Robert W. Behle

Sunlight Persistence and Rainfastness of Spray-Dried Formulations of Baculovirus Isolated From Anagrapha falcifera (Lepidoptera: Noctuidae)

Abstract 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

Abstract 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 ≈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 2× 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 2× rather than technical powder, caused 62.5% mortality (corn flour-based formulations), and 72.3% mortality (lignin + PCF), versus 53.4% for Dipel 2× after rain. When tested after simulated sunlight, formulations killed 95% of the larvae (average of both formulations) versus 82% for Dipel 2×. 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 2× for 4 d after treatment, but was significantly better than Dipel 2× 7 d after application. A lignin and PCF-based formulation showed significantly higher residual activity than Dipel 2×, 4 and 7 d after application.

Field Activity and Storage Stability of Anagrapha falcifera Nucleopolyhedrovirus (AfMNPV) in Spray-Dried Lignin-Based Formulations

A multiple-embedded nucleopolyhedrovirus isolated from Anagrapha falcifera (Kirby) (AfMNPV) has potential to be developed into a microbial bioinsecticide because the host range includes several economic pests. We tested spray-dried AfMNPV formulations after storage for insecticidal activity based on bioassays with neonate Trichoplusia ni (Hübner). Eight experimental lignin-based spray-dried formulations, a glycerin-based formulation, and an unformulated sample were made with virus stock from three commercial production lots. Samples of these formulations were stored at 30 degrees C in individually sealed sample containers for destructive sampling after 1, 3, and 6 mo whereas the remaining product was stored in glass jars under refrigeration for up to 30 mo. Spray drying did not significantly reduce the initial LC50s of AfMNPV in experimental formulations compared with unformulated virus that was not spray dried. Refrigerated storage for 6 mo did not significantly lower virus activity of formulated samples compared with the unformulated AfMNPV stored frozen, while samples stored for 30 mo had higher LC50 values determined by both droplet and leaf feeding assays. When stored at 30 degrees C, most formulations (22 of 24) maintained insecticidal activity for 3 mo, but most (21 of 24) lost significant activity after 6 mo of storage. The glycerin-based formulation also lost activity within 6 mo of storage at 30 degrees C when compared with frozen unformulated virus, but did not lose activity when stored refrigerated for up to 30 mo. These formulations were evaluated after 7 mo at 4 degrees C for residual insecticidal activity when applied to field grown cabbage. Insecticidal activity was determined against T. ni neonates for treated leaf samples collected at 3, 7, 27, and 51 h after application of 2.5 x 10(12) obs/ha. Field tests showed no differences in activity among samples of stored formulations and one freshly made formulation. Spray-dried formulations had significantly higher insecticidal activity (67.5% mortality) compared with the unformulated treatment (30% mortality) sampled 3 h after application. At 3, 7, and 27 h after application, the spray-dried formulations had higher residual activity (67%, 59%, and 42% mortality, respectively), compared with the commercial glycerin-based formulation (61%, 38%, and 23% mortality, respectively). These experiments demonstrated that AfMNPV in lignin-based spray-dried formulations had a shelf-life of up to 3 mo at 30 degrees C and up to 30 mo at 4 degrees C, and with longer residual insecticidal activity in the field compared with unformulated or a glycerin formulation.

Storage stability of Anagrapha falcifera nucleopolyhedrovirus in spray-dried formulations.

A multiply embedded nucleopolyhedrovirus isolated from Anagrapha falcifera (Kirby) (AfMNPV) can lose insecticidal activity during months of dry storage in ambient room conditions. We tested the spray-dried AfMNPV formulations after storage for up to 1 year at room temperatures for insecticidal activity against neonate Trichoplusia ni (Hübner). Experimental formulations were made using combinations of corn flours, lignin, and sucrose, and were selected based on previous work which demonstrated that these formulations resisted solar degradation in field experiments. Twelve experimental formulations (organized in three groups of four formulations) compared the effect of (1) the ratio of formulation ingredients (lignin and corn flour) to virus concentration, (2) different sources of lignin, or (3) different corn flours and sugar. Based on a single-dose plant assay with these 12 formulations, none of the formulations lost significant activity due to the drying process, when compared with the unformulated wet AfMNPV. Samples of the 12 dried formulations were stored at room (22+/-3 degrees C) and refrigerated (4 degrees C) temperatures. Insecticidal activity (LC(50)) was determined with a dosage-response assay for neonates fed on treated cotton-leaf disks. After 6 (or 9) and 12 months storage, refrigerated samples maintained insecticidal activity better than corresponding samples stored at room temperatures with LC(50)s that averaged 2.0 x 10(6) polyhedral inclusion bodies per milliliter (pibs/ml) for refrigerated samples and 5.4 x 10(6) pibs/ml for samples stored at room temperatures. Compared with unformulated stock virus stored frozen, six formulations stored at room temperature and 10 formulations stored in the refrigerator did not lose significant insecticidal activity after 1 year based on overlapping 90% confidence intervals. Changing the ratio of virus to formulation ingredients did not provide a clear trend over the range of concentrations tested, and may be less important for shelf-life of virus activity compared with formulations made with different ingredients. Two of the four formulations made with different lignins were about 15 times less active after 1 year at room temperature compared with refrigerated samples, indicating that specific formulation ingredients can affect storage stability. Formulations that contained sugar generally maintained activity during storage better than formulations without sugar. Unformulated virus stock maintained insecticidal activity (ranged from 0.20 to 2.5 x 10(6) pibs/ml) better during storage than dried formulations with LC(50)s that ranged from 0.39 to 27 x 10(6) pibs/ml. Unformulated virus stock, which is essentially a suspension of virus occlusion bodies in homogenized insect cadavers, did not lose activity when stored at refrigerated or room temperature. We believe that stability of AfMNPV insecticidal activity during storage as dry formulations is related to the general composition of the formulation and that sugar may play a critical role in maintaining insecticidal activity.

Repellency of a Wax-Based Catnip-Oil Formulation against Stable Flies

Stable flies, Stomoxys calcitrans (L.), are one of the most serious livestock pests, which cause significant economic loss in the cattle industry. Current practices for managing stable flies are limited to costly sanitation techniques and unsustainable insecticide applications. The present study reports the initial efforts using catnip essential oil as a spatial repellent and the results of field trials using a wax-based formulation to repel stable flies in the cattle feedlot. Electroantennograms showed that catnip oil and its ingredient compounds elicit significant antennal responses from both sexes of stable flies. Catnip oil and ZE- and EZ-nepetalactone showed repellent activity in a single cage olfactormeter study. No behavioral activity was observed from another ingredient compound, caryophyllene. A laboratory dispersal bioassay also showed that stable flies avoided areas treated with catnip oil. Using a solid phase microextraction (SPME) method, the atmospheric concentration of catnip active ingredient compounds (nepetalactones) absorbed by SPME fiber in treated areas was detected at 4 times higher than those in control areas. Application of wax-based catnip pellets in cattle feedlots resulted in >99% repellency of stable flies in treated areas, compared with that in nontreated areas. However, the repellent efficacy of the formulation only lasted 3 h. This is the first study demonstrating the potential application of a plant-based repellent formulation that may be used as an alternative method against stable flies.

Evaluation of Soyscreen in an Oil-Based Formulation for UV Protection of Beauveria bassiana Conidia

ABSTRACT Soyscreen oil was studied as a formulation ingredient to protect Beauveria bassiana (Balsamo) Vuillemin conidia from UV degradation. Feruloylated soy glycerides, referred to as Soyscreen oil, are biobased UV-absorbing molecules made by combining molecules of soybean oil with ferulic acid. Conidia stored in Soyscreen oil for 28 wk at 25, 30, and 35°C retained viability as well as conidia stored in sunflower oil, demonstrating that Soyscreen did not adversely affect viability with prolonged storage. For samples applied to glass and exposed to simulated sunlight (xenon light), conidia in sunflower oil with or without sunscreens (Soyscreen or oxyl methoxycinnimate) had similar conidia viability after exposure. These oil formulations retained conidia viability better than conidia applied as an aqueous treatment. However, the 10% Soyscreen oil formulation applied to field grown cabbage (Brassica oleracea L.) and bean (Phaseolus vulgaris L.) plants, did not improve residual insecticidal activity compared with aqueous applications of unformulated conidia or two commercial formulations when assayed against Trichoplusia ni (Hübner) larvae. Our results suggest that the oil applications lose UV protection because the oil was absorbed by the leaf. This conclusion was supported in subsequent laboratory exposures of conidia in oil-based formulations with UV screens applied to cabbage leaves or balsa wood, which lost protection as measured by decreased viability of conidia when exposed to simulated sunlight. As a result, additional formulation techniques such as encapsulation to prevent separation of the protective oil from the conidia may be required to extend protection when oil formulations are applied in the field.

Importance of Direct Spray and Spray Residue Contact for Infection of Trichoplusia ni Larvae by Field Applications of Beauveria bassiana

Commercial formulations and unformulated conidia of Beauveria bassiana strain GHA were applied to field-grown plants and artificially infested with Trichoplusia ni (Hübner) larvae to compare the relative insecticidal activity resulting from direct spray contact with insecticidal activity due to contact with dry spray residue. In general, applications to cabbage, Brassica oleracea L., resulted in nearly equal mortalities when comparing insects exposed to direct spray contact with those exposed by spray residue, suggesting a potential benefit by improving formulations to extend residual activity. For applications to beans, Phaseolus vulgaris L., direct spray contact provided significant insect mortality, but mortality due to residual contact was generally not different than the untreated control. In contrast to the differences observed for larvae exposed in the field, larvae exposed in laboratory bioassays to leaf disks collected from the same treated cabbage and bean plants (residual contact exposure) resulted in nearly identical mortalities. Field applications of Beauveria showed rapid loss of activity, expressed as a loss of conidia viability and loss of insecticidal activity during the first 8 h after application. Evidence of significant mortality by residual contact and the rapid loss of insecticidal activity with field exposure support additional research to improve formulations to extend the residual activity of fungal biopesticides.

Calibration of a Sunlight Simulator for Determining Solar Stability of Bacillus thuringiensis and Anagrapha falcifera Nuclear Polyhedrovirus

Abstract The effect of light on survival of entomopathogens is well described and efforts are underway to develop formulations that may protect an entomopathogen from damage by sunlight. The availability of solar simulators allows for year-round testing of solar protectants. A commercial formulation of Bacillus thuringiensis Berliner and an unformulated baculovirus isolated from Anagrapha falcifera (Kirby) were exposed to various amounts of light from a solar simulator or the sun to determine the relative effect of each source on loss of insecticidal activity. Rate of pathogen degradation was essentially the same for both light sources when original activity remaining was regressed against total energy (as measured by joules/m2). The amount of time required to reduce activity was different, however, because of a difference in total energies produced by the solar simulator and natural sunlight. Virus was approximately two times more sensitive to light than bacteria. To obtain 50% reduction of virus activity, exposure to 1.8 × 107 joules was required, whereas 3.2 × 107 joules was necessary to achieve a similar loss of activity for B. thuringiensis. The importance of reporting energy levels from various solar simulators is discussed.

Comparison of Application Methods for Suppressing the Pecan Weevil (Coleoptera: Curculionidae) with Beauveria bassiana Under Field Conditions

Abstract The pecan weevil, Curculio caryae (Horn), is a key pest of pecans. The entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin is pathogenic to C. caryae. One approach to managing C. caryae may be application of B. bassiana directed toward adult weevils as they emerge from the soil to attack nuts in the tree canopy. Our objective was to compare different application methods for suppression of C. caryae adults. Treatments included direct application of B. bassiana (GHA strain) to soil under the tree canopy, soil application followed by cultivation, soil application in conjunction with a cover crop (Sudan grass), direct application to the tree trunk, and application to the trunk with an UV radiation–protecting adjuvant. The study was conducted in a pecan orchard in Byron, GA, in 2005 and 2006. Naturally emerging C. caryae adults, caught after crawling to the trunk, were transported to the laboratory to determine percentage mortality and signs of mycosis. When averaged over the 15-d sampling period, weevil mortality and signs of mycosis were greater in all treatments than in the nontreated control in 2005 and 2006; >75% average mortality was observed with the trunk application both years and in the trunk application with UV protection in 2005. Results indicated trunk applications can produce superior efficacy relative to ground application, particularly if the ground application is followed by cultivation. Efficacy in the cover crop treatment, however, did not differ from other application approaches. Future research should focus on elucidating the causes for treatment differences we observed and the extent to which B. bassiana–induced C. caryae mortality reduces crop damage.

Formulations of Entomopathogens as Bioinsecticides

Abstract Developing a proper formulation is a necessary component for commercialization of entomopathogenic microbes as biological insecticides. The objective of this chapter is to present broad-ranging information about formulations to foster research toward developing commercial microbial-based insecticides. There are numerous combinations of pathogens, ingredients, processes, formulation types, and application environments to consider for potential commercial biopesticides. Suitable combinations can be identified by a thorough understanding of the entire system beginning with the microbe through various manufacturing processes to the application environment. Key factors can then be addressed by applying specific formulation technologies. Formulation research generally focuses on biological (e.g. viability, efficacy) and physical (e.g. form, mixing, application) aspects of potential biopesticide products. Formulation scientists also need to consider how processes and ingredients affect worker safety, manufacturing hazards, and product registration. Such is the breadth of information presented in this chapter.