Bruce C. Campbell

Synergism of Antifungal Activity between Mitochondrial Respiration Inhibitors and Kojic Acid

Co-application of certain types of compounds to conventional antimicrobial drugs can enhance the efficacy of the drugs through a process termed chemosensitization. We show that kojic acid (KA), a natural pyrone, is a potent chemosensitizing agent of complex III inhibitors disrupting the mitochondrial respiratory chain in fungi. Addition of KA greatly lowered the minimum inhibitory concentrations of complex III inhibitors tested against certain filamentous fungi. Efficacy of KA synergism in decreasing order was pyraclostrobin > kresoxim-methyl > antimycin A. KA was also found to be a chemosensitizer of cells to hydrogen peroxide (H2O2), tested as a mimic of reactive oxygen species involved in host defense during infection, against several human fungal pathogens and Penicillium strains infecting crops. In comparison, KA-mediated chemosensitization to complex III inhibitors/H2O2 was undetectable in other types of fungi, including Aspergillus flavus, A. parasiticus, and P. griseofulvum, among others. Of note, KA was found to function as an antioxidant, but not as an antifungal chemosensitizer in yeasts. In summary, KA could serve as an antifungal chemosensitizer to complex III inhibitors or H2O2 against selected human pathogens or Penicillium species. KA-mediated chemosensitization to H2O2 seemed specific for filamentous fungi. Thus, results indicate strain- and/or drug-specificity exist during KA chemosensitization.

A pear-derived kairomone with pheromonal potency that attracts male and female codling moth, Cydia pomonella (L.)

Abstract. Ethyl (2E, 4Z)-2,4-decadienoate, a pear-derived volatile, is a species-specific, durable, and highly potent attractant to the codling moth (CM), Cydia pomonella (L.), a serious pest of walnuts, apples, and pears worldwide. This kairomone attracts both CM males and virgin and mated females. It is highly attractive to CM in both walnut and apple orchard contexts, but has shown limited effectiveness in a pear orchard context. Rubber septa lures loaded with ethyl (2E, 4Z)-2,4-decadienoate remained attractive for several months under field conditions. At the same low microgram load rates on septa, the combined gender capture of CM in kairomone-baited traps was similar to the capture rate of males in traps baited with codlemone, the major sex pheromone component. The particular attribute of attracting CM females renders this kairomone a novel tool for monitoring population flight and mating–ovipositional status, and potentially a major new weapon for directly controlling CM populations.

A New Rickettsia from a Herbivorous Insect, the Pea Aphid Acyrthosiphon pisum (Harris)

Abstract. An undescribed, maternally heritable, rod-shaped bacterium (or “tertiary symbiont”) was detected by microscopy in hemolymph of about half (59/122) of pea aphid [Acyrthosiphon pisum (Harris)] clones collected from widely separated locations in California. On the basis of molecular phylogenetic analysis of 16S rDNA sequences, the bacterium was clearly placed among other Rickettsia in the α-subgroup of Proteobacteria, close to Rickettsia bellii—a rickettsia found in ticks. A PCR assay was developed to detect this bacterium in pea aphid clones with specific 16S rDNA PCR primers. Results of PCR-based assays completely correlated with detection by microscopy. This is the first confirmed detection of a Rickettsia in a herbivorous insect.

The eubacterial endosymbionts of whiteflies (homoptera: Aleyrodoidea) constitute a lineage distinct from the endosymbionts of aphids and mealybugs

Whiteflies (superfamily Aleyrodoidea) contain eubacterial endosymbionts localized within host cells known as mycetocytes. Sequence analysis of the genes for the 16S rRNA of the endosymbionts ofBemisia tabaci, Siphoninus phillyreae, andTrialeurodes vaporariorum indicates that these organisms are closely related and constitute a distinct lineage within the γ-subdivision of theProteobacteria. The endosymbionts of whiteflies are unrelated to the endosymbionts of aphids and mealybugs, which are in two separate lineages.

Elucidation of the functional genomics of antioxidant-based inhibition of aflatoxin biosynthesis

Caffeic acid (3,4-dihydroxycinnamic acid, 12 mM) added to a fat-based growth medium reduces >95% of aflatoxin production by Aspergillus flavus NRRL 3357, without affecting fungal growth. Microarray analysis of caffeic acid-treated A. flavus indicated expression of almost all genes in the aflatoxin biosynthetic cluster were down-regulated, ranging from a log2 ratio of caffeic acid treated and untreated of -1.12 (medium) to -3.13 (high). The only exceptions were genes norB and the aflatoxin pathway regulator-gene, aflJ, which showed low expression levels in both treated and control fungi. The secondary metabolism regulator-gene, laeA, also showed little change in expression levels between the fungal cohorts. Alternatively, expression of genes in metabolic pathways (i.e., amino acid biosynthesis, metabolism of aromatic compounds, etc.) increased (log2 ratio >1.5). The most notable up-regulation of A. flavus expression occurred in four genes that are orthologs of the Saccharomyces cerevisiae AHP1 family of genes. These genes encode alkyl hydroperoxide reductases that detoxify organic peroxides. These increases ranged from a log2 ratio of 1.08 to 2.65 (moderate to high), according to real-time quantitative reverse transcription-PCR (qRT-PCR) assays. Based on responses of S. cerevisiae gene deletion mutants involved in oxidative stress response, caffeic, chlorogenic, gallic and ascorbic acids were potent antioxidants under oxidative stress induced by organic peroxides, tert-butyl and cumene hydroperoxides. Differential hypersensitivity to these peroxides and hydrogen peroxide occurred among different mutants in addition to their ability to recover with different antioxidants. These findings suggest antioxidants may trigger induction of genes encoding alkyl hydroperoxide reductases in A. flavus. The possibilities that induction of these genes protects the fungus from oxidizing agents (e.g., lipoperoxides, reactive oxygen species, etc.) produced during host-plant infection and this detoxification attenuates upstream signals triggering aflatoxigenesis are discussed.

Competence to Respond to Floral Inductive Signals Requires the Homeobox Genes PENNYWISE and POUND-FOOLISH

The transition from vegetative to reproductive development establishes new growth patterns required for flowering. This switch is controlled by environmental and/or intrinsic developmental cues that converge at the shoot apical meristem (SAM). During this developmental transition, floral inductive signals cause the vegetative meristem to undergo morphological changes that are essential for flowering. Arabidopsis plants containing null mutations in two paralogous BEL1-like (BELL) homeobox genes, PENNYWISE (PNY) and POUND-FOOLISH (PNF), disrupt the transition from vegetative to reproductive development. These double mutants are completely unable to flower even though the SAM displays morphological and molecular changes that are consistent with having received floral inductive signals. These studies establish a link between the competence to receive floral inductive signals and restructuring of the SAM during floral evocation.

TOXICITY AND FEEDING DETERRENCY OF HYDROXAMIC ACIDS FROM GRAMINEAE IN SYNTHETIC DIETS AGAINST THE GREENBUG, SCHIZAPHIS GRAMINUM

2,4‐Dihydroxy‐7‐methoxy‐1,4‐benzoxazin‐3‐one (DIMBOA), the main hydroxamic acid isolated from maize extracts, increased the mortality of Schizaphis graminum when fed in artificial diets. Electrically‐monitored feeding assays showed that DIMBOA acted as a feeding deterrent at concentrations as low as 1 mM. On 12 mM DIMBOA diets, feeding by aphids was completely inhibited. Additional feeding experiments showed that when DIMBOA was ingested there was an increase in aphid mortality relative to that of aphids which did not ingest the compound. Thus, the deleterious effects of DIMBOA on aphids are due to feeding deterrency and toxicity. The 2‐ß‐0 glucoside of DIMBOA (DIMBOA‐Glc), the form in which DIMBOA naturally occurs in Gramineae, had a slight effect on lowering aphid survival and an appreciable feeding‐deterrent effect on diet‐fed aphids. The relevance of the effects of DIMBOA and DIMBOA‐Glc on aphids to resistance of certain graminaceous crops against aphids is discussed.

PROBING BEHAVIOR OF THE GREENBUG (SCHIZAPHIS GRAMINUM, BIOTYPE C) ON RESISTANT AND SUSCEPTIBLE VARIETIES OF SORGHUM

The probing behavior of biotype C of the greenbug, Schizaphis graminum (Rondani) on susceptible and resistant lines of sorghum, Sorghum bicolor (L.) Moench., was electronically monitored. Waveforms corresponding to salivation, phloem ingestion and non‐phloem ingestion are described. The results of a parallel study revealed that the rate of population growth of S. graminum was significantly greater on susceptible lines of sorghum [i.e., NC + 70X. SC423 (Purple). SC423 (Tan)] than on resistant lines (i.e., TAM 2567, IS 809). Aphids probing the resistant lines of sorghum showed a significantly reduced imbibition of phloem sap compared with those aphids which fed on susceptible varieties. Also, increased numbers of separate probes and increased duration of non‐probing were associated with greenbugs feeding on resistant lines. Greenbugs monitored on the nonhost plant, rice, exhibited non‐phloem ingestion, but not phloem ingestion.

Evolutionary origin of whiteflies (Hemiptera: Sternorrhyncha: Aleyrodidae) inferred from 18S rDNA sequences

Phylogenetic analysis of 18S rDNA nucleotide sequences of hemipteran exemplars shows Sternorrhyncha (psyllids, whiteflies, aphids and scales) is monophyletic and forms a sister group to all other hemipterans (Euhemiptera). Whiteflies form a sister group to aphids and scales. Psyllids form a sister group to all other Sternorrhyncha. Primary structures of 18S rDNAs of all sternorrhynchans are exceptionally long (˜2200 to ˜2500 bp) due to internal expansions. These expansions are a synapomorphy of Sternorrhyncha; other hemipterans possess shorter 18S rDNAs (˜1900 to ˜1925 bp). The 18S rDNA of whiteflies is the longest recorded to date and has a base substitution rate of ˜3 times greater than Euhemiptera taxa examined. The relevance of these findings to the fossil record, feeding strategies, reproductive biologies, and geoclimatic distribution is discussed.

Examination of fungal stress response genes using Saccharomyces cerevisiae as a model system: targeting genes affecting aflatoxin biosynthesis by Aspergillus flavus Link

Saccharomyces cerevisiae served as a model fungal system to examine functional genomics of oxidative stress responses and reactions to test antioxidant compounds. Twenty-two strains of S. cerevisiae, including a broad spectrum of singular gene deletion mutants, were exposed to hydrogen peroxide (H2O2) to examine phenotypic response to oxidative stress. Responses of particular mutants treated with gallic, tannic or caffeic acids, or methyl gallate, during H2O2 exposure, indicated that these compounds alleviated oxidative stress. These compounds are also potent inhibitors of aflatoxin biosynthesis in Aspergillus flavus. To gain further insights into a potential link between oxidative stress and aflatoxin biosynthesis, 43 orthologs of S. cerevisiae genes involved in gene regulation, signal transduction (e.g., SHO1, HOG1, etc.) and antioxidation (e.g., CTT1, CTA1, etc.) were identified in an A. flavus expressed sequence tag library. A successful exemplary functional complementation of an antioxidative stress gene from A. flavus, mitochondrial superoxide dismutase (sodA), in a sod2Δ yeast mutant further supported the potential of S. cerevisiae deletion mutants to serve as a model system to study A. flavus. Use of this system to further examine functional genomics of oxidative stress in aflatoxigenesis and reduction of aflatoxin biosynthesis by antioxidants is discussed.