Patrick F. Dowd

Enzymatic oxidation products of allelochemicals as a basis for resistance against insects: effects on the corn leafhopper Dalbulus maidis.

Oxidation products of com allelochemicals generated by peroxidases or tyrosinases were tested in 10% sucrose solutions for effects on the corn leafhopper Dalbulus maidis. Some reduction in feeding was noted with hydrogen peroxide (a cofactor for peroxidase). Significant reduction in feeding was noted with chlorogenic acid, ferulic acid, p-coumaric acid, and 6-methoxybenzoxazolinone (MBOA), but not rutin at 400 ppm in solution. Oxidation products of these compounds all caused significantly less feeding by the leafhoppers compared to the original compound. Oxidation products generated by peroxidase from ferulic acid and 6-methoxybenzoxazolinone caused significant mortality to the leafhoppers within 5 days. Thus, provided conditions are such that oxidizing enzymes and allelochemicals can interact due to damage by insects, resistance may be significantly enhanced by the oxidized products as opposed to the effects of the allelochemicals alone.

Aggregation pheromone of driedfruit beetle,Carpophilus hemipterus Wind-tunnel bioassay and identification of two novel tetraene hydrocarbons

A male-produced aggregation pheromone was demonstrated inCarpophilus hemipterus (L.) (Coleoptera: Nitidulidae) using a wind-tunnel bioassay. Both sexes responded to the pheromone, but the beetles flew in the wind tunnel only after they had been starved for at least several hours. The attractiveness of the pheromone was greatly enhanced by volatiles from a food source, and combinations of pheromone and food volatiles typically attracted 3–10 times more beetles than either source by itself. A variety of food-related sources of volatiles were effective. These included apple juice; a mixture of bakers yeast plus banana; the pinto bean diet used for rearing this beetle; the chemicals propyl acetate, ethanol; and a mixture of acetaldehyde, ethyl acetate, and ethanol. The pheromonal activity resided with a series of 10 male-specific, unsaturated hydrocarbons of 13, 14, and 15 carbon atoms. These were partially separated by HPLC. No single compound was absolutely required for pheromonal activity to be observed, and various subsets of these compounds were active. The most abundant component was (2E,4E,6E,8E)-3,5,7-trimethyl-2,4,6,8-decatetraene. One minor component was (2E,4E,6E,8E)-3,5,7-trimethyl-2,4,6,8-undecatetraene. These structures were proven by synthesis. Together, the synthetic compounds were as active in the wind tunnel as the beetle-derived pheromone.

Toxicity and Interactions of Some Fusarium Graminearum Metabolites to Caterpillars

Determination de la toxicite relative et de la toxicite de combinaisons choisies (desoxyrivalenol, sambucinol, dihydroxycalonectrines, culmorine, 8-hydroxycalonectrine, dihydrodesoxyrivalenol) vis-a-vis de Spodoptera frugiperda et de Heliothis zea

The contribution of symbiotic yeast to toxin resistance of the cigarette beetle (Lasioderma serricorne)

Representative plant allelochemicals were tested for toxicity to larvae of the cigarette beetle Lasioderma serricorne (F.) that were either untreated, treated with fungicides, or treated to render them free of symbiotic yeast (‘aposymbiotic’) through surface sterilization of eggs. Insects rendered symbiont‐free had higher mortality and/or developmental rates than controls when fed diets containing flavone, resorcinol or tannic acid. Fungicides reduced symbiont populations and/or caused morphological abnormalities. Two hydrolytic enzymes, which made up a significant portion of mycetome hydrolytic activity, were absent when symbionts were not present, as indicated by gel electrophoresis. This information indicates symbionts do contribute to the survival of their host by detoxifying toxins.

The structure of tubingensin B: A cytotoxic carbazole alkaloid from the sclerotia of Aspergillus tubingensis

Tubingensin B (4), a cytotoxic carbazole alkaloid with a novel ring system, was isolated from the sclerotia of the fungus . The structure was assigned primarily on the basis of selective INEPT, homonuclear decoupling, and COSY experiments.

Engineered Saccharomyces cerevisiae strain for improved xylose utilization with a three-plasmid SUMO yeast expression system

A three-plasmid yeast expression system utilizing the portable small ubiquitin-like modifier (SUMO) vector set combined with the efficient endogenous yeast protease Ulp1 was developed for production of large amounts of soluble functional protein in Saccharomyces cerevisiae. Each vector has a different selectable marker (URA, TRP, or LEU), and the system provides high expression levels of three different proteins simultaneously. This system was integrated into the protocols on a fully automated plasmid-based robotic platform to screen engineered strains of S. cerevisiae for improved growth on xylose. First, a novel PCR assembly strategy was used to clone a xylose isomerase (XI) gene into the URA-selectable SUMO vector and the plasmid was placed into the S. cerevisiae INVSc1 strain to give the strain designated INVSc1-XI. Second, amino acid scanning mutagenesis was used to generate a library of mutagenized genes encoding the bioinsecticidal peptide lycotoxin-1 (Lyt-1) and the library was cloned into the TRP-selectable SUMO vector and placed into INVSc1-XI to give the strain designated INVSc1-XI-Lyt-1. Third, the Yersinia pestis xylulokinase gene was cloned into the LEU-selectable SUMO vector and placed into the INVSc1-XI-Lyt-1 yeast. Yeast strains expressing XI and xylulokinase with or without Lyt-1 showed improved growth on xylose compared to INVSc1-XI yeast.

Lycotoxin-1 insecticidal peptide optimized by amino acid scanning mutagenesis and expressed as a coproduct in an ethanologenic Saccharomyces cerevisiae strain

New methods of safe biological pest control are required as a result of evolution of insect resistance to current biopesticides. Yeast strains being developed for conversion of cellulosic biomass to ethanol are potential host systems for expression of commercially valuable peptides, such as bioinsecticides, to increase the cost‐effectiveness of the process. Spider venom is one of many potential sources of novel insect‐specific peptide toxins. Libraries of mutants of the small amphipathic peptide lycotoxin‐1 from the wolf spider were produced in high throughput using an automated integrated plasmid‐based functional proteomic platform and screened for ability to kill fall armyworms, a significant cause of damage to corn (maize) and other crops in the United States. Using amino acid scanning mutagenesis (AASM) we generated a library of mutagenized lycotoxin‐1 open reading frames (ORF) in a novel small ubiquitin‐like modifier (SUMO) yeast expression system. The SUMO technology enhanced expression and improved generation of active lycotoxins. The mutants were engineered to be expressed at high level inside the yeast and ingested by the insect before being cleaved to the active form (so‐called Trojan horse strategy). These yeast strains expressing mutant toxin ORFs were also carrying the xylose isomerase (XI) gene and were capable of aerobic growth on xylose. Yeast cultures expressing the peptide toxins were prepared and fed to armyworm larvae to identify the mutant toxins with greatest lethality. The most lethal mutations appeared to increase the ability of the toxin α‐helix to interact with insect cell membranes or to increase its pore‐forming ability, leading to cell lysis. The toxin peptides have potential as value‐added coproducts to increase the cost‐effectiveness of fuel ethanol bioproduction. Copyright

Comparative toxicity of allelochemicals and their enzymatic oxidation products to maize fungal pathogens, emphasizing Fusarium graminearum

A series of stable quinones and their precursors, and enzymatic oxidation products of plant allelochemicals were tested for their effect on maize fungal pathogens, primarily Fusarium graminearum. Benzoquinone was typically significantly more toxic than hydroquinone, while 1,2-naphthoquinone was typically significantly more toxic than 1,2-dihydroxynaphthalene. Aspergillus flavus was the most resistant fungus to these compounds, while Phoma medicaginis was the most susceptible. Applying tyrosinase in conjunction with several phenolic compounds only increased the toxicity of gallic acid to Fusarium graminearum. Applying peroxidase generally increased toxicity of all compounds tested to this fungus in a dose-dependent fashion. Ferulic acid was generally the most toxic compound, both alone and when combined with peroxidase and H2O2, followed by coumaric acid. These results suggest that enzymatic oxidation of plant allelochemicals may result in the generation of products that either are directly toxic to maize pathogens, or indirectly inhibitory due to their ability to tie up nutrients.

Relative Resistance of Transgenic Tomato Tissues Expressing High Levels of Tobacco Anionic Peroxidase to Different Insect Species

Different parts of genetically transformed tomato (Lycopersicon esculentum L.) plants that express the tobacco anionic peroxidase were compared for insect resistance with corresponding wild type plants. Leaf feeding by first instar Helicoverpa zea and Manduca sexta was often significantly reduced on intact transgenic plants and/or leaf disks compared to wild type plants, but the effect could depend on leaf age. Leaves of transgenic plants were generally as susceptible to feeding damage by third instar Helicoverpa zea (Boddie) and Manduca sexta (L.) as wild type plants. Green fruit was equally susceptible to third instar larvae of H. zea in both type plants, but fruit of transgenic plants were more resistant to first instar larvae as indicated by significantly greater mortality. Basal stem sections were more resistant to neonate larvae of H. zea and adults of Carpophilus lugubris Murray compared to wild type plants as indicated by significantly greater mortality and/or reduced feeding damage. Thus, tobacco anionic peroxidase activity can increase plant resistance to insects in tomato, a plant species closely related to the original source plant species, when expressed at sufficiently high levels. However, the degree of resistance is dependent on the size of insect and plant tissue involved.

New paspalinine derivatives with antiinsectan activity from the sclerotia of Aspergillus nomius

Abstract 14-Hydroxypaspalinine ( 3 ) and 14-(N,N-Dimethyl-L-valyloxy)paspaline ( 4 ) have been isolated from the sclerotia of Aspergillus nomius , and identified by analysis of 2D NMR data. Both compounds cause ca . 90% reduction in weight gain in assays against the corn earworm Helicoverpa zea at the 100 ppm (dry weight) dietary level. Paspaline causes no effect at this concentration.