Addendum in pocket. Biblography: leaves 162-173. Phytophagous sucking insects, aphids in particular, are common pests of plants. These insects secrete salivary enzymes into their food material. One plant defence mechanism is the induction and/or accumulation of deterrent phytochemicals. In the present study, a model enzyme system was chosen to mimic the oxidative activity of insect saliva. Isolation and sructural identification of the products of plant allelochemicals was achieved for several substrates. Insects were also exposed to individual plant chemicals in feeding "choice" tests. Preliminary examinations suggest that plants containing increased levels of phenolic allelochemicals show correlated increases in resistance to attack by aphids.

Competitive exclusion has been the mechanism hypothesized to account for the biological control of fire blight disease of pear and apple by the bacterium Pseudomonas fluorescens A506 (A506). Recent laboratory assays demonstrated, however, that A506 produces an antibiotic that is toxic to the fire blight pathogen, Erwinia amylovora, when cultured on media amended with iron (Fe����� or Fe�����). This study investigated this iron-dependent antibiosis by A506 by: 1) examining bioavailability of iron to A506 on blossom surfaces, 2) mutagenizing A506 to disrupt genes involved in antibiotic production, and 3) evaluating suppression of fire blight by A506 when co-treated with an iron chelate (FeEDDHA). Bioavailability of iron on blossoms was investigated with an iron biosensor [iron-regulated promoter (pvd) fused to an ice nucleation reporter gene (inaZ)] in A506. A506 (pvd-inaZ) expressed high ice nucleation activity (INA) on blossoms indicating a low-iron environment unlikely to induce antibiosis by A506. Spraying blossoms with FeEDDHA at concentrations ���0.1 mM significantly suppressed INA by A506 (pvd-inaZ). Transposon mutagenesis was used to generate and select mutants of A506 exhibiting altered antibiotic production profiles. One antibiotic-deficient mutant, A506 Ant���, was recovered; this mutant showed reduced epiphytic fitness on blossoms of apple and pear trees compared to the parent stain, A506. Another mutant, A506 Ant���, lost the characteristic fluorescent phenotype and exhibited iron-independent antibiotic production in defined culture media. A506 Ant��� established high populations on blossoms of apple and pear trees, similar to populations attained by A506, and reduced incidence of fire blight between 20 to 40%, levels comparable to A506 in orchard trials. In orchard trials, A506 was co-treated with FeEDDHA and fire blight suppression was evaluated. Bacterial strains established high populations on blossoms when co-treated with 0.1 mM FeEDDHA or in water. Significantly enhanced suppression of fire blight incidence by antibiotic producing strains of A506 amended with 0.1 mM FeEDDHA was observed in 2 of 5 trials, providing some evidence that iron-induced antibiosis can be a contributing mechanism in disease control. Lack of disease control by the antibiotic deficient strain, A506 GacS, and by 0.1 mM FeEDDHA alone added support to this hypothesis. / Graduation date: 2004

Aphids are a widespread family of plant pests, whose abilities to suppress shoot and root growth are well documented for many terrestrial plants. Only a few studies have been conducted on conifer aphids of the genus Cinara. Cinara pseudotsugae are known to attack Douglas-fir seedlings, an important crop in the Pacific Northwest. Douglas-fir are most susceptible to aphid damage as seedlings, especially in nurseries where conditions favor aphid outbreaks. A parasitoid wasp (Pauesia sp.) attacks C. pseudotsugae, and may be useful as a biological control agent. Studies of its natural history and host interactions are needed to assess its potential as a bio-control agent. This study examined the effects of an experimental range of aphid densities on the growth of total shoot and root volume and biomass and shoot morphology of Douglas-fir seedlings. Eighteen-week tests explored short term effects of different aphid feeding intensities in both the greenhouse and field. Long term effects were tested by exposing greenhouse seedlings to 16 months of aphid feeding. The ability of the plants to recover was tested by allowing one set of seedlings to grow aphid free for one year, after being exposed to aphid feeding for 18 weeks. The success rate of parasitoids over an 18 week period was compared to aphid density in both greenhouse and field tests. Increasing aphid destiny was significantly related to decreasing root and shoot dry weights in greenhouse tests. Growth suppression increased slightly during the second year of testing, regardless of whether or not aphid feeding continued. The results for root and shoot volumes were highly variable. However, root tissue density was significantly reduced after the second year of testing. Few shoot characteristics showed consistently significant aphid effects among the trials. Stem diameter and height decreased and needle density of new buds increased significantly with aphid feeding in most tests. Root and shoot growth of field plants did not show any significant aphid effects. Percentage of parasitoid success was independent of aphid density except at the lowest aphid densities. There was a block effect on parasitoid success in the field test, that may have been a result of varying environmental conditions. These results indicate that even short term aphid feeding can have long lasting effects on plant growth and structure. The effect on shoot and root growth was small, but there were no signs of recovery. The long term effects of the reduced root tissue density on Douglas-fir is unknown. / Graduation date: 1998

Graduation date: 1994

Differences in plant structure and host abundance, distribution and density within and between plants may affect host finding success of searching parasitoids. The main objective of this research consisted in developing and validating a model that can predict parasitism by Trichogramma evanescens on various plant structures. Also, we evaluated the effect of both artificial and natural plant structures on host encountering success and on searching behavior of two species of Trichogramma. / Size (S), heterogeneity (H) and connectivity (C) define plant structure. The development of the model of parasitism was based on laboratory experiments using three dimensional artificial plants of different combinations of S, H and C. The model was then validated with experiments, within greenhouse, using natural cruciferous plants of different structures. Significant regressions of observed values of parasitism as a function of those predicted by the model were obtained. / The effect of plant structure on two species of Trichogramma was studied by using three species of Lepidoptera and three structurally different but closely related crucifers. Also the distribution pattern of parasitized eggs according to leaf side and plant height was characterized. A repeated measure ANOVA in time where plant ages defined the repeated measures showed that all three main effects (plant, host, parasitoid) had significant effect on parasitism and only the parasitoid x plant structure interaction was significant. Mean percent of parasitism was higher on cabbage, intermediate on broccoli and lower on Brussels sprouts whereas cabbage appeared to be intermediate in plant structure, broccoli appeared to be the most simple and Brussels sprouts the most complex plant structure. On simple plant structure, both wasp species performed well on the three varieties of plant but T. evanescens outperformed T. pretiosum more often on the various plant structures. A doubly repeated measures ANOVA in space revealed significant effects of leaf side and plant height on parasitism, being greatest under leaf surface and at the base of the plant. The interaction between those two variables was not significant. / The effect of plant structure on searching behavior of T. evanescens was determined by direct observation of individual females searching on simple and complex plant structures during 1 hour. Time, frequence and sequence associated to activities and plant parts explored were obtained and analyzed. Plant structure had significant effect on time budget associated to activities (walking, resting, flying) and plant parts explored. / This study demonstrates that plant structure mediates ecological interactions by affecting host finding success and this may have important implications in population dynamics, evolution of hosts and parasitoids but also in biological control programs.

Six pathogenic fungal species were isolated from naturally-infected Echinochloa species and evaluated as biological control agents for E. crus-galli, E. colona, and E. glabrescens in rice. Bipolaris sacchari, Curvularia geniculata, and Exserohilum monoceras were non-pathogenic to rice and caused high mortality of Echinochloa species. E. monoceras was selected for further study. Under regulated greenhouse conditions, an inoculum dose of 2.5 $ times$ 10$ sp7$ conidia/m$ sp2$ killed E. crus-galli and E. glabrescens seedlings while 5.0 $ times$ 10$ sp7$ conidia/m$ sp2$ caused 100% mortality of E. colona seedlings. The 1.5-leaf stage was the most susceptible growth stage for all three Echinochloa species. E. glabrescens was most susceptible to E. monoceras infection, E. crus-galli had an intermediate susceptibility, and E. colona was least susceptible. The optimum temperature for 100% mortality was between 20 and 30 C for all Echinochloa species, whereas the minimum dew period for 100% mortality was 16 h for E. colona, 12 h for E. crus-galli, and 8 h for E. glabrescens. Under screenhouse conditions and in the absence of an artificial dew period, over 90% of Echinochloa seedlings were killed when inoculum was sprayed in an oil emulsion or when applied as a dry powder to the water surface of a simulated paddy field. Maximum conidia production occurred on V-8 juice agar or centrifuged V-8 juice agar, at 28 C in the dark. No conidia were produced in liquid media. Of various agricultural products tested as solid substrates, the highest sporulation (1.81 $ times$ 10$ sp6$ conidia/g dry weight) occurred on corn leaves. Host range tests on 54 plant species in 43 genera and 19 families showed that Rottboellia cochinchinensis, was also highly susceptible to this fungus. Of the crops tested, only corn seedlings were lightly infected under optimum greenhouse conditions but no disease occurred on corn under field conditions. Bipolaris sacchari, Exserohilum monoceras, and E. oryzae

A culture system was established for mass rearing of the Russian knapweed nematode, Subanguina picridis (Kirjanova) Brzeski. This system consisted of two parts; a shoot culture system used as the host plant source for the nematode culture, and a monoxenic nematode culture system. The nematode developed and reproduced in this system. Galls were induced on the leaves, stems, and shoot tips of cultured Russian knapweed (Acroptilon repens (L.) DC.) shoots. After 3 months in culture, the nematode number per petri dish increased from the initial 50 inoculated to 7,000-10,000, a 140 to 200 fold increase. This study represents the first time that an above-ground gall forming nematode has been propagated in vitro. It also represents a unique monoxenic nematode culture system to mass rear above-ground endoparasitic plant nematodes. / Various factors, including tissue type, tissue age, medium, and temperature, which affect the formation and development of galls, were examined. The nematode failed to reproduce in callus tissues, and it could not develop beyond the 4th stage in excised root cultures. The optimum incubation conditions determined were: 60-80 $ mu$molm$ sp{-2}$s$ sp{-1}$ light intensity, 20 C temperature, and 4-8 mm for shoot length. / The virulence of cultured nematodes was tested in the greenhouse on Russian knapweed seedlings and vegetative shoots from root segments. The results demonstrated the feasibility and application of this novel mass production system. Nematodes produced in this system were virulent and the growth rate of infested Russian knapweed were reduced.

There has been little documentation of the success of introduced agents for classical weed biological control. Field evaluation of an insect's establishment, spread and early host impact within its new environment must be performed before agent success can either be doucmented or predicted. Population attributes of the ednophagous biological control agent, Mecinus janthinus Germar (Coleoptera: Curculionidae), and interactions with its target weed, Dalmation toadflax, (Linaria dalmatica (L.) Mill.) (Scrophulariaceae), were explored across variable levels of resource availability and insect abundance. Patterns of population growth and impact of this biocontrol agent were very consistent throughout this study. Within four years of release, populations of M. janthinus achieved outbreak population levels and virtually eliminated the seed producing shoots from toadflax stands. There is a tight but flexible relationship between oviposition site selection and offspring performance in this endophagous herbivore, maximizing offspring survival even under moderate to high M. janthinus densities. These attributes allow M.janthinus to be an effective biocontrol agent under changing levels of resource availability. / ix, 134 leaves : ill. ; 28 cm.

Strains of Bacillus thuringiensis active against Coleoptera were evaluated for toxicity against the adult stage of the carrot weevil, Listronotus oregonensis. Mortality and frass bioassays using a suitable semi-artificial diet showed strains A30, A429 and BTT to be highly toxic. Mortality persisted after initial exposure to the bacteria with the survivors not resuming normal feeding. Attempts to reisolate B. thuringiensis from the insects revealed B. thuringiensis-like organisms in the gut and in/on other structures. At the midgut pH of the insect (pH 8.0), the crystals of the toxic strains were significantly more soluble in vitro than were crystals of the less toxic strain A311. Proteolytic activation of the crystals with gut extracts yielded a protein band (66-67 kDa) for strains A30 and A429 which was similar to he apparent molecular weight of the toxin protein for BTT. Evidence suggests that the low toxicity of strain A311 might be due, in part, to the absence of the toxic moiety of the $ delta$-endotoxin.

Apolipophorin-III is a hemolymph protein known for its role in lipid transport. Apolipophorin-III isolated from the hemolymph of last instar larvae of Galleria mellonella bound to the surface of the insect pathogenic Gram-negative bacterium Xenorhabdus nematophilus and to the lipid A moiety of its lipopolysaccharide. This binding reduced the toxicity of the lipopolysaccharide to hemocytes and decreased the inhibitory effect of the lipopolysaccharide on phenoloxidase. Apolipophorin-III also bound to the Gram-positive bacterium Micrococcus lysodeikticus; this enhanced the activity of hen egg lysozyme on the organism as well as the lytic activity of G. mellonella cell-free hemolymph. / The involvement of apolipophorin-III in the immune responses of G. mellonella larvae to lipoteichoic acids, surface components of Gram-positive bacteria, was examined. Lipoteichoic acids from Bacillus subtilis, Enterococcus hirae and Streptococcus pyogenes caused a dose- and time-dependent drop in the total counts of circulating hemocytes and a partial or complete depletion of plasmatocytes depending on the species of lipoteichoic acid. All lipoteichoic acids tested activated phenoloxidase in vitro; however, in vivo, only B. subtilis lipoteichoic acid elevated the phenoloxidase activity while the other two suppressed it. Binding of apolipophorin-III to lipoteichoic acids was demonstrated. Apolipophorin-III prevented the complete depletion of plasmatocytes and depressed the activation of phenoloxidase by lipoteichoic acid from B. subtilis. The concentration of apolipophorin-III in hemolymph two hours post injections of lipopolysaccharides or lipoteichoic acids into larvae of G. mellonella did not change with respect to control insects that received phosphate-buffered saline. The concentration of apolipophorin-III in hemolymph at the end of the feeding larval stage was 8--12 mg/mL of hemolymph. Apolipophorin-III was present in significant amounts in the prepupal, pupal and adult stages. The protein was detected immunologically in hemocyte lysates, plasma and fat body. Non-denaturing polyacrylamide gels and immunoblots of fresh hemolymph suggested that apolipophorin-III is associated with a 77 kDa protein.