin F was the major component of the mixture fromT. geodes with the order of abundance of the six efrapeptins detected being F >G>DE>H>C. Efrapeptin D was the major component fromT. niveum with the order of abundance of the six efrapeptins detected being D >E>F>CG>H. Efrapeptin F diifers from efrapeptin D by a single amino acid residue, efrapeptin F having an alanine where efrapeptin D has a glycine. Addition of alanine to the culture medium increased the relative abundance of efrapeptin F in the profile of both species. Conversely, addition of glycine increased the relative abundance of efrapeptin D in the profile of both species.Mention of a commercial or proprietary product does not constitute an endorsement by the USDA.     

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.     

Odorant-binding proteins from a primitive termite

Hitherto, odorant-binding proteins (OBPs) have been identified from insects belonging to more highly evolved insect orders (Lepidoptera, Coleoptera, Diptera, Hymenoptera, and Hemiptera), whereas only chemosensory proteins have been identified from more primitive species, such as orthopteran and phasmid species. Here, we report for the first time the isolation and cloning of odorant-binding proteins from a primitive termite species, the dampwood termite, Zootermopsis nevadensis nevadensis (Isoptera: Termopsidae). A major antennae-specific protein was detected by native PAGE along with four other minor proteins, which were also absent in the extract from control tissues (hindlegs). Multiple cDNA cloning led to the full characterization of the major antennae-specific protein (ZnevOBP1) and to the identification of two other antennae-specific cDNAs, encoding putative odorant-binding proteins (ZnevOBP2 and ZnevOBP3). N-terminal amino acid sequencing of the minor antennal bands and cDNA cloning showed that olfaction in Z. n. nevadensis may involve multiple odorant-binding proteins. Database searches suggest that the OBPs from this primitive termite are homologues of the pheromone-binding proteins from scarab beetles and antennal-binding proteins from moths.     

Geranium defensive agents. III. Structural determination and biosynthetic considerations of anacardic acids of geranium

Ozonolysis, dithioether derivatization, and EI and CI mass spectrometry were used to establish the location of the double bond in the side chain of the two major anacardic acids in geranium (Pelargonium hortorum) trichome exudate. The point of unsaturation was shown to be between C-5 and C-6 counting from the methyl end of the side chain, contradicting the earlier hypothesis that the olefmic bond was probably at the 9–10 position based upon expected biosynthetic considerations. The two major components are thus 6-[(Z)-10-pentadecenyl]salicylic acid (C₂₂H₃₄O₃) and 6-[(Z)-12-heptadecenyl] salicylic acid (C₂₄H₃₈O₃). This may indicate that the precursor of these anacardic acids is a saturated fatty acid since the location of a double bond at the 5–6 position is unusual among the unsaturated fatty acids. Capillary GLC and HPLC of the trichome exudate indicated the presence of small amounts of other anacardic acid analogs possessing such features as odd numbers of carbon atoms and saturated side chains.     

Sex pheromone biosynthesis of (E,E)-8,10-dodecadienol in codling mothCydia pomonella involvesE9 desaturation

Sex pheromone biosynthesis in the codling mothCydia pomonella (Lepidoptera; Tortricidae) was studied by topical application of deuterated fatty acids in DMSO to pheromone glands. The incorporation of deuterium label into fatty acids and alcohols in the pheromone gland was monitored by gas chromatography with flame ionization detection and mass spectrometry in the selected ion monitoring mode. Dodecanol, (E)-9-dodecenol, (E,E)-8,10-dodecadienol, tetradecanol, and hexadecanol were found in gland extracts. The application of [12,12,12-²H₃]dodecanoic acid resulted in labeled dodecanol, (E)-9-dodecenol, and (E,E)-8,10-dodecadienol, as well as the corresponding labeled acids. No label was incorporated into tetradecanol or hexadecanol or any acid with more than 12 carbon atoms. The application of labeled tetradecanoic or hexadecanoic acid introduced label not only into the 12-carbon alcohols, but also into tetradecanol, or tetradecanol and hexadecanol, respectively. The application of (E)-[11, 11,12,12,12,-²H₅]9-dodecen-oic acid, whose facile synthesis is described, resulted in labeled (E)-9-do-decenol and (E,E)-8,10-dodecadienol. The (E,E)-8,10-dodecadienol so produced was characterized by an ion atm/z 186, equivalent to [M]⁺ of a dienol labeled with four deuterons. Thus, one deuterium label is lost when the labeled (E)-9-monoene is converted to the (E,E)-8,10-diene. We conclude that (E,E)-8,10-dodecadienol is synthesized by chain shortening (-oxidation) of palmitic acid to dodecanoic acid, followed by an unusualE9 desaturation and subsequent conversion of this intermediate into the conjugated precursor, which is finally reduced to the pheromone alcohol. The evolutionary significance ofE9 desaturation being responsible for pheromone production in an Olethreutinae species is discussed.     

Growth analysis of corn and soybean response to allelopathic effects of weed residues at various temperatures and photosynthetic photon flux densities

The effects of redroot pigweed (Amaranthus retroflexus L.) and yellow foxtail [Setariaglauca (L.) Beauv.] residues on corn (Zea mays L.) and soybeans [Glycine max (L.) Merr.] were evaluated at various temperatures and photosynthetic photon flux densities (PPFD) in a Biotron. Mathematical growth analysis techniques were used for the evaluation. Redroot pigweed markedly reduced leaf area duration (LAD), leaf weight ratio (LWR), and total dry matter production in both crops. Yellow foxtail residue inhibited total dry matter production in corn and soybeans 20 and 30 days after planting (DAP). It also reduced growth rate (GR) and LAD in corn and biomass increment ( W) in soybeans. Biomass increment was more closely correlated to LAD than net assimilation rate (NAR) in soybeans, whereas in corn NAR contributed more to W than LAD. The 30/20 ° C temperature with a PPFD of 380 E/m²/sec produced a larger W with a greater NAR and larger LAD in corn, resulting in maximum dry matter accumulation than 20/10 ° C and other levels of PPFD. Under similar conditions, soybeans showed little or no response to the changes. The results demonstrate the allelopathic effects of weed residues on growth and dry matter production, affecting LAD, LWR, and GR. The results also show that environmental temperature and PPFD may alter the allelopathic effects of weeds on crops. The possible interference with photosynthesis and the partitioning of biomass into leaf component relative to the total biomass produced by the plant may be the inhibitory effects of allelochemicals present in redroot pigweed and yellow foxtail residues.