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.     

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.     

Evaluating conditions for producing spray-dried formulations of Anagrapha falcifera nucleopolyhedroviruses (AfMNPV)

We investigated how altering parameters during the production of spray-dried lignin formulations affected the insecticidal activity of a baculovirus (AfMNPV), which was originally isolated from celery looper, Anagrapha falcifera (Kirby). Exposure to high temperature, varied pH of the dryer feedstock, substitution of formulation ingredients, and different commercial production lots of virus were evaluated for their effect on the insecticidal activity of AfMNPV. Insecticidal activities of treatments were determined by droplet-feeding assays using neonate Trichoplusia ni (Hübner) for dosage response or single-dosage comparisons. Unformulated virus exposed to 68^oC for 2 h showed no loss of insecticidal activity, whereas exposure to 90^oC for 30 min caused >20% loss of activity. Thus, residence at higher temperatures in drying systems could adversely affect virus activity. Spray-dried formulations made with Indulin AT lignin (pH 8.5, 9.5, and 10.5) lost insecticidal activity with increasing alkalinity of the dryer feedstock. In contrast to Indulin AT, the same formulations made with PC-1307 lignin did not lose insecticidal activity with increased alkalinity. Adding corn flour to spray-dried Indulin AT-based formulations improved insecticidal activity of the virus. These experiments demonstrated the importance of carefully selecting feed-stock ingredients and processing conditions when spray drying AfMNPV.     

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.     

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.     

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.     

Laboratory and field evaluations of the efficacy of a fast-killing baculovirus isolate from Spodoptera frugiperda

Three biopesticide parameters were evaluated for a fast-killing isolate (3AP2) and a wild-type isolate (Sf3) of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV). Both isolates were evaluated for virus production using in vivo methods, for speed of kill based on bioassay of applications to glasshouse-grown and field-grown plants, and for residual insecticidal activity of unformulated virus and an encapsulating formulation to provide UV protection. Two inoculation rates comparing relative in vivo production of the isolates demonstrated 3AP2 inoculated larvae were significantly smaller than Sf3 inoculated larvae at death. At the lower inoculation rate, Sf3 inoculated larvae produced approximately twofold more occlusion bodies as the 3AP2 inoculated larvae. A model system of applications to cabbage plants and a bioassay to observe mortality of neonate S. frugiperda (J.E. Smith) after feeding on samples of treated leaves was used to evaluate speed of kill and residual insecticidal activity. The LT(50) for the 3AP2 isolate was at least 30 h less than the LT(50) for the Sf3 isolate when applied to either glasshouse-grown or field-grown plants. The spray-dried lignin encapsulating formulation provided similar benefits to both virus isolates when exposed to simulated sunlight in the laboratory and to natural sunlight in the field. For treatment applications to field grown cabbage in June, the half-life for efficacy of unformulated virus was <7.5 h compared with a half-life of >26.7 h for encapsulated virus. These results demonstrate that improved technologies can be combined to address characteristics which otherwise can limit the commercial potential of microbial-based biological insecticides.     

Effect of optical brighteners on the insecticidal activity of a nucleopolyhedrovirus in three instars of Spodoptera frugiperda

Certain optical brighteners are effective UV protectants, and can improve the insecticidal activity of baculoviruses. We evaluated the effect of 10 optical brighteners, from four chemically different groups, on the insecticidal activity of a nucleopolyhedrovirus (SfMNPV) in third instar Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae). The most effective optical brighteners were Blankophor BBH and Calcofluor M2R, both of which are stilbenes. The distyryl‐biphenyl derivative, Tinopal CBS, had no effect, whereas the stilbenes, Blankophor CLE and Leucophor SAC and the styryl‐benzenic derivative, Blankophor ER, resulted in a decrease in virus induced mortality compared to larvae infected with SfMNPV alone. Mixtures of SfMNPV + 0.1% Calcofluor M2R had relative potencies of 2.7, 6.5, and 61.6 in the second, third, and fourth instars, respectively. The mean time to death differed with instar, but was not affected by the addition of 0.1% Calcofluor M2R. Analysis of published studies indicated that the concentration of Calcofluor M2R‐related stilbenes was positively correlated with the relative potency observed in mixtures with homologous NPVs. The average magnitude of optical brightener activity did not differ significantly between early instars of 10 species of Lepidoptera. We conclude that virus formulations containing optical brighteners may be valuable for control of late instar lepidopteran pests.     

荧光增白剂对伊朗核型多角体病毒分离株对棉铃虫幼虫的杀虫活性的增效作用（英文）

在室内条件下调查了几种荧光增白剂对采自伊朗East Azarbaijan的棉铃虫Helicoverpa armigera核型多角体病毒两个地域株(EAZ-I 和EAZ-II)对棉铃虫2龄幼虫的杀虫活性, 以提高这两个地域株的生物学活性。结果表明：与EAZ-II相比, EAZ-I的杀虫活性强,其对棉铃虫幼虫的LC₅₀和 LT₅₀ 值低（分别为1.98×103 OB/mL和122.7 h）。本研究所用的所有荧光增白剂均能有效增强病毒的生物学活性,特别是0.2% 的Tinopal F-3543与EAZ-I 混用对棉铃虫幼虫的LC50值最低(5.16×102 OB/mL),与病毒单独应用相比活性增强了3.84倍。幼虫致死的相对速率测定结果表明,荧光增白剂提高了菌株的LT₅₀值,其中Tinopal F-3543 的效果最佳。这些结果说明,在害虫综合治理中影响围食膜通透性的荧光增白剂与核型多角体病毒制剂混用是一种可供选择的重要方法。     

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.     

Coating Beauveria bassiana with lignin for protection from solar radiation and effects on pathogenicity to Lygus lineolaris (Heteroptera: Miridae)

The entomopathogenic fungus, Beauveria bassiana, is highly susceptible to the damaging effects of solar radiation. This study demonstrates protection from simulated solar radiation by coating B. bassiana (GHA) spores with lignin and effects on pathogenicity to Lygus lineolaris (Palisot de Beauvois) adults in direct spray and contact uptake bioassays. Spores were coated with either lignin or cross-linked lignin by spray drying to produce spore coatings of high and low water solubility, respectively. Non-coated spores and the two spore coating treatments were suspended in either water (0.04% Silwet L77) or oil (Orchex 692) to produce six formulations. Rates of loss in spore viability under simulated solar radiation were approximately ten times lower for the three formulations in which spores remained coated in suspension (cross-linked lignin in water, cross-linked lignin in oil, and lignin in oil). However, these three formulations were the least pathogenic. Estimates of LC₅₀ and LC₇₅ values for the most pathogenic formulation (non-coated in oil) were 5 and 12 times lower, respectively, than the least pathogenic formulation (cross-linked lignin in oil), but these differences were not significant. The three lignin-coated formulations that provided the greatest UV protection were also the least pathogenic based on LT values, which were significant. Overall, L. lineolaris mortality was approximately 80 times less when exposed to treated broccoli rather than sprayed directly, based on LC₅₀ values. If the contribution of spore uptake from plant surfaces to L. lineolaris mortality is similarly low under field conditions, then improving persistence may be less important for improving efficacy. However, under control conditions where solar radiation significantly impacts mycoinsecticide efficacy, the improved persistence of lignin coating formulations may outweigh negative effects on pathogenicity.     

Effect of different additives on the persistence and insecticidal activity of native strains of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The persistence and insecticidal activity of native strains of Bacillus thuringiensis was evaluated in formulations containing different additives such as arrow-root powder, carboxy methyl cellulose (CMC), gum acacia, non-food grade (NFG) starch, and soluble starch. Persistence of B. thuringiensis varied with different additives used in the formulations. Among the different additives used, NFG starch provided maximum protection to B. thuringiensis and native strain 42 showed maximum persistence (83%) which was higher than that obtained in commercial formulation. In commercial formulation, the persistence of B. thuringiensis was 47% only after 3 d of spray. The feeding trials conducted on second instar larvae of H. armigera using leaves sprayed with NFG starch formulation revealed 70% larval mortality while commercial formulation showed 50% mortality during the same period.     

Effects of stilbene optical brighteners on the insecticidal activity of Bacillus thuringiensis and a single nucleopolyhedrovirus on Helicoverpa armigera

The incorporation of certain stilbene optical brighteners into virus-based formulations has been demonstrated to increase viral pathogenicity (as indicated by reduced LD/LC₅₀ values) but their effect on Bacillus thuringiensis activity has been scarcely investigated. We determined the effect of nine optical brighteners on the insecticidal activity of B. thuringiensis ser. kurstaki HD-1 strain (Bt HD-1) on Helicoverpa armigera and also compared the effect of two optical brighteners on the insecticidal activity of Bt HD-1 and occlusion bodies (OBs) of a Spanish isolate of H. armigera single nucleocapsid nucleopolyhedrovirus (HearNPV-SP1). Blankophor CLE, Blankophor DRS, Blankophor ER, and Leucophor SAC significantly increased the pathogenicity of Bt HD-1. In contrast, Tinopal UNPA-GX, Tinopal CBS, Blankophor BA, Leucophor AP, and Leucophor UO had an adverse or no effect on its insecticidal activity. Mixtures of HearNPV-SP1 OBs with Tinopal UNPA-GX or Leucophor UO resulted in 31.4- and 11.4-fold increases in pathogenicity, respectively, at 1%, and 11.4- and 6.3-fold increases in pathogenicity, respectively, at 0.1%, compared to the OBs alone. However, none of these brighteners increased Bt HD-1 activity. These results appear consistent with the hypothesis that the enhancement of HearNPV-SP1 pathogenicity and the null or antagonistic effects observed in Bt HD-1 against H. armigera were due to optical brightener-mediated degradation of the peritrophic membrane, but additional systematic studies involving a broad range of brighteners and electron microscope observations are required to confirm this premise.     

The Role of Formulation Additives in Increasing the Potency of Cydia pomonella Granulovirus for Codling Moth Larvae,in Laboratory and Field Experiments

Studies were undertaken to improve the biological efficacy of the granulovirus (CpGV) of the codling moth, Cydia pomonella , by evaluating the performance of some formulation additives that might improve virus persistence and/or virus uptake by first instar larvae. Laboratory studies, using a leaf disc bioassay, demonstrated that 15% cane molasses incorporated within a formulation of purified CpGV dramatically reduced the median lethal exposure time (LET 50 ) to CpGV for neonate larvae at a CpGV dosage rate of 10 7 occlusion bodies (OBs) ml -1 . Screening of a range of other compounds showed that sucrose, fructose and sorbitol (at 10% concentrations) and extracts of apple flesh and skin also gave significant reductions in the LET 50 of CpGV formulations containing these ingredients. Pectin, malic acid and &#102 -farnesene did not significantly reduce the LET 50 . In a field trial, molasses included at 15% (v/v) in a CpGV formulation, containing a dosage rate of 10 12 OBs ha -1 , gave as good control of codling moth damage as virus formulations containing the 'sticker' 0.2% skimmed milk at higher dosage rates of 10 13 and 10 14 OBs ha -1 . Studies of CpGV persistence on foliage revealed no significant improvement of virus persistence on apple foliage using 10% or 15% molasses formulations. A second field trial demonstrated that 10% molasses, 10% sorbitol or 0.08% &#102 -farnesene significantly reduced codling moth deep damage to fruit when these ingredients were added to formulations of pure CpGV. Substantial sooty-mould growth ( Cladosporium spp.) was observed on apple foliage treated with formulations containing molasses, indicating that this formulation additive has secondary consequences that would need to be taken into account if molasses was to be used in commercial CpGV formulations. Nonetheless, these studies clearly demonstrate that major biological improvements in CpGV performance can be achieved by the incorporation of formulation additives, including molasses and several other compounds, that probably function as attractants and/or feeding stimulants for codling moth larvae.     

Effects of stilbene optical brighteners on the insecticidal activity of Bacillus thuringiensis and a single nucleopolyhedrovirus on Helicoverpa armigera

The incorporation of certain stilbene optical brighteners into virus-based formulations has been demonstrated to increase viral pathogenicity (as indicated by reduced LD/LC₅₀ values) but their effect on Bacillus thuringiensis activity has been scarcely investigated. We determined the effect of nine optical brighteners on the insecticidal activity of B. thuringiensis ser. kurstaki HD-1 strain (Bt HD-1) on Helicoverpa armigera and also compared the effect of two optical brighteners on the insecticidal activity of Bt HD-1 and occlusion bodies (OBs) of a Spanish isolate of H. armigera single nucleocapsid nucleopolyhedrovirus (HearNPV-SP1). Blankophor CLE, Blankophor DRS, Blankophor ER, and Leucophor SAC significantly increased the pathogenicity of Bt HD-1. In contrast, Tinopal UNPA-GX, Tinopal CBS, Blankophor BA, Leucophor AP, and Leucophor UO had an adverse or no effect on its insecticidal activity. Mixtures of HearNPV-SP1 OBs with Tinopal UNPA-GX or Leucophor UO resulted in 31.4- and 11.4-fold increases in pathogenicity, respectively, at 1%, and 11.4- and 6.3-fold increases in pathogenicity, respectively, at 0.1%, compared to the OBs alone. However, none of these brighteners increased Bt HD-1 activity. These results appear consistent with the hypothesis that the enhancement of HearNPV-SP1 pathogenicity and the null or antagonistic effects observed in Bt HD-1 against H. armigera were due to optical brightener-mediated degradation of the peritrophic membrane, but additional systematic studies involving a broad range of brighteners and electron microscope observations are required to confirm this premise.     

Comparative activity of baculoviruses against the codling moth Cydia pomonella and three other tortricid pests of tree fruit

The granulovirus of Cydia pomonella (L.) (CpGV) offers potential for selective control of codling moth. Two major limitations of CpGV are its narrow host range and lack of persistence in the orchard agroecosystem. The nucleopolyhedroviruses of the alfalfa looper Autographa californica (Speyer) (AcMNPV) and those of the celery looper Anagrapha falcifera (Kirby) (AfMNPV) have broad host ranges. Comparative assays of CpGV, AcMNPV, and AfMNPV against codling moth neonate larvae revealed a 54-93-fold greater susceptibility of codling moth to the granulovirus than to the two nucleopolyhedroviruses based on the LC(50) values for each virus. The LC(50)s for CpGV, AfMNPV, and AcMNPV were 32.7 capsules/mm(2), 1.77 x 10(3) occlusion bodies (OBs)/mm(2), and 3.05 x 10(3)OBs/mm(2), respectively. The LT(50) determined for AfMNPV using an approximate LC(95) of the virus against neonate larvae was 3.6 days. Histological examination of tissues in moribund codling moth larvae that had been treated with AfMNPV revealed the presence of nonoccluded and unenveloped virus rods in midgut tissue. Neither OBs nor signs of infection were detected in other tissues. The activity of AfMNPV was also evaluated in three other tortricid apple pests (obliquebanded leafroller, Choristoneura rosaceana (Harris); Pandemis leafroller, Pandemis pyrusana Kearfott; and the oriental fruit moth, Grapholitha molesta (Busck)). Codling and Oriental fruit moths were significantly more susceptible to AfMNPV than were the two leafroller species.     

Sprayable self-encapsulating starch formulations for Bacillus thuringiensis

Under glasshouse conditions, various cornstarches and adjuvants were examined as encapsulating agents in sprayable formulations for Bacillus thuringiensis subsp. kurstaki Berliner. When these materials were suspended in water and tested for physical factors such as viscosity, clumping, and retention on plant surfaces, a range of responses was observed. All formulations tested had acceptable viscosity, but some formulations clumped. When applied to cotton leaves, certain formulations remained less than 4 d, whereas others remained greater than 2 wk. One of these formulations containing equal parts of Mira-sperse (a commercially available pregelatinized cornstarch) and sucrose (6% total solids) was selected for testing persistence of B. thuringiensis insecticidal activity against Ostrinia nubilalis Hübner neonate larvae. When encapsulating agents were present, insecticidal activity decreased over a 2-wk period significantly more slowly than when encapsulating agents were absent. This indicates an enhanced persistence of B. thuringiensis under glasshouse conditions. Implications of a sprayable self-encapsulating formulation for B. thuringiensis are discussed.     

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.     

重组病毒杀虫剂应用研究进展

应用分子生物学技术可以将昆虫特异性的毒素基因、某些酶基因等外源基因插入昆虫病毒基因组,或通过改造昆虫病毒基因组等方法构建重组病毒杀虫剂,提高杀虫效果。温室及田间释放实验证实,重组病毒杀虫剂可以显著提高现场防治效果。连续多代抗性筛选实验表明,宿主被诱导产生对重组病毒杀虫剂抗性的速度低于野生型病毒杀虫剂。采用在剂型中添加光增白剂等保护剂、在基因组中插入具有增效作用的基因、应用病毒增强蛋白等技术可以提高重组病毒杀虫效果。随着基因工程技术的发展和安全性研究的深入,以重组杆状病毒为主的重组昆虫病毒杀虫剂的应用研究正面临着突破。     

Comparing formulations for a mixed-microbial biopesticide with Bacillus thuringiensis var. kurstaki and Beauveria bassiana blastospores

Developing a dual agent biopesticide formulation made with blastospores of Beauveria bassiana (Balsamo) Vuillemin strain GHA (Bb) at the ratio of 1:1 mixed with traditional fermentation production of Bacillus thuringiensis kurstaki (Btk) spores and crystals were studied. Twelve potential formulation ingredients were screened for their impact on insecticidal activity, microbe viability, UV protection and product suspensibility of a spray dried Bt/Bb (1:1) mixture applications to cabbage plants against cabbage looper, Trichoplusia ni. Based on the results, Spray-dried mixtures of Bt/Bb made with biochar, chitosan and skim milk powder formulations gave the greatest efficacy and higher solar stability when exposed to simulated sunlight. They therefore can be used as a formulation that combines Bt with Bb in one product. However, the formulation made with aitkin component had significantly more colony forming units (CFUs) of Bt, whereas, formulations made with albumin, sodium carbonate and pharmamedia had more Bb CFUs than other formulations. Albumin, pharmamedia, skim milk powder and biochar had significantly better suspension in water (less settling) than others. These results support the concept of formulating the two biocontrol agents, B. thuringiensis and B. bassiana blastospores for greater efficacy, more viability and longer field persistence as an environmentally friendly sustainable pest control tool.

LSID:urn:lsid:zoobank.org:pub:568D6FE6-E876-417B-BE39-C51F7CBAD6AB