Bioactivity of cedarwood oil and cedrol against arthropod pests.

Heartwood samples from Juniperus virginiana L. were extracted with liquid carbon dioxide, and the bioactivity of carbon dioxide-derived cedarwood oil (CWO) toward several species of ants and cedrol toward ticks was determined. Repellency was tested for ants, and toxicity was tested for ticks. Ants in an outdoor bioassay were significantly repelled by the presence of CWO on a pole leading to a sugar-water solution. Similarly, CWO was a significant repellent barrier to red imported fire ants and prevented them from finding a typical food source. Black-legged tick nymphs exhibited dosage-dependent mortality when exposed to cedrol and at the highest dosage (i.e., 6.3 mg/ml) tested, the cedrol killed 100% of the ticks. These repellency and toxicity results together demonstrate a clear potential for the use of CWO as a pest control agent.

Nepetalactones from essential oil of Nepeta cataria represent a stable fly feeding and oviposition repellent.

The stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), is one of the most serious pests to livestock. It feeds mainly on cattle and causes significant economic losses in the cattle industry. Standard stable fly control involving insecticides and sanitation is usually costly and often has limited effectiveness. As we continue to evaluate and develop safer fly control strategies, the present study reports on the effectiveness of catnip (Nepeta cataria L.) oil and its constituent compounds, nepetalactones, as stable fly repellents. The essential oil of catnip reduced the feeding of stable flies by >96% in an in vitro bioassay system, compared with other sesquiterpene-rich plant oils (e.g. amyris and sandalwood). Catnip oil demonstrated strong repellency against stable flies relative to other chemicals for repelling biting insects, including isolongifolenone, 2-methylpiperidinyl-3-cyclohexen-1-carboxamide and (1S,2'S)-2-methylpiperidinyl-3-cyclohexen-1-carboxamide. The repellency against stable flies of the most commonly used mosquito repellent, DEET, was relatively low. In field trials, two formulations of catnip oil provided >95% protection and were effective for up to 6 h when tested on cattle. Catnip oil also acted as a strong oviposition repellent and reduced gravid stable fly oviposition by 98%.

Developing wax-based granule formulations for mating disruption of oriental beetles (Coleoptera: Scarabaeidae) in turfgrass.

Oriental beetle, Anomala orientalis Waterhouse (Coleoptera: Scarabaeidae), is a pest of turfgrass that may be controlled by applications of synthetic pheromone (Z)-/ (E)-7-tetradecen-2-one to disrupt mating. Laboratory experiments were conducted to determine release profiles of pheromone from experimental wax-based granules, a proprietary wax granule, and rubber septa commonly used in pheromone traps. Rubber septa loaded with 10, 100, and 300 microg per septum provided steady rates of pheromone release (zero-order) over 4 wk of laboratory evaluation (total = 1.1, 9.0, and 26.9 microg/4 wk, respectively). Septa with 1,000 microg per septum had a significant decline in the rate of pheromone release for this 4-wk exposure time (total = 119 microg/4 wk). A large proprietary wax granule (44 mg per granule, 25% wt:wt pheromone) provided a steady rate of pheromone release (total = 2,347 microg/4 wk per granule). Experimental granules (16 mg per granule) made of soywax with higher pheromone loads (10% wt:wt) approached zero-order release (steady state) (total = 69 microg/4 wk per granule), whereas smaller granules (4 mg per granule) with less pheromone (0.1% wt:wt) provided first-order release profiles (decreasing rate with longer exposure time) (total = 0.35 microg/4 wk per granule). A field trial in turfgrass demonstrated the potential of selected granular formulations to provide effective mating disruption for up to 4 wk, as measured by pheromone trap shutdown. Documenting pheromone release profiles for these experimental granules and rubber septa provides valuable information that will support future field evaluations of mating disruption as a control strategy.

Evaluation of spray-dried lignin-based formulations and adjuvants as solar protectants for the granulovirus of the codling moth, Cydia pomonella (L).

Commercial formulations of the codling moth, Cydia pomonella L., granulovirus (CpGV) are limited by their short residual activity under orchard conditions in the Pacific Northwest. We evaluated spray-dried lignin-encapsulated formulations of CpGV for improved solar stability based on laboratory bioassays with a solar simulator and in field tests in an infested apple orchard. In laboratory tests, aqueous lignin formulations containing a high dosage of 3 x 10(10) occlusion bodies (OB)/L, with and without the additives titanium dioxide (TiO(2)) and sugar, provided significant solar protection of virus, i.e., mortality of codling moth exposed to lignin formulations that had been irradiated with 9.36 x 10(6) joules/m(2) was 92-94%, compared with 66-67% from a glycerin-stabilized product (Cyd-X) or suspension of pure unformulated virus at the same rates. By comparison, a lower dosage of the lignin formulation (3 x 10(8)OB/L) did not provide significant solar protection. Equivalent dosage-dependent patterns in solar protection were observed in further tests with the lignin formulation, when an intermediate (3 x 10(9)OB/L) as well as the low dosage provided no solar protection. Equivalent rates of a blank lignin formulation (containing no virus) did not affect larval mortality, suggesting a protective effect of the lignin on the virus at the high rate. The use of several spray adjuvants, 'NuFilm-17' and 'Organic Biolink' (sticker-spreaders at 0.06% v/v), 'Raynox' (sunburn protectant at 5% v/v), and 'Trilogy'(neem oil at 1% v/v) did not provide solar protection of a commercial CpGV preparation in laboratory tests. In season long orchard tests (Golden Delicious), the lignin formulation of CpGV applied at 6.57 x 10(12)OB/ha did not significantly improve control of codling moth or protection of fruit compared with Cyd-X at equivalent rates. Our studies show that lignin-based CpGV formulations provided solar protection at relatively high virus dosages. The testing of lignin formulations containing reduced virus concentrations may allow virus solar protection to be achieved at more economical rates.

Formulation of Bacillus spp. for Biological Control of Plant Diseases.

ABSTRACT Maximizing the potential for successfully developing and deploying a biocontrol product begins with a carefully crafted microbial screening procedure, proceeds with developing mass production protocols that optimize product quantity and quality, and ends with devising a product formulation that preserves shelf-life, aids product delivery, and enhances bioactivity. Microbial selection procedures that require prospective bio-control agents to possess both efficacy and amenability to production in liquid culture increase the likelihood of selecting agents with enhanced commercial development potential. Scale-up of biomass production procedures must optimize product quantity without compromise of product efficacy or amenability to stabilization and formulation. Formulation of Bacillus spp. for use against plant pathogens is an enormous topic in general terms but limited in published specifics regarding formulations used in commercially available products. Types of formulations include dry products such as wettable powders, dusts, and granules, and liquid products including cell suspensions in water, oils, and emulsions. Cells can also be microencapsulated. Considerations critical to designing successful formulations of microbial biomass are many fold and include preserving biomass viability during stabilization, drying, and rehydration; aiding biomass delivery, target coverage, and target adhesion; and enhancing biomass survival and efficacy after delivery to the target. Solutions to these formulation considerations will not necessarily be compatible. Data from several biocontrol systems including the use of B. subtilis OH 131.1 (NRRL B-30212) to reduce Fusarium head blight of wheat are used to illustrate many of these issues. Using our recently described assay for efficiently evaluating biomass production and formulation protocols, we demonstrate the effectiveness, in vitro, of UV protectant compounds lignin (PC 1307) and Blankophor BBH in reducing OH 131.1 morbidity when cells were exposed to UV light from artificial sunlight.

Effect of transgenic plants expressing high levels of a tobacco anionic peroxidase on the toxicity of Anagrapha falcifera nucleopolyhedrovirus to Helicoverpa zea (Lepidoptera: Noctuidae).

Wild type and corresponding transgenic tomato (Lycopersicon esculentum Miller) and two tobacco (Nicotiana spp.) plants that express high levels of a tobacco anionic peroxidase were used to determine what type of interactions occurred between peroxidase altered plant chemistry and the baculovirus Anagrapha falcifera nucleopolyhedrovirus (AfMNPV) for control of neonate corn earworms, Helicoverpa zea (Boddie). Transgenic plants expressed approximately five to 400 times higher peroxidase activity than corresponding tissues of wild type plants. The H. zea larvae typically fed 1.5 times less on transgenic compared with wild type leaf disks. There was only one experiment (of three with tomato leaves) where the larvae that fed on transgenic leaves were less susceptible to the virus based on nonoverlapping 95% confidence intervals for LC50 values. When the exposure dose was corrected for reduced feeding on the transgenic leaf disks, the insecticidal activity of the virus was not significantly different for larvae fed on transgenic versus wild type plants. Eight other experiments (with tomato and two species of tobacco) indicated either no significant effect or enhanced susceptibility (when corrected for feeding rates) to the virus of larvae fed on the transgenic leaves. These results indicate enhanced insect resistance in plants expressing high levels of a specific anionic peroxidase may be compatible with applications of AfMNPV. Potential reasons for this compatibility are discussed.

Comparative field stability of selected entomopathogenic virus formulations.

Nucleopolyhedroviruses originally isolated from Anagrapha falcifera (Kirby) and Autographa californica (Speyer) were formulated with various ingredients using a spray dry method and tested for residual field activity in Illinois and Mississippi. In Mississippi, field tests were conducted on cotton in 1997, whereas in Illinois tests were conducted on cabbage in 1997 and 1998. Within 24 h, significant differences were observed among formulations in all tests. Unformulated virus had significantly less insecticidal activity than formulated virus and formulations containing lignin retained activity significantly longer than other formulations. Relatively small amounts of Blankophor BBH, when encapsulated within the formulation, did not greatly enhance (>10x) insecticidal activity based on LC50 determinations nor prolong insecticidal activity based on field evaluations. In most tests, >50% activity remained in formulations containing lignin, whereas unformulated virus retained <50% activity within 24 h after application.

Formulation of entomopathogenic nematode-infected cadavers.

Entomopathogenic nematodes are commercially applied in aqueous suspension. These biocontrol agents may also be applied in nematode-infected insect cadavers, but this approach may entail problems in storage and ease of handling. We determined the feasibility of formulating nematode-infected insect cadavers to overcome these hindrances. All experiments were conducted with Heterorhabditis bacteriophora Poinar and Galleria mellonella (L.). Nonformulated cadavers were used as controls. Of 19 formulations tested (including combinations of starches, flours, clays, etc.) 1 (starch-clay combination) was found to adhere to the cadaver and to have no significant deleterious effects on nematode reproduction and infectivity; other formulations exhibited poor adhesion or reduced nematode reproduction. Two formulations enabled cadavers to be partially desiccated without affecting reproduction; other formulations and nonformulated cadavers exhibited reduced reproduction upon desiccation. Four-day-old cadavers were more amenable to desiccation than 8-day-old cadavers. Formulated cadavers were more resistant to rupturing and sticking together during agitation than nonformulated cadavers.

Consumption of residue containing cucurbitacin feeding stimulant and reduced rates of carbaryl insecticide by western corn rootworm (Coleoptera: Chrysomelidae).

Application of insecticide at a reduced rate with a cucurbitacin-based feeding stimulant is a viable alternative to a broadcast insecticide application for control of adult western corn rootworms, Diabrotica virgifera virgifera, LeConte. Because of the small amount of material applied, it is conceivable that a high density of beetles could consume all of the spray residue before economic control is achieved. A laboratory experiment was conducted to determine the amount of cucurbitacin-based spray residue consumed by beetles. Dried residue of four treatments were exposed to three groups of 10 rootworm beetles for 1 h each. Treatments consisted of a cucurbitacin-based adjuvant (Cidetrak CRW, Trécé, Salinas, CA) with carbaryl insecticide (Sevin XLR Plus, Rhone Poulenc, Research Triangle Park, NC) mixed at 0, 0.12, 1.2, and 12 g (AI)/liter. For the treatment with cucurbitacin adjuvant only (no insecticide), beetles consumed 0.029 mg beetle(-1) h(-1) of exposure. Approximately 54% of the beetles were recorded as feeding at any given time during the 60-min feeding period. However, when the spray residue contained carbaryl, no weight loss of treatment residue was measured, though the beetles were observed to feed from the residue during the first few minutes of exposure. When residue included insecticide, beetles quickly ceased feeding (within 20 min), and toxicity behavior was observed 30 min after initial exposure for up to 75% of the beetles, which were classified as moribund (unable to stand upright). Beetle mortality was recorded 24 h after exposure and demonstrated that male beetles (53% dead for three insecticide treatments) were more susceptible to carbaryl toxicity than female beetles (28% dead for three insecticide treatments). Regression analysis showed a significant positive relationship between mortality of female beetles and ovarian development. Based on the measurements of this experiment, it is unlikely that realistic beetle densities would consume enough spray residue to prevent economic control of the beetle population.

Multiacreage evaluation of aerially applied adherent malathion granules for selective insect control and indirect reduction of mycotoxigenic fungi in specialty corn.

Aerially applied adherent corn flour granules containing 1% malathion were more often as, or more, effective than 15% chlorpyrifos (Lorsban 15G) granules in controlling caterpillars and sap beetles in high amylose corn in 1997 than 1996. Use of malathion granules corresponding closely in size to chlorpyrifos granules in the second year of the study apparently increased relative efficacy. Significantly less corn borer damage occurred on plants (1996) or ears (1997) within 2 wk of application for both types of insecticide granules compared with untreated plots. In 1997, there were sixfold fewer milk stage ears with more than 20 kernels damaged per ear in the malathion-treated plots compared with chlorpyrifos-treated plots, and severity of caterpillar damage was also less in malathion versus chlorpyrifos-treated plots at harvest. Control of beetles (corn rootworm adults and sap beetles) for both treatments was less effective compared with caterpillars. Significant corn rootworm adult control was noted for both chlorpyrifos and malathion in 1996 and significant sap beetle control was noted for the malathion granules in 1997. Significantly fewer live lady beetles, and more dead lady beetles were present in chlorpyrifos-treated plots compared with malathion-treated or untreated plots in 1996. The incidence and severity of Fusarium mold on ears at harvest was often indirectly reduced by both malathion treatments and chlorpyrifos treatments, with the malathion treatment significantly better than the chlorpyrifos treatment in one case.

Effect of light energy on alkali-released virions from Anagrapha falcifera nucleopolyhedrovirus.

We compared the insecticidal activities of occluded and nonoccluded AfMNPV baculovirus obtained by dissolving the occlusion bodies (OB) with sodium carbonate. Droplet feeding and cotton leaf feeding bioassay techniques were used to determine the dose response against neonate Trichoplusia ni (Hübner) and loss of insecticidal activity when the virus was exposed to simulated sunlight from a xenon light source. Using droplet bioassays to determine a dose response, nonoccluded virus (NOV) was 20 times more active (LC(50) = 4.8 x 10(3) OB/ml, dissolved) than occluded virus (LC(50) = 9.6 x 10(4) OB/ml) when the samples remained wet. However, NOV lost activity when air dried before being tested by droplet (LC(50) > 1.0 x 10(6) OB/ml) or leaf feeding (LC(50) > 3.0 x 10(6) OB/ml) bioassays. Adding sucrose to NOV prevented the loss of insecticidal activity when samples were dried. The activity of NOV with 2% sucrose was similar to that of occluded virus samples, with or without sucrose, in both droplet feeding and leaf feeding assays. These results indicate that the OB protected the insecticidal activity of virions from the detrimental effects of drying. The OB also provided some protection from the detrimental effects of simulated sunlight (xenon) exposure. NOV samples exposed to xenon light had significantly greater loss of insecticidal activity than did similar samples of occluded virus. Without advancement in technologies, such as formulations, possible benefits of increased insecticidal activity from the use of nonoccluded virus is probably not sufficient to offset the rapid loss of activity due to drying or light exposure.

Sunlight persistence and rainfastness of spray-dried formulations of baculovirus isolated from Anagrapha falcifera (Lepidoptera: Noctuidae).

Nuclear polyhedrosis viruses such as the one isolated from the celery looper, Anagrapha falcifera (Kirby) (AfMNPV), have the potential to be successful bioinsecticides if improved formulations can prevent rapid loss of insecticidal activity from environmental conditions such as sunlight and rainfall. We tested 16 spray-dried formulations of AfMNPV to determine the effect of different ingredients (e.g., lignin, corn flour, and so on) on insecticidal activity after simulated rain and simulated sunlight (at Peoria, IL) and natural sunlight exposures (at Tifton, GA). The most effective formulation contained pregelatinized corn flour and potassium lignate, which retained more than half of its original activity after 5 cm of simulated rain, and almost full activity after 8 h of simulated sunlight. In Georgia, formulations made with and without lignin were compared for persistence of insecticidal activity when exposed to natural sunlight. In addition, the effect of fluorescent brighteners as formulation components and spray tank additives was tested. Results showed that the formulations with lignin had more insecticidal activity remaining after sunlight exposure than formulations without lignin. The inclusion of brighteners in the formulation did not improve initial activity or virus persistence. However, a 1% tank mix significantly enhanced activity and improved persistence. Scanning electron micrographs revealed discreet particles, and transmission electron micrographs showed virus embedded within microgranules. Results demonstrated that formulations made with natural ingredients could improve persistence of virus-based biopesticides.

Assessment of microencapsulated formulations for improved residual activity of Bacillus thuringiensis.

Bacillus thuringiensis Berliner is a highly efficacious bioinsecticide used to control lepidopteran pests in the field. Unfortunately, it has limited residual activity on plants because sunlight inactivates spores and crystals and they can be washed off by rain. To minimize loss of activity, formulations must contain UV protectants, stickers, or both. We tested approximately 80 formulations and determined optimal combinations of ingredients and spray drying conditions for improving B. thuringiensis residual activity after simulated rain and simulated sunlight. B. thuringiensis stability, after simulated sunlight (xenon light/8 h) and rain (5 cm/50 min), was improved using formulations based on lignin, corn flours, or both, with up to 20% of the active ingredient, when compared with technical powder or Dipel 2x in laboratory assays. Two formulations, made with corn flours or lignin + pregelatinized corn flour (PCF), killed 51.6 and 75.3% of Ostrinia nubilalis (Hübner) neonates after rain, respectively, versus 27% for technical powder. When the insecticidal activity was tested after simulated sunlight, corn flour-based formulations killed 78.5% of test larvae, and the lignin + PCF formulation killed 70.4%, in contrast to technical powder which caused an average of 29% mortality. Formulations made with Dipel 2x rather than technical powder, caused 62.5% mortality (corn flour-based formulations), and 72.3% mortality (lignin + PCF), versus 53.4% for Dipel 2x after rain. When tested after simulated sunlight, formulations killed 95% of the larvae (average of both formulations) versus 82% for Dipel 2x. In a field test, formulations were applied to cabbage and insecticidal activity was determined against Trichoplusia ni (Hübner) neonates exposed to treated leaves. Insecticidal activity of the corn flour-based formulations was comparable to Dipel 2x for 4 d after treatment, but was significantly better than Dipel 2x 7 d after application. A lignin and PCF-based formulation showed significantly higher residual activity than Dipel 2x, 4 and 7 d after application.

Detection of Salmonella enteritidis in feces from poultry using booster polymerase chain reaction and oligonucleotide primers specific for all members of the genus Salmonella.

Salmonella enteritidis was identified in feces from hens using the polymerase chain reaction (PCR) and oligonucleotide primers specific for all members of the genus Salmonella. Feces from specific-pathogen-free Leghorn hens were determined to be negative for Salmonella by microbiological culture and by the PCR. Fecal samples were inoculated with known numbers of colony-forming units of S. enteritidis. The DNA was extracted from fecal samples and amplified by the PCR using genus-specific primers. Salmonella were detected in all samples known to be positive; the sensitivity of the assay extended to 1 cfu of S. enteritidis/g feces. Feces that were not inoculated with Salmonella were negative. Microbiological culture was less sensitive than the PCR assay; results of culture of feces with less than 10(2) cfu/g were negative. Although S. enteritidis was used in this study, the oligonucleotide primers used in this study have been previously demonstrated to be genus-specific for Salmonella.

Genus-specific detection of salmonellae using the polymerase chain reaction (PCR).

Oligonucleotide primers for the polymerase chain reaction (PCR) that enable genus-specific detection of members of the genus Salmonella were developed. The primers amplify a 496-bp genetic sequence of members of the genus Salmonella. Amplification of DNA extracted from all other genera of the family Enterobacteriaceae and various other gram-positive aerobic and anaerobic bacteria yielded negative results. Applications of the PCR using these genus-specific primers are discussed.