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The anther culture response of diploid Solanum chacoense (chc) - S. phureja (phu) hybrids and the regeneration potential of anther-derived monoploids was evaluated. In vivo evaluation of interspecific hybrids was also performed. Three hybrids were anther cultured to observe the effects of reduced nitrogen source on androgenesis. Anthers were distributed to five reduced nitrogen sources. The N concentration was 30 mM. No tested reduced nitrogen source proved superior to the control. Genotype significantly affected embryo production. Eleven monoploid genotypes were included in a leaf disc regeneration procedure utilizing three separate transfers to fresh medium differing by growth regulator composition; six genotypes responded. Silver thiosulfate (STS) at either of two steps in the process proved detrimental to diploid recovery. Hybrids between phu and chc involving six phu clones and eight chc clones or accessions, all resistant to Colorado potato beetle (CPB), were used. No inter-family differences for germination, fruit/pollination, or seed/fruit were observed. Substantial mortality, ascribed to the phenomenon of “hybrid breakdown”, occurred in three families by month four of the study. Field plantings revealed adequate CPB resistance, while Ambush (147 g/ha) application increased total tuber weight per plant and average tuber weight. Hybrids produced less total tuber weight than S. tuberosum (tbr), while chc genotypes produced the smallest average tuber size. Interspecific hybrids produced tuber sizes intermediate between chc and phu. Tbr tubers were the largest. Chc families, regardless of selection for leptine glycolakaloids, suffered the least CPB damage and phu parental clones and hybrid families suffered the most. / Master of Science

Within-field variation in mortality of Colorado potato beetle, Leptinotarsa decemlineata (Say), in a filter-paper insecticide bioassay was determined in three Virginia potato fields in 1989 and five fields in 1990. Bioassays were performed for each of three different insecticides on Colorado potato beetle larvae collected from 12-16 different locations (equal sized blocks) per field. Comparisons of 95% mortality confidence intervals between all block combinations per field indicated that field size had no influence on variation in Colorado potato beetle mortality in the bioassay. No apparent relationship existed between the level of Colorado potato beetle insecticide susceptibility (LC₉₀ value) and percentage of mortality confidence interval overlap among field blocks. Bioassay data from the sample fields indicated that a single bioassay (of potato beetles from one location per field) will yield at least a 0.50 probability (> 0.90 in six of the eight fields) of estimating the mean mortality response (± 5%) of Colorado potato beetle in the entire field. The probability of sampling potato beetles from one location which differed in mean mortality (P ≤ 0.05) from individuals collected in another location within the same field decreased as the number of locations sampled increased (i.e., the probability is 0.12 if three locations are sampled). Filter paper insecticide bioassays were performed on Colorado potato beetles collected from three commercial potato fields to determine the most cost-effective number of bioassay sample units (filter paper disks, 10 larvae per disk). Relative net precision values from three different insecticide bioassays were used as an indication of sample size efficiency, and were based on variation in larval mortality and sample cost. Greatest sample size efficiency in all insecticide bioassays was achieved from a sample of two to five filter paper disk sample units (20-50 total larvae). Additional insecticide bioassays were performed on Colorado potato beetle larvae collected at different times during the potato growing season (from a commercial potato field and from experiment station plots) to determine whether the larval generation sampled or previous insecticide application affected results of the bioassay. Although trends in mortality were not always consistent among first generation larvae sampled on different dates (from insecticide-treated and untreated plots), first generation larvae exhibited significantly (P ≤0.05) greater mortality in the bioassay compared with second generation larvae. Based on these results, we recommend that bioassays to estimate the effectiveness of a particular insecticide against Colorado potato beetle be performed on the target generation immediately before the planned insecticide treatment. PIES (potato insect expert system) is a knowledge-based system for Colorado potato beetle insecticide management in commercial potato fields on the Eastern Shore of Virginia. PIES was written using VP-Expert, a rule-based expert system shell, and uses Colorado potato beetle lifestage, potato growth stage, percent defoliation, and other factors to decide if an insecticide application is necessary to prevent tuber yield loss due to Colorado potato beetle pressure. Field tests at the Eastern Shore Agricultural Experiment Station compared PIES with conventional spray thresholds based on the number of Colorado potato beetles per stem. Tuber yields were not significantly different (P ≤ 0.05) between the two methods, while PIES recommended, on average, 3.7 insecticide applications and conventional thresholds required six insecticide applications. An additional benefit of PIES is that scouting requirements are simpler and quicker than the conventional stem counts. Supercooling points determined for Colorado potato beetle populations from Maine, Washington, and Prince Edward Island, Canada, suggest these populations are freeze-tolerant, that is, they can sustain ice formation within their body fluids. Colorado potato beetles collected from Virginia supercooled at a significantly (P ≤ 0.05) lower temperature than the other Colorado potato beetle populations. The temperature at which Virginia Colorado potato beetle supercooled is indicative of freeze-sensitive species and may indicate divergence in the mechanisms Colorado potato beetle are using for cold tolerance. Two polyhydric alcohols (polyols) were isolated in the hemolymph of Colorado potato beetle using high performance thin layer chromatography. The polyols were tentatively identified as inositol and xylitol. / Ph. D.

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The Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), is the most devastating foliage-feeding pest of potatoes in the United States. Potential biological control agents include the nematodes Heterorhabditis marelatus Liu & Berry and Steinernema riobrave Cabanillas, Poinar & Raulston, which provided nearly 100% CPB control in previous laboratory trials. In the present study, laboratory assays tested survival and infection by the two species under the soil temperatures CPB are exposed to, from 4-37°C. H. marelatus survived from 4-31°C, and S. riobrave from 4-37°C. Both species infected and developed in waxworm hosts from 13-31°C, but H. marelatus rarely infected hosts above 25°C, and S. riobrave rarely infected hosts below 19°C. H. marelatus infected an average of 5.8% of hosts from 13- 31°C, whereas S. riobrave infected 1.4%. Although H. marelatus could not survive at temperatures as high as S. riobrave. H. marelatus infected more hosts so is preferable for use in CPB control. Heterorhabditis marelatus rarely reproduced in CPB. Preliminary laboratory trials suggested the addition of nitrogen to CPB host plants improved nematode reproduction. Field studies testing nitrogen fertilizer effects on nematode reproduction in CPB indicated that increasing nitrogen from 226 kg/ha to 678 kg/ha produced 25% higher foliar levels of the alkaloids solanine and chaconine. However, the increased alkaloids did not affect nematode infection of, nor reproduction in, CPB prepupae. Nematodes applied to field plot soil at 50 infective juveniles/cm² reduced adult CPB by 50%, and increased numbers of dead prepupae in soil samples up to five times more than in non-nematode plots. Laboratory studies of H. marelatus and its symbiotic bacteria in CPB hemolymph indicated that immune responses did not limit nematode reproduction. A 58kD CPB hemolymph protein apparently caused the symbiotic bacteria to switch to the secondary form, which does not produce antibiotics and enzymes necessary for nematode growth and reproduction. Despite heat denaturation of the protein, the nematodes did not reproduce unless lipids were added to the hemolymph. Therefore, while H. marelatus may provide high levels of CPB control, nutritional constraints on the nematode and its bacteria inhibit reproduction in CPB and limit long-term multi-generation control. / Graduation date: 2003

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