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.     

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.     

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.     

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.     

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.     

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.     

Screening of insecticidal activity of plant essential oils and extract-based formulations against four agricultural insect pests and their risk assessment

Frankliniella occidentalis (Pergande), Myzus persicae (Sulzer), Plutella xylostella (L), and Spodoptera exigua (Hübner) are notorious agricultural insect pests worldwide. Due to the rapid development of resistance to the conventional insecticides, the number of available insecticides might be reduced in a near future, and botanical insecticides can be considered as one of the potential candidates to solve this problem. In the present study, test formulations were prepared using plant essential oils and extracts as main active ingredients, and their insecticidal activity and phytotoxicity were examined. Among the combinations tested, two oil-in-water formulations of NRS-13 containing thyme white oil and NRS-24 containing basil and rosemary oils were selected based on their notable insecticidal activity and low phytotoxicity. Chemical analyses on the active essential oils, the quantitative analysis of the major components in the formulations, insecticidal activities under pot and field conditions, their acute toxicity on mammals, and ecotoxicity on fish were also examined. The NRS-13 (BaechooSafer^TM) and NRS-24 (JindiOut^TM) formulations showed species-specific insecticidal activity against P. xylostella and M. persicae, respectively. In addition, both formulations produced low acute oral and dermal toxicity, skin and eye irritation, and fish toxicities, which suggests those formulations based on essential oils could be considered as insecticides for pest control in small-scale environments in organic farming.     

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.     

Laboratory and field performance of consumer bait products for German cockroach (Dictyoptera: Blattellidae) control.

Bait formulations containing boric acid, chlorpyrifos, or hydramethylnon were evaluated in field and laboratory experiments against the German cockroach, Blattella germanica (L.). In continuous exposure tests, chlorpyrifos formulations produced 100% mortality in less than 24 h, boric acid formulations had LT50's of approximately 5.5 d, and hydramethylnon had an LT50 of 0.86 d. LT50's estimated in Ebeling choice boxes were 1.6 to 10.8 times greater than those from continuous exposure tests for chlorpyrifos formulations and 2.5 times greater for the hydramethylnon formulation. The same LT50's were produced by choice box and continuous exposure tests for boric acid formulations. Relative repellency, measured as the mean percentage of live cockroaches in the light side of the choice box, was greatest for chlorpyrifos formulations, followed by boric acid formulations, then hydramethylnon. A performance index (PI) that combined choice box repellency and mortality data indicated that hydramethylnon (Combat) had the greatest potential for field effectiveness. Only hydramethylnon significantly reduced German cockroach populations in the field. Apartments treated with the IT WORKS formulation of boric acid bait had significantly greater cockroach trap catches. The PI is an accurate predictor of the field performance of toxic baits for German cockroach control.     

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 evaluation of commercial repellent formulations against mosquitoes (Diptera: Culicidae) in Queensland, Australia

Abstract  Laboratory tests of commercial repellent formulations were conducted against Anopheles farauti Laveran, Culex annulirostris Skuse, Ochlerotatus vigilax (Skuse) and Stegomyia aegypti (L.). The majority of repellent formulations tested contain N,N ,-diethyl- 3 -methylbenzamide (also known as diethyl- m -toluamide, commonly called deet). Two formulations containing picaridin (1-piperidinecarboxylate acid, 2-(2-hydroxyethyl)-1-methylpropylester, also known as KBR 3023), one containing ethyl butylacetylaminopropionate (EBAP) and two formulations containing essential oils (e.g. Citronella oil) were also tested. In the laboratory tests, repellent formulations containing deet provided the best protection, and picaridin and EBAP also provided good protection. Citronella oil provided only limited protection. Two field trials to compare commercially available repellent formulations containing picaridin and deet against mosquitoes at Redcliffe, Queensland, were conducted. In the first, Autan Repel, containing 9.3% picaridin, RID, containing 10% deet, and Bushman Ultra, containing 80% deet in a gel, were compared. In the second, Autan Repel Army 20, containing 19.2% picaridin, OFF! Skintastic, containing 7% deet, and Aerogard, containing 12% deet, were compared. The predominant mosquito in both tests was Cx. annulirostris . Bushman provided >95% protection against all mosquitoes for at least 8 h when tests ceased. The other deet repellents also provided good protection against mosquitoes, with RID providing 5 h, Skintastic 4 h and Aerogard 2 h protection. The Autan repel (9.3% picaridin) provided >95% protection for 3 h, and Autan Repel Army (19.2% picaridin) provided 4 h protection. These studies have shown that commercial formulations of both deet and picaridin provide good protection against Cx. annulirostris , an important vector of arboviruses in Australia.     

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.     

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.     

Effect of spray dryer processing parameters on the insecticidal activity of two encapsulated formulations of baculovirus

The effect of spray dryer processing parameters on the product yield and insecticidal activity of baculovirus was evaluated. Spray-dried samples of a granulovirus (GV) from Pieris rapae (L.) and a multiple nucleopolyhedrovirus (MNPV) from Anagrapha falcifera (Kirby) were prepared using two dryer-atomiser configurations (rotary atomiser and two-fluid spray atomiser), four drying temperatures (50–100°C outlet temperatures) and two encapsulating formulations (lignin and methacrylic acid polymer). The samples were evaluated based on yield and insecticidal activity under laboratory conditions. The two atomising configurations produced similar outlet temperatures for dryer stock feed rates of 4.12 and 20 ml/min when processed using increasing inlet temperatures. The atomiser selection significantly affected the physical properties like the product yield; the microparticles produced with a two-fluid spray atomiser had lower product yields (57.8 ± 18.80% – 74.6 ± 4.26%) when compared with paired samples produced with a rotary-disc atomiser (58.1 ± 7.13% – 82.6 ± 3.12%). Spray drying reduced insecticidal activity of the GV but did not significantly reduce insecticidal activity of the MNPV when compared with samples that were not dried. Among dried samples, the spray dryer processing parameters (atomiser, drying temperatures and formulation) had minimal effect on the insecticidal activity of either baculovirus. The versatility of spray drying for processing baculoviruses was demonstrated by identifying parameters that improve process yield while having minimal impact on insecticidal activity.     

Activity and residual effect of two formulations of lambdacyhalothrin sprayed on palm leaves to Rhodnius prolixus

The insecticidal activity and residual effect of two formulations of lambdacyhalothrin were evaluated with Rhodnius prolixus; laboratory and field tests were conducted in the State of Chiapas, Mexico. The results indicate that the lethal concentrations of the active ingredient of SC (LC50 = 2.37 and LC90 = 8.5 mg, a.i./m2) were 4-8 times than those with the insecticide WP applied on R. prolixus bugs in palm leaves, a common building material for thatched roofs. Other investigators in South America recommended applying 30 mg a.i./m2 in porous materials; we obtained that the products WP and SC were 3.5 and 16 times more effective on palm leaves. Regarding the evaluation of the residual effects in field spraying, there was up to 15 months persistence after the application of WP in two doses (8.6 mg a.i./m2 and 3.752 mg a.i./m2) with SC. We consider R. prolixus highly susceptible to the employed pyrethroids; they could be used to control this vector in the state of Chiapas, Mexico.     

Lignin peroxidase H2 from Phanerochaete chrysosporium: purification, characterization and stability to temperature and pH

The wood-destroying fungus Phanerochaete chrysosporium secretes extracellular enzymes known as lignin peroxidases that are involved in the biodegradation of lignin and a number of environmental pollutants. Several lignin peroxidases are produced in liquid cultures of this fungus. However, only lignin peroxidase isozyme H8 has been extensively characterized. In agitated nutrient nitrogen-limited culture, P. chrysosporium produces two lignin peroxidases in about equal proportions. The molecular weights of these two major proteins (H2 and H8) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 38,500 (H2) and 42,000 (H8). The isoelectric points of these enzymes were 4.3 for H2 and 3.65 for H8. All subsequent experiments in this study were performed with H2 as it contributed the most (42%) to total activity and had the highest specific activity (57.3 U/mg). The Km values of lignin peroxidase H2 for H2O2 and veratryl alcohol were calculated to be 47 microM and 167 microM at pH 3.5, respectively. The pH optima for veratryl alcohol oxidase activity were pH 2.5 at 25 degrees C, pH 3.0 at 35 degrees C, and pH 3.5 at 45 degrees C. In the same manner the temperature optimum shifted from 25 degrees C at pH 2.5 to 45 degrees C at pH 3.5 and approximately 45-60 degrees C at pH 4.5. During storage the resting enzyme was relatively stable for 48 h up to 50 degrees C. Above this temperature the enzyme lost all activity within 6 h at 60 degrees C. At 70 degrees C all activity was lost within 10 min. The resting enzyme retained approximately 80% of its initial activity when stored at 40 degrees C for 21 h at a pH range of 4.0-6.5. Above pH 7.5 and below 4.0, the enzyme lost all activity in less than 5 h. During turnover the enzyme remained active at pH 5.5 for over 2 h whereas the enzyme activity was lost after 45 min at pH 2.5. The oxidation of veratryl alcohol was inhibited by EDTA, azide, cyanide, and by the catalase inhibitor 3-amino-1,2,4-triazole, but not by chloride. In the absence of another reducing substrate incubation of lignin peroxidase H2 with excess H2O2 resulted in partial and irreversible inactivation of the enzyme. The spectral characteristics of lignin peroxidase H2 are similar to those of other peroxidases. The suitability of lignin peroxidases for industrial applications is discussed.     

Detergent Formulations for Wool Domestic Washings Containing Immobilized Enzymes

The stability of immobilized and native Esperase, a commercial serine protease, was studied by incubating the enzymes in four formulations containing the same amount of anionic and non-ionic surfactants. The results show that the activity of the immobilized enzyme is not affected by the presence of detergents while the native enzyme lost 50% of activity after 20 min of incubation in these four formulations. The washing performance of the detergents prepared with the immobilized Esperase was studied on cotton and wool fabric samples stained with human blood and egg yolk, using as control the detergent containing native Esperase. The best stain removal for cotton samples stained with human blood was achieved using the detergent with immobilized Esperase. Several physical tests confirmed that wool keratin was not degraded by the immobilized Esperase, validating the ability to use formulated detergents containing this immobilized enzyme for safe wool domestic washing.     

Can pesticide formulations significantly influence pest behavior?: the case of Tetranychus urticae and dicofol

Pesticide formulations profoundly influenced the encounter of pest with pesticide. In four differently formulated treatments of identical dicofol concentration, total time spent on treated areas of a checkerboard residue immediately following treatment varied dramatically from 13.3% on Modified Formulation residues to 42.1 % on 50 Wettable Powder residues. With 24 h old checkerboard residues, total time spent on treated areas varied from a low of 30.1% with Modified Formulation residues to a high of 45.1% with Emulsifiable Concentrate residues. Different formulations exhibited dramatically different decay of repellency over a 24 h period. The greatest loss of repellency was found on Modified Formulation residues, although this formulation was still the most repellent at 24 h. Observations were made of three behavior patterns with both 0 h and 24 h old residues of four formulations of dicofol. Our results indicate that the constituents of some pesticide formulations may elicit behavior that greatly reduces pest encounter with pesticide. To study this phenomenon it is necessary to observe pest behavior with discontinuous residues.     

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.     

Knockdown, residual, and antifeedant activity of pyrethroids and home landscape bioinsecticides against Japanese beetles (Coleoptera: Scarabaeidae) on Linden foliage

Residual toxicity and leaf protection capability of five pyrethroids, professional and home garden azadirachtin formulations, and six other bioinsecticides for the home landscape were evaluated against the Japanese beetle, Popillia japonica Newman (Coleoptera: Scarabaeidae), on linden, Tilia cordata L. Capacity of intoxicated beetles to recover and subsequently feed and disperse also was evaluated to provide insight on activity characteristics of the different compounds. Intact shoots were sprayed and left in the field for varying intervals before being challenged with beetles in no-choice and choice tests. All pyrethroids except permethrin gave greater leaf protection, knockdown, and kill than did carbaryl, the standard, after 14 d of weathering. Deltamethrin, cyfluthrin, bifenthrin, and lamda-cyhalothrin gave a high level of protection for at least 19 d, and azadirachtin (Azatin XL) deterred feeding in choice tests for as long as 14 d. Home garden formulations containing pyrethrins in canola oil (Pyola) or azadiractin (Neem-Away) gave good short-term (< 3-d) protection. Formulations of capsaicin, rotenone + pyrethrins, kaolin particle film, D-limonene, or garlic extract were ineffective, the latter two formulations being highly phytotoxic to linden. Results of this study should help support updating of guidelines for insecticidal control of Japanese beetles.