Control of arthropod infestation

Abstract

A dry powder formulation comprising spores of an entomopathogenic fungus that has activity against arthropods that infest dry commodity storage areas, wherein the spores are present in an amount of 2 to 5% w/w of the formulation; particles of an industrial mineral in an amount of 80 to 88% w/w of the formulation and having a volume mean diameter of 5 m; and electret particles in an amount of 10 to 15% w/w of the formulation, and uses thereof.

Claims

1. A dry powder formulation comprising: i) spores of an entomopathogenic fungus that has activity against arthropods that infest dry commodity storage areas, wherein the spores are present in an amount of 2 to 5% w/w of the formulation; ii) mineral-based particles of an industrial mineral in an amount of 80 to 88% w/w of the formulation and having a volume mean diameter of 5 m; and iii) electret particles, different from said mineral-based particles, in an amount of 10 to 15% w/w of the formulation.

2. The formulation of claim 1, wherein the spores of i) are present in an amount of 2 to 4.5% w/w; the mineral-based particles of ii) are present in an amount of 82.5 to 87% w/w; and the electret particles of iii) are present in an amount of 11 to 13% w/w of the formulation.

3. The formulation of claim 1, wherein the spores of i) are present in an amount of 2.10 to 4.17% w/w; the mineral-based particles of ii) are present in an amount of 83.0 to 85.0% w/w and the electret particles of iii) are present in an amount of 12.5 to 12.80% w/w of the formulation.

4. The formulation of claim 1, wherein the spores are present in an amount of at least 110.sup.9 CFU/gram dry weight.

5. The formulation of claim 1, wherein the spores are present in an amount of at least 110.sup.10 CFU/gram dry weight.

6. The formulation of claim 1, wherein the spores are present in an amount of at least 110.sup.11 CFU/gram dry weight.

7. The formulation of claim 1, wherein the industrial mineral is selected from clays, sand, gravel, diatomite, diatomaceous earth, kaolin, bentonite, silica, barite, gypsum, and talc or a mixture of two or more thereof.

8. The formulation of claim 1, wherein the industrial mineral is selected from kaolin and talc or a mixture thereof.

9. The formulation of claim 1, wherein the arthropods comprise Sitophilus oryzae, Sitophilus granarius, Sitophilus zeamais, Rhyzopertha dominica, Ahasverus advena, Oryzaephilus surinamensis, Prostephanus truncatus and Cryptolestes ferrugineus.

10. The formulation of claim 1, wherein the arthropods comprise Oryzaephilus surinamensis, Sitophilus granarius and Cryptolestes ferrugineus.

11. A method of using the formulation of claim 1, comprising controlling arthropod infestation in a dry commodity storage area (facility) with the formulation of claim 1.

12. A method of using the formulation of claim 1, comprising controlling, with the formulation of claim 1, arthropod infestation within and/or on a dry commodity selected from whole grains and seeds for sowing, whole grains selected from wheat, rice, rye, oats, barley and corn for use as food and/or in the manufacture of processed foods, pulses, beans, lentils and products made therefrom from or made with dried commodities, dry processed goods including pasta, grain flours, couscous, cereals, dried herbs, breakfast cereals, semolinas, breads, nuts (ground, flaked and/or whole nuts), snacking food selected from sweet and savoury biscuits, potato crisps, vegetable crisps, pretzels, dried wafers, domestic livestock feed, timber lumber, planking, articles made of timber including roof scaffolding for buildings, panelling, doors and door frames, lintels, skirting boards, hardboards, plywoods, furniture, and woodchips.

13. A method of controlling dry commodity arthropod infestation in a dry commodity storage area, wherein the formulation of claim 1 is presented to the surfaces of a dry commodity storage area by i) collecting the dry powder formulation in a dusting apparatus; and ii) releasing the said dry powder formulation from the said dusting apparatus and into the said dry commodity storage area.

14. A method of controlling dry commodity arthropod infestation in a dry commodity storage area, wherein the formulation of claim 1 is presented to the surfaces of a dry commodity storage area.

15. A dry powder formulation comprising: i) spores of an entomopathogenic fungus that has activity against an arthropod selected from Oryzaephilus surinamensis, Sitophilus granarius and Cryptolestes ferrugineus, wherein the spores are present in an amount of 2 to 5% w/w of the formulation, and in an amount of at least 110.sup.10 CFU/gram dry weight; ii) mineral-based particles of an industrial mineral selected from kaolin, talc, and a mixture of kaolin and talc, in an amount of 80 to 88% w/w of the formulation and having a volume mean diameter of 5 m; and iii) electret particles, different from said mineral-based particles, in an amount of 10 to 15% w/w of the formulation.

16. A method of producing a dry powder formulation comprising: i) spores of an entomopathogenic fungus comprising Beauveria bassiana that has activity against arthropods that infest dry commodity storage areas, wherein the spores are present in an amount of 2 to 5% w/w of the formulation; ii) mineral-based particles of an industrial mineral in an amount of 80 to 88% w/w of the formulation and having a volume mean diameter of 5 m; and iii) electret particles, different from said mineral-based particles, in an amount of 10 to 15% w/w of the formulation, comprising the steps of i) micronising dry electret particles; ii) admixing dry spores of the entomopathogenic fungus comprising Beauveria bassiana with said electret particles, wherein the spores are present in an amount of 2 to 5% w/w of the formulation and the electret particles are present in an amount of 10 to 15% w/w of the formulation; and iii) admixing dry industrial mineral earth particles, having a volume mean diameter of 5 m in an amount of 80 to 88% w/w of the formulation, with the product of step ii).

17. A dry powder formulation, comprising: i) spores of an entomopathogenic fungus comprising Beauveria bassiana that has activity against arthropods that infest dry commodity storage areas, wherein the spores are present in an amount of 2 to 5% w/w of the formulation; ii) mineral-based particles of an industrial mineral in an amount of 80 to 88% w/w of the formulation and having a volume mean diameter of 5 m; and iii) electret particles in an amount of 10 to 15% w/w of the formulation, wherein the formulation is produced by the method of claim 16.

Description

DESCRIPTION OF THE EXPERIMENT

(1) Adults of P. truncatus were reared in the laboratory at Plant Protection and Regulatory Service of Ghana (PPRSD) under ambient conditions (282 C., 655% rh). One month post adult eclosion insects were cultured on whole maize grains. Maize grains (1250 g each) were thoroughly mixed with the various concentrations (10.sup.8, 10.sup.9, 10.sup.10 and 10.sup.11 CFU) of B. bassiana IMI 398548. Each of the four treatments was then divided into five equal parts to represent 5 replications (250 g) each and placed in 500 mL Kilner jars. Adult P. truncatus (50) were then introduced into the treatments after 24 hours. In order to determine the viability of the fungus another 1250 g of maize was treated with B. bassiana at 10.sup.11 CFU and colony fuming units (CFU) were determined at the beginning and end of the trial. There was a negative control treatment in which no B. bassiana was added. All treatments were held at 252 C. and 655% rh. Mortality of P. truncatus was assessed at 7-day intervals for 3 weeks (days 7, 14 and 21). Post-mortem was done on cadavers of the insects to determine if death was as a result of mycosis caused by B. bassiana, IMI 398548.

Methods

4. Test Item Details

(2) Materials provided by Exosect for dose response trial were: Four sachets, each containing 3 g of formulation A (10.sup.8 CFU per kg maize). Four sachets, each containing 3 g of formulation B (10.sup.9 CFU per kg maize). Four sachets, each containing 3.2 g of formulation C (10.sup.10 CFU per kg maize). Four sachets, each containing 4.5 g of formulation D (10.sup.11 CFU per kg maize).
A: 2 g kaolin+0.3 g Entostat+0.001 g spores/Kg maize
B: 2 g kaolin+0.3 g Entostat+0.01 g spores/Kg maize
C: 2 g kaolin+0.3 g Entostat+0. g spores/Kg maize
D: 2 g kaolin+0.3 g Entostat+1.0 g spores/Kg maize
Source of Insects

(3) The one month post adult eclosion of P. truncatus used for the trial, were reared on whole maize grains at 282 C. and 655% rh in the Entomology laboratory of the Biocontrol Unit, Plant Protection and Regulatory Service of MOFA, Accra.

(4) Determination of Concentrations of Beauveria bassiana, IMI 398548 for the Protection of Stored Maize Against Prostephanus truncatus

(5) To determine the most effective concentration of B. bassiana, IMI 398548 formulation for stored maize protection against P. truncatus, a laboratory study was undertaken with four concentrations (10.sup.8, 10.sup.9, 10.sup.10 and 10.sup.11 CFU per kg maize) of B. bassiana, IMI 398548.

(6) Maize (Obatanpa cultivar) was stored in a freezer, at 4 C. for 2 days. The maize was then removed, oven dried for a day at 50 C. and left for a further day to cool. Maize (2500 g) was treated with 4.5 g of B. bassiana (110.sup.11 CFU/kg maize) and divided into 10 jars containing aliquots of 250 g each. Five of these jars were used for mortality assessment whilst, the other five were used for initial and final viability tests. For the other concentrations, a 1,250 g of maize each was mixed thoroughly with 3.2 g of B. bassiana at 10.sup.10 CFU/kg maize, 3 g of B. bassiana, at 10.sup.8 and 10.sup.9 CFU and divided into five equal parts (250 g) representing five replications. There was a negative control in which no B. bassiana was added. The grains were left for 24 hours after which 50 unsexed P. truncatus were introduced into each jar. The treatments were left at laboratory conditions of 252 C. and 655% rh. The temperature and relative humidity in the Entomology laboratory of the Department of Crop Science, Legon where this bioassay was carried out was monitored using a data logger.

(7) After the insects were introduced, 5 g of maize grains was sampled from each replicate of B. bassiana at 110.sup.11 CFU/kg maize (which had no insects) and washed in 10 mL sterile 0.05% Tween 80. The suspension was agitated slightly by shaking gently for some few minutes before performing a 10-fold serial dilution resulting in 8 dilutions. A 200 l aliquots of each dilution was inoculated in duplicate on Sabouraud Dextrose Agar (SDA) using the spread plate technique and incubated for 4 days to determine the number of colony forming units (CFU) per mL

(8) Mortality of P. truncatus was recorded at 7-day intervals for three weeks by empting each jar on laboratory trays. Dead insects were removed at each assessment time and surface sterilized in 2% sodium hypochlorite for 1 minute, followed by two rinses in sterile distilled water for 1 minute. The cadavers from each treatment were then transferred onto Whatman filter papers moistened with 1 mL sterile distilled water, placed on 9 cm Petri dishes and sealed with Parafilm. Insects on moistened filter papers were kept apart and incubated at ambient conditions and examined after 6 days for growth of B. bassiana, IMI 398548.

(9) On day 21, fungal viability was determined as done on the first day of the experiment by determining CFU count per mL. Grains were sieved to separate grain powder from the kernel. The weight of the powder, kernel and number of live P. truncatus in each jar were recorded. Percentage weight loss (L) was calculated using the formula below
L=[(WiWf)/Wi)]100,
where, Wi is the initial weight of grains, and Wf is the final weight of grains.

(10) The cumulative percentage mortality data and percent mycosis were arcsine transformed; Additional data collected were the total number live P. truncatus at day 21, weight of kernel and powder at the end of the experiment, number of colonies formed by the highest concentration at the start and end of the trial.

(11) Data were subjected to analysis of variance (ANOVA). Least significant difference (LSD.sub.5%) was used to separate means. The statistical analysis was accomplished using Genstat statistical software (9.sup.th) edition.

5. Test System

(12) All insects totaling 1000 of unsexed adult P. truncatus were supplied by Plant Protection and Regulatory Service Ghana (PPRSD). The insects were reared on whole maize grains at the Biocontrol Unit of PPRSD under ambient conditions (282 C. and 655% rh). Insects used were approximately one month post adult eclosion.

6. Test Location

(13) The dose response bioassay was conducted in the Entomology laboratory of the Department of Crop Science, Legon at an average room temperature of 252 C. and 655% rh. Insect cadavers were incubated at 262 C. and 655% rh respectively in the Pathology laboratory of the Department of Crop Science, University of Ghana.

7 Experimental Design

(14) A completely randomized design (CRD) was used with 5 treatments (P. truncatus infested with four concentrations (10.sup.8 to 10.sup.11 CFU/kg maize) of B. bassiana, IMI 398548 spore powder and a negative control which had no B. bassiana added with five replicates. Each replicate contained 50 adult P. truncatus.

8. Application Details and Regime

(15) The weight of maize kernel and powder at the end of the experiment were weighed using an electronic balance.

9. Statistical Analysis

(16) Control mortality at the end of the trial (21 days) was relatively low (<10%), consequently, cumulative percentage mortality data were not corrected for the corresponding control mortality (Abbott, 1925).

(17) The cumulative percentage mortality and percent mycosis data were arcsine transformed; Additional data collected were the total number of live P. truncatus at day 21, weight of kernel and powder at the end of the experiment, number of colonies formed from B. bassiana at 10.sup.11 CFU/kg maize (which had no insects) at the start and end of the trial (Appendix 3-10).

(18) Data were subjected to analysis of variance (ANOVA). Least significant difference (LSD.sub.5%) was used to separate means. The statistical analysis was accomplished using Genstat statistical software (9.sup.th) edition.

10. Protocol Deviations

(19) The proposed protocol was strictly adhered to except that, there was no mention of control which we deemed fit and therefore included in this trial.

11. Results

(20) Response of Prostephanus truncatus to four concentrations (CFU/kg maize) of B. bassiana, IMI 398548 in the laboratory.

(21) The response of P. truncatus to B. bassiana, IMI 398548 was evaluated by applying four concentrations of B. bassiana (10.sup.8, 10.sup.9, 10.sup.10 and 10.sup.11 CFU) per kg maize for 21 days to determine their effect on the mortality of P. truncatus. Quantification of the mortality of the one month post adult eclosion P. truncatus was made at 7, 14 and 21 days after infestation. For all concentrations, the mortality of P. truncatus increased with increasing days of exposure. Beauveria bassiana, IMI 398548 at 10.sup.10 and 10.sup.11 CFU per kg maize resulted in significantly (P<0.05) higher mortality of adult P. truncatus compared to 10.sup.8 and 10.sup.9 CFU per kg maize. For these concentrations (10.sup.10 and 10.sup.11), mortality of P. truncatus was 64.7-68.0% after 7 days of exposure reaching 96.0% and 98.7% respectively after 14 days. In contrast, mortality of adult P. truncatus in B. bassiana, IMI 398548 at 10.sup.8 and 10.sup.9 CFU per kg maize was <13% after 7 days of exposure and remained less than 30%, 21 days after exposure. Mortality of P. truncatus in the control treatment (where no B. bassiana was added) was <10% at the end of 21 days (Table 1).

(22) TABLE-US-00001 TABLE 1 Cumulative percentage mortality (%) of P. truncatus infested with spore powder of B. bassiana (CFU per kg maize) and incubated for 7, 14 and 21 days at 25 2 C. and 65 5% rh in the laboratory. Dose of B. bassiana Cumulative mortality (%) at days after treatment (CFU/per kg maize) 7 14 21 1 10.sup.8 5.33 10.00 14.67 1 10.sup.9 12.67 22.00 28.00 1 10.sup.10 64.67 96.00 100.00 1 10.sup.11 68.00 98.67 100.00 Control 1.33 4.67 9.33 LSD (p < 0.05) 6.97 4.20 3.87
Mean Percentage Weight Loss of Kernel after Infestation by P. truncatus Treated with B. bassiana (10.sup.8 to 10.sup.11 CFU) Per Kg Maize and Incubated at 252 C. and 655% Rh for 21 Days.

(23) The percentage weight loss of maize kernel caused by P. truncatus treated with B. bassiana at 10.sup.8 and 10.sup.9 CFU per kg maize were not significantly different from the control treatment. The weight loss (%) of kernel produced by P. truncatus on B. bassiana at 10.sup.10 and 10.sup.11 CFU per kg maize were similar but differed significantly (P<0.05) from the control. A significantly (P<0.05) higher grain weight loss was recorded on maize grains treated with B. bassiana, IMI 398548 at 10.sup.8 and 10.sup.9 CFU per kg maize compared with 10.sup.10 and 10.sup.11 CFU per kg maize after infestation by P. truncatus for 21 days (Table 2).

(24) TABLE-US-00002 TABLE 2 Mean (%) weight loss of kernel after infestation by P. truncatus infested with B. bassiana (10.sup.8 to 10.sup.11 CFU) per kg maize and incubated for 21 days at 25 2 C. and 65 5% rh in the laboratory. Dosage (CFU/kg maize) % weight loss of kernel 1 10.sup.8 1.554 1 10.sup.9 1.460 1 10.sup.10 0.439 1 10.sup.11 0.400 Control 1.528 LSD (p < 0.05) 0.2927 Viability and persistence of B. bassiana, IMI 398548 at 10.sup.11 CFU per kg maize

(25) The number of colonies formed by the highest concentration (10.sup.11 CFU per kg maize) at day 1 was not significantly different from those at day 21 (t=0.10, t-prob.=0.922, df=4). Post-mortem mycelia and conidial growth showed that most insects had died from infection by the fungus with percent mycosis for the four concentrations (10.sup.8, 10.sup.9, 10.sup.10 and 10.sup.11 CFU per kg maize) of B. bassiana being 97.1, 98.6, 99.6 and 99.6 respectively (Table 3).

(26) TABLE-US-00003 TABLE 3 Mean (%) mycosis of P. truncatus, infested with B. bassiana (10.sup.8 to 10.sup.11 CFU) per kg maize and incubated for 4 days at 26 2 C. and 65 5% rh in the laboratory. Dosages (CFU/kg maize) Percentage mycosis 1 10.sup.8 97.1 1 10.sup.9 98.6 1 10.sup.10 99.6 1 10.sup.11 99.6 LSD (p < 0.05) 4.9 Analysis of variance was made on arcsine percentage sporulated data

12. Discussion

(27) This is the first report of a new isolate (IMI 398548) of B. bassiana for the control of P. truncatus. B. bassiana, IMI 398548 at 10.sup.10 and 10.sup.11 CFU per kg maize resulted in higher mortality of adult P. truncatus compared to 10.sup.8 CFU and 10.sup.9 per kg maize. In the present study the highest mortality (64.7-68.0%) of P. truncatus was achieved in B. bassiana at 10.sup.10 and 10.sup.11 CFU per kg maize respectively at 252 C. and 655% rh after 7 days of exposure reaching 96.0% and 98.7% respectively 14 days after treatment.

(28) In the current study, viability of conidia of B. bassiana at 10.sup.11 CFU per kg maize persisted though out the study period for 21 days. This demonstrates the requirement that, mycopesticide formulation should persist in the environment for a considerable time after application (Burges, 1998). A similar result is envisaged under semi field conditions since conditions under grain storage in Ghana are more stable and similar to the conditions observed in the study.

(29) One of the enormous advantages of using microbial control systems is that disease infection cycling occurs when infected and dead insects increase the amount of inocula after sporulation to effectively increase the persistence of the mycopesticide (Hidalgo et al., 1998). During this study insect cadavers were consistently removed from maize grains, however, B. bassiana mycelia and spore sporulation appeared 3-4 days after incubation and completely covered the cadavers of P. truncatus, indicating the existence of an adequate dose transfer from treated maize grains. The present study indicated the possibility of successfully controlling P. truncatus on stored and infested maize using spore powder of B. bassiana at 110.sup.10 CFU per kg maize in the laboratory.

13. References

(30) Abbott, W. S. (1925). A method of computing the effectiveness of an insecticide. Journal of Economic Entomology. 18: 265-267. Burges H. D. (1998). Formulation of mycoinsecticides, In H. D. Burges, (ed.), Formulation of microbial biopesticides: beneficial microorganisms, nematodes and seed treatments: Kluwer Academic Publisher, Dordrecht pp 132-185. Hidalgo, E., D. Moore & G. Le Patourel. (1998). The effect of different formulations of Beauveria bassiana on Sitophilus zeamais in stored maize. Journal of Stored Product Research 34: 171-179.

Experiment 2

Pilot Scale Trial of Biopesticide Formulations in the Grain Store

1. Aim

(31) To assess dry conidia of an isolate of the entomopathogenic fungus Beauveria bassiana at different concentrations against three species of insect when applied to arenas made of galvanized steel and stored in the grain store environment.

2. Introduction

(32) The aim of this study was to conduct a range finding test to determine the effective dose of dry conidia powder of B. bassiana IMI398548 (a biologically pure culture of a novel isolate of Beauveria bassiana deposited with CABI, Bakeham Lane, Egham, Surrey, TW20 9TY, UK on 11 May 2010 in accordance with the Budapest Treaty for the deposit of microorganisms and accorded the deposit number of IMI 398548) against three beetle species when applied to steel arenas in the Fera grain store. Entostat was not included in this study as the aim was to evaluate the effect of the conidia in the absence of any material that may enhance the effect.

3. Materials

(33) 3.1 Fungal Isolate.

(34) The B. bassiana isolate (IMI398548) was produced by Somycel S. A. using a mass production method and was checked using in-house quality control (QC) procedures. The final product contained 9.310.sup.10 conidia/g. The isolate was designated as TA 2645 for the purposes of this study and was stored in a refrigerator at 4-8 C.

(35) 3.2 K-Obiol

(36) K-Obiol EC25 (TA 2644) (containing 25 g/l active ingredient) was obtained from Killgerm Chemicals and was used as the positive control. Prior to treatment the pesticide was stored in a secure cabinet situated in a laboratory with a mean temperature of 20 C.0.5 and ambient relative humidity (r.h.). A solution containing the recommended field application rate was made up in water, according to the manufacturer's recommendations, on the day of use.

(37) 3.3 Insects

(38) Three species of insect were tested. These were Oryzaephilus surinamensis strain Tram (saw-toothed grain beetle), Sitophilus granarius strain Gainsborough (grain weevil) and Cryptolestes ferrugineus strain Stow (rust-red grain beetle). Insects used were of mixed age and sex. All three species were used simultaneously within the arena. Insects were provided by the Invertebrate Supply Unit at Fera and reared according to Fera Standard Operating Procedures (ISU/018 and ISU/034 revision 3).

(39) 3.4 Construction of Arenas

(40) The arenas were constructed within Fera's grain storage facility. Insects were confined to squares of galvanized steel (5005000.8 mm) within circular galvanized steel rings (approx 450 mm diameter, 150 mm high). The inside surface of the steel ring was coated with Fluon (Whitford Plastics, UK) to prevent escape of the insects. The rings were sealed to the steel sheet using decorators caulk so that insects could not get under the ring. A refuge made from a piece of electrical conduit (2516100 mm), containing kibbled wheat to provide food for the insects, was placed in the centre of each ring approximately 1 hour after introduction of the insects.

4. Methods

(41) 4.1 Treatment of Arenas

(42) The treatments were as follows:

(43) No treatment (negative control)

(44) Water (control for K-Obiol)

(45) K-Obiol (positive control) applied at 0.05 liters/m.sup.2

(46) 110.sup.9 conidia/m.sup.2

(47) 110.sup.10 conidia/m.sup.2

(48) 110.sup.11 conidia/m.sup.2

(49) Treatments were assigned to each arena using a randomized block design. There were five replicate rings for each treatment.

(50) The conidia were weighed out on to small pieces of aluminium foil, which was folded to prevent loss of material during transportation to the grain store. The appropriate amount of material was evenly distributed across the floor area of the assigned rings using a small brush.

(51) The K-Obiol and water controls were treated by spraying the solution to achieve a coverage of 0.05 liters/m.sup.2. A known volume (12.5 ml) of pesticide solution or water was applied to the steel squares using a hand held De Vilbis laboratory sprayer (EBGF-047).

(52) The spray gun was held approximately 20 cm above the surface of the square and the pesticide or water was sprayed onto the surface, working progressively across and down the square in zigzag movements, in order to obtain an even treatment across the surface.

(53) 4.2 Addition of Insects

(54) Insects were counted in the laboratory, transported in vials and released into the steel rings 24 hours after treatment. All three species were present within each of the steel rings and 50 insects of each species were used in each replicate. The refuge containing kibbled wheat was added to the arena approximately one hour after the introduction of the insects.

(55) 4.3 Assessment of Insects

(56) Insect mortality was assessed after 14 and 28 days. Insects were collected from the rings into labelled glass tubes with the aid of an artist's paintbrush. Refuges were removed from the rings and placed in labelled self-seal plastic bags. The numbers of live and dead insects of each species within each steel ring and the refuge was recorded. Live insects were returned to the rings from which they were removed after the 14-day assessment for re-assessment of mortality after 28 days. The refuges were also returned to the rings from which they had been removed.

(57) 4.4 Monitoring of Environmental Conditions

(58) Temperature was monitored throughout the trial using calibrated thermocouples (Type-T with a beaded tip and PTFE insulation (50 to +250 C.)) linked to a Squirrel (model no. 1045) temperature logger (EBGF 121/122). Temperatures were logged every 60 minutes. The data was downloaded and analysed using SquirrelView software. Temperature and humidity were also monitored using Tinytag dataloggers (TGP 1500, Gemini Dataloggers Ltd, UK) (OPA0/AppEnt 001-003) positioned in three of the arenas. Lights within the store were switched on during treatment and assessments, but remained off at all other times.

(59) 4.5 Statistical Analysis

(60) The difference in mortality between treatments was analysed by a generalized linear model (GLM) with a logit link function. Post-hoc tests were used to test for differences between the treatments by comparison of the least significant difference for the treatments.

5. Results

(61) 5.1 Environmental Conditions

(62) The average temperature recorded in the grain store throughout the trial was 14.3 C. with a minimum of 7.2 C. and a maximum of 20.9 C. There was very little variation in temperature observed between the arenas. The average humidity during the trial recorded using the Tinytag dataloggers was 81.7% with a minimum of 51.2% and a maximum of 98.2%.

(63) 5.2 Insect Mortality Data

(64) Application of K-Obiol at the recommended rate resulted in 100% mortality of all three beetle species tested after 14 days exposure. Although an accurate assessment was not made, observation of the arenas after 24 hours indicated that many, if not all, of the beetles in the arenas treated with K-Obiol were dead. As the aim of this experiment was to determine the effective application rate of the B. bassiana isolate, the K-Obiol treatment and the water control were not included in the statistical analysis.

(65) There was a highly significant effect of treatment with the different doses of conidia on the mortality for all three beetle species after 14 days exposure (GLM F.sub.3.19=79.76, P<0.001, F.sub.3.19=31.34, P<0.001 and F.sub.3.19=106.64, P<0.001 for S. granarius, O. surinamensis and C. ferrugineus respectively (Table 1). There was no significant difference in mortality compared with the control at the lowest application rate tested (110.sup.9 conidia/m.sup.2) for S. granarius and O. surinamensis (P>0.05). There was a significant difference in mortality compared with the control at the highest application rate tested (110.sup.11 conidia/m.sup.2) for all three beetle species.

(66) The intermediate application rate (110.sup.10 conidia/m.sup.2) resulted in significant mortality of S. granarius and C. ferrugineus after 14 days exposure. Application of 110.sup.11 conidia/m.sup.2 resulted in the highest levels of mortality for all three beetle species, and this mortality was significantly greater than that with 110.sup.10 conidia/m.sup.2.

(67) TABLE-US-00004 TABLE 1 Derived mean % mortality of insects 14 days after exposure to different treatments. % mortality is expressed in terms of the number of insects recovered for each species. Figures in parentheses are the derived 95% confidence intervals. S. granarius O. surinamensis C. ferrugineus Control 1.2a 2.6a 5.2a (0.2, 7.8) (0.4, 17.0) (2.3, 11.7) .sup.1 10.sup.9 conidia/m.sup.2 4.7a 3.6a 22.1b (1.8, 11.8) (0.7, 16.4) (15.2, 31.1) 1 10.sup.10 conidia/m.sup.2 31.6b 13.6a 68.1c (22.7, 42.1) (5.9, 28.0) (58.5, 76.4) 1 10.sup.11 conidia/m.sup.2 86.0c 77.5b 98.8d (77.0, 91.9) (62.2, 87.8) (93.2, 99.8) In each column means followed by the same letter are not significantly different (GLM; P > 0.05).

(68) There was a highly significant effect of treatment with the different doses of conidia on the mortality for all three beetle species after 28 days exposure (GLM F.sub.3.19=171.61, P<0.001, F.sub.3.19=87.14, P<0.001 and F.sub.3.19=54.89, P<0.001 for S. granarius, O. surinamensis and C. ferrugineus respectively (Table 2). All three application rates of conidia resulted in significant levels of mortality for S. granarius and O. surinamensis compared with the control (P<0.05). The intermediate and highest application rates resulted in significant mortality of O. surinamensis compared with the control (P<0.05). Application of 110.sup.11 conidia/m.sup.2 resulted in the highest levels of mortality for all three beetle species, and this mortality was significantly greater than that with 110.sup.10 conidia/m.sup.2.

(69) TABLE-US-00005 TABLE 2 Derived mean % mortality of insects 28 days after exposure to different treatments. % mortality is expressed in terms of the cumulative number of dead insects recovered after 14 and 28 days divided by the total of the number of insects recovered after 28 days and the number of dead insects after 14 days. Figures in parentheses are the derived 95% confidence intervals. S. granarius O. surinamensis C. ferrugineus Control 1.6a 14.7a 25.5a (0.4, 5.6) (9.0, 23.2) (16.4, 37.5) .sup.1 10.sup.9 conidia/m.sup.2 13.1b 10.8a 45.4b (8.6, 19.6) (6.2, 18.1) (34.1, 57.2) 1 10.sup.10 conidia/m.sup.2 63.1c 38.7b 92.8c (54.9, 70.6) (29.9, 48.2) (83.9, 96.9) 1 10.sup.11 conidia/m.sup.2 97.6d 96.1c 99.6c (93.4, 99.1) (89.9, 98.5) (86.3, 99.9) In each column means followed by the same letter are not significantly different (GLM; P > 0.05).

6. Discussion

(70) The trial was designed to determine the effective application rate of a dry conidia powder of B. bassiana IMI398548 for three beetle species when applied as a surface treatment in a grain store under typical UK conditions. A dose response was observed for all three beetle species and this was particularly evident after 28 days exposure to the treated surface. The highest application rate used (110.sup.11 conidia/m.sup.2) resulted in significant mortality of all three beetle species after 14 and 28 days compared with the control. The lowest application rate 110.sup.9 conidia/m.sup.2 did not result in significant mortality of S. granarius or O. surinamensis after 14 days exposure and no significant mortality of O. surinamensis after 28 days exposure. Of the three beetle species tested, C. ferrugineus was the most susceptible to treatment with isolate IMI398548.

(71) In Conclusion:

(72) An application rate greater than 110.sup.9 conidia/m.sup.2 of IMI398548 is needed for effective control of the three beetle species tested. An application rate of 110.sup.11 conidia/m.sup.2 resulted in mortalities greater than 96% for all three beetle species after 28 days exposure.

Experiment 3

(73) Pilot Scale Trial of Bio-Pesticide Formulations in the Grain StoreFormulation with Kaolin

1. Aim

(74) To assess the formulation of an isolate of the entomopathogenic fungus Beauveria bassiana mixed with kaolin at different concentrations against two species of insect when applied to arenas made of galvanized steel and stored in the grain store environment.

2. Introduction

(75) The aim of this study was to determine the effectiveness of a powder formulation of B. bassiana IMI398548 mixed with kaolin and Entostat against two beetle species when applied to steel arenas in the Fera grain store.

3. Materials

(76) 3.1 Fungal Isolate

(77) The B. bassiana isolate (IMI398548) was produced by Somycel S. A. using a mass production method and was checked using in-house quality control (QC) procedures. The final product contained 3.810.sup.10 conidia/g. The isolate was designated as TA 2654 for the purposes of this study and was stored in a refrigerator at 4-8 C.

(78) 3.2 Kaolin and Entostat

(79) The kaolin used was produced by Opal Omega and was supplied to Fera by Exosect Ltd. The Entostat was supplied by Exosect Ltd. Both the kaolin and Entostat were kept at room temperature.

(80) 3.3 Insects

(81) Two species of insect were tested. These were Oryzaephilus surinamensis strain Tram (saw-toothed grain beetle), and Cryptolestes ferrugineus strain Stow (rust-red grain beetle). Insects used were of mixed age and sex. Both species were used simultaneously within the arena. Insects were provided by the Invertebrate Supply Unit at Fera and reared according to Fera Standard Operating Procedures (ISU/018 revision 4 and ISU/034 revision 3).

(82) 3.4 Construction of Arenas

(83) Insects were confined to squares of galvanized steel (5005000.8 mm) within circular galvanized steel rings (approx 450 mm diameter, 150 mm high). The inside surface of the steel ring was coated with Fluon (Whitford Plastics, UK) to prevent escape of the insects. The rings were sealed to the steel sheet using decorators caulk so that insects could not get under the ring. A refuge made from a piece of electrical conduit (2516100 mm), containing kibbled wheat to provide food for the insects, was placed in the centre of each ring approximately 1 hour after introduction of the insects.

4. Methods

(84) 4.1 Treatment of Arenas

(85) The treatments were as follows:

(86) 1. No treatment (control)

(87) 2. Entostat+kaolin (weight to equal that used in the IMI398548 510.sup.9 conidia/m.sup.2+Entostat+kaolin (1:3:20) formulation)=Carrier low

(88) 3. Entostat+kaolin (weight to equal that used in the IMI398548 110.sup.10 conidia/m.sup.2+Entostat+kaolin (1:3:20) formulation)=Carrier high

(89) 4. IMI398548 510.sup.9 conidia/m.sup.2

(90) 5. IMI398548 110.sup.10 conidia/m.sup.2

(91) 6. IMI398548 510.sup.9 conidia/m.sup.2+Entostat+kaolin (1:3:20)

(92) 7. IMI398548 510.sup.9 conidia/m.sup.2+Entostat+kaolin (1:6:40)

(93) 8. IMI398548 110.sup.10 conidia/m.sup.2+Entostat+kaolin (1:3:20)

(94) Treatments were assigned to each arena using a randomized block design. There were five replicate rings for each treatment.

(95) The formulations were made up by weighing out the individual components into a glass vial and mixing using a vortex mixer. The conidia and formulations required to treat the arenas were weighed out on to small pieces of aluminium foil, which were then carefully folded to protect the contents during transfer to the grain store. The appropriate amount of material was evenly distributed across the floor area of the assigned rings using a small brush. The calculated amount of material added to the arenas for each treatment is shown in Table 1.

(96) TABLE-US-00006 TABLE 1 Calculated amount of material added to the arenas for each treatment. Conidia Kaolin Treatment (g) Entostat (g) (g) Total (g) Control 0 0 0 0 Carrier low 0 0.0627 0.418 0.4807 Carrier high 0 0.1254 0.836 0.9614 .sup.5 10.sup.9 conidia/m.sup.2 0.0209 0 0 0.0209 1 10.sup.10 conidia/m.sup.2 0.0418 0 0 0.0418 .sup.5 10.sup.9 conidia/m.sup.2 1:3:20 0.0209 0.0627 0.418 0.5016 .sup.5 10.sup.9 conidia/m.sup.2 1:6:40 0.0209 0.1254 0.836 0.9823 1 10.sup.10 conidia/m.sup.2 1:3:20 0.0418 0.1254 0.836 1.0032
4.2 Addition of Insects

(97) Insects were counted in the laboratory, transported in vials and released into the steel rings 24 hours after treatment. Both species were present within each of the steel rings and 50 insects of each species were used in each replicate. The refuge containing kibbled wheat was added to the arena approximately one hour after the introduction of the insects.

(98) 4.3 Assessment of Insects

(99) Insect mortality was assessed after 7 days. Insects were collected from the rings into labelled glass tubes with the aid of a battery operated suction device. Refuges were removed from the rings and placed in labelled self-seal plastic bags. The numbers of live and dead insects of each species within each steel ring and the refuge was recorded.

(100) 4.4 Monitoring of Environmental Conditions

(101) Temperature and humidity were monitored using Tinytag dataloggers (TGP 1500, Gemini Dataloggers Ltd, UK) (OPA0/AppEnt 002 and 003) located in two positions around the arenas. Lights within the store were switched on during treatment and assessments, but remained off at all other times.

(102) 4.5 Statistical Analysis

(103) The difference in mortality between treatments was analysed by a generalized linear model (GLM) with a logit link function. Post-hoc tests were used to test for differences between the treatments by comparison of the least significant difference for the treatments. When treatment mortalities were 0% or 100% a least significant difference value could not be obtained and post-hoc comparisons based on the least significant difference were therefore not possible for these treatments.

5. Results

(104) 5.1 Environmental Conditions

(105) The average temperature recorded in the grain store throughout the trial was 15.8 C. with a minimum of 13.1 C. and a maximum of 19.5 C. The average humidity during the trial recorded using the Tinytag dataloggers was 81.9% with a minimum of 61.9% and a maximum of 98.8%.

(106) 5.2 Insect Mortality Data

(107) There was a highly significant effect of treatment on the mortality of O. surinamensis and C. ferrugineus after 7 days exposure (GLM F.sub.7.36=11.11, P<0.001 and F.sub.7.36=7.98, P<0.001 respectively). See Table 2, below.

(108) Mortality in the treatments with the conidia alone at application rates of 5109 or 11010 conidia/m.sup.2 was not significantly different to the mortality in untreated (control) arenas for either O. surinamensis or C. ferrugineus (Table 2). The carrier used alone at the same application rate as in the 510.sup.9 conidia/m.sup.2 6:40 and 110.sup.10 conidia/m.sup.2 3:20 (Carrier high) resulted in significant mortality of O. surinamensis and C. ferrugineus (P<0.05) compared with the control (Table 2). The carrier used alone at the same application rate as in the 510.sup.9/m.sup.2 3:20 (Carrier low) resulted in significant mortality of O. surinamensis compared with the control treatments. The highest mortalities for both O. surinamensis and C. ferrugineus were seen in the treatments with IM1398548, Entostat and kaolin (Table 2).

(109) TABLE-US-00007 TABLE 2 Derived mean % mortality of insects 7 days after exposure to different treatments. % mortality is expressed in terms of the number of insects recovered for each species. Figures in parentheses are the derived 95% confidence intervals. O. surinamensis C. ferrugineus Control 22.6a 13.83a (8.2, 48.7) (3.0, 45.5) Carrier low 58.6b 49.3a, b (36.7, 77.7) (25.3, 73.6) Carrier high 79.4b, c 86.2b, c (55.3, 92.3) (54.7, 97.0) .sup.5 10.sup.9/m.sup.2 19.7a 20.4a (7.7, 42.0) (6.5, 48.5) 1 10.sup.10/m.sup.2 17.2a 28.4a (6.0, 40.5) (11.1, 55.7) .sup.5 10.sup.9/m.sup.2 6:40 91.3b, c 87.6b, c (56.9, 98.8) (52.2, 97.8) .sup.5 10.sup.9/m.sup.2 3:20 93.4c 90.8c (68.3, 98.9) (62.3, 98.3) 1 10.sup.10/m.sup.2 3:20 100 100 In each column means followed by the same letter are not significantly different (GLM; P > 0.05).

6. Conclusions

(110) High levels of mortality (87-100%) were observed for O. surinamensis and C. ferrugineus in treatments with B. bassiana IMI398548, Entostat and kaolin after 7 days exposure in treated arenas. The Entostat and kaolin carrier alone resulted in statistically significant levels of mortality for O. surinamensis at both application rates compared with the control and conidia only treatments. The greater amount of the carrier gave the higher level of mortality. The Entostat and kaolin carrier alone at the higher application rate resulted in a significant increase in mortality of C. ferrugineus compared with the control and conidia only treatments. The lower application rate of Entostat and kaolin caused nearly 50% mortality of C. ferrugineus, but this was not significantly different from the mortality in control treatments (13.8%).
List of Standard Operating Procedures

(111) ISU 018 revision 3 General culturing procedure for stored product beetles

(112) ISU 034 revision 3 Food preparation and general culture requirements within the ISU

Experiment 4

(113) Pilot Scale Trial of Biopesticide Formulations in the Grain StoreFormulation with Talc

1. Aim

(114) To assess the formulation of an isolate of the entomopathogenic fungus Beauveria bassiana mixed with talc at different concentrations against two species of insect when applied to arenas made of galvanized steel and stored in the grain store environment.

2. Introduction

(115) The aim of this study was to determine the effectiveness of a powder formulation of B. bassiana I Ml 398548 mixed with talc and Entostat against two beetle species when applied to steel arenas in the Fera grain store.

3. Materials

(116) 3.1 Fungal Isolate

(117) The B. bassiana isolate (IMI398548) was produced by Somycel S. A. using a mass production method and was checked using in-house quality control (QC) procedures. The final product contained 3.810.sup.10 conidia/g. The isolate was designated as TA 2654 and was stored in a refrigerator at 4-8 C.

(118) 3.2 Magnesium Silicate (Talc) and Entostat

(119) The magnesium silicate (talc) used was supplied by Alfa Aesar, UK. The Entostat was supplied by Exosect Ltd. Both the talc and Entostat were kept at room temperature (approximately 20 C.).

(120) 3.3 Insects

(121) Two species of insect were tested. These were Oryzaephilus surinamensis strain Tram (saw-toothed grain beetle), and Cryptolestes ferrugineus strain Stow (rust-red grain beetle). Insects used were of mixed age and sex. Both species were used simultaneously within the arena. Insects were provided by the Invertebrate Supply Unit at Fera and reared according to Fera Standard Operating Procedures (ISU/018 revision 4 and ISU/034 revision 3).

(122) 3.4 Construction of Arenas

(123) Insects were confined to squares of galvanized steel (5005000.8 mm) within circular galvanized steel rings (approx. 450 mm diameter, 150 mm high). The inside surface of the steel ring was coated with Fluon (Whitford Plastics, UK) to prevent escape of the insects. The rings were sealed to the steel sheet using decorators caulk so that insects could not get under the ring. A refuge made from a piece of electrical conduit (2516100 mm), containing kibbled wheat to provide food for the insects, was placed in the centre of each ring approximately 1 hour after introduction of the insects.

4. Methods

(124) 4.1 Treatment of Arenas

(125) The treatments were as follows:

(126) 1. No treatment (control)

(127) 2. Entostat+talc (weight to equal that used in the IMI398548 510.sup.9 conidia/m.sup.2+Entostat+talc (1:3:20) formulation)=Carrier low

(128) 3. Entostat+kaolin (weight to equal that used in the IMI398548 110.sup.10 conidia/m.sup.2+Entostat+talc (1:3:20) formulation)=Carrier high

(129) 4. IMI398548 510.sup.9 conidia/m.sup.2

(130) 5. IMI398548 110.sup.10 conidia/m.sup.2

(131) 6. IMI398548 510.sup.9 conidia/m.sup.2+Entostat+talc (1:3:20)

(132) 7. IMI398548 510.sup.9 conidia/m.sup.2+Entostat+talc (1:6:40)

(133) 8. IMI398548 110.sup.10 conidia/m.sup.2+Entostat+talc (1:3:20)

(134) Treatments were assigned to each arena using a randomized block design. There were five replicate rings for each treatment.

(135) The formulations were made up by weighing out the individual components into a glass vial and mixing using a vortex mixer. The conidia and formulations required to treat the arenas were weighed out on to small pieces of aluminium foil, which were then carefully folded to protect the contents during transfer to the grain store. The appropriate amount of material was evenly distributed across the floor area of the assigned rings using a small brush. The calculated amount of material added to the arenas for each treatment is shown in Table 1

(136) TABLE-US-00008 TABLE 1 Calculated amount of material added to the arenas for each treatment. Entostat Total Treatment Conidia (g) (g) Talc (g) (g) Control 0 0 0 0 Carrier low 0 0.0627 0.418 0.4807 Carrier high 0 0.1254 0.836 0.9614 .sup.5 10.sup.9 conidia/m.sup.2 0.0209 0 0 0.0209 1 10.sup.10 conidia/m.sup.2 0.0418 0 0 0.0418 .sup.5 10.sup.9 conidia/m.sup.2 1:3:20 0.0209 0.0627 0.418 0.5016 .sup.5 10.sup.9 conidia/m.sup.2 1:6:40 0.0209 0.1254 0.836 0.9823 1 10.sup.10 conidia/m.sup.2 1:3:20 0.0418 0.1254 0.836 1.0032
4.2 Addition of Insects

(137) Insects were counted in the laboratory, transported in vials and released into the steel rings 24 hours after treatment. Both species were present within each of the steel rings and 50 insects of each species were used in each replicate. The refuge containing kibbled wheat was added to the arena approximately one hour after the introduction of the insects.

(138) 4.3 Assessment of Insects

(139) Insect mortality was assessed after 7 days. Insects were collected from the rings into labelled glass tubes with the aid of a battery operated suction device. Refuges were removed from the rings and placed in labelled self-seal plastic bags. The numbers of live and dead insects of each species within each steel ring and the refuge was recorded.

(140) 4.4 Monitoring of Environmental Conditions

(141) Temperature and humidity were monitored using Tinytag dataloggers (TGP 1500, Gemini Dataloggers Ltd, UK) (OPA0/AppEnt 002 and 003) located in two positions around the arenas. Lights within the store were switched on during treatment and assessments, but remained off at all other times.

(142) 4.5 Statistical Analysis

(143) The difference in mortality between treatments was analysed by a generalized linear model (GLM) with a logit link function. Post-hoc tests were used to test for differences between the treatments by comparison of the least significant difference for the treatments. When treatment mortalities were 0% or 100% a least significant difference value could not be obtained and post-hoc comparisons based on the least significant difference were therefore not possible for these treatments.

5. Results

(144) 5.1 Environmental Conditions

(145) The average temperature recorded in the grain store throughout the trial was 15.0 C. with a minimum of 9.3 C. and a maximum of 19.2 C. The average humidity during the trial recorded using the Tinytag dataloggers was 77.6% with a minimum of 51.1% and a maximum of 95.6%.

(146) 5.2 Insect Mortality Data

(147) There was a significant effect of treatment on the mortality of both beetle species tested after 7 days exposure (GLM F.sub.7.36=2.42, P=0.045, F.sub.7.36=44.56, P<0.001 and F.sub.7.36=88.61, P<0.001 for O. surinamensis and C. ferrugineus, respectively) (Table 2).

(148) The highest mortalities for O. surinamensis were seen in the treatments with IMI398548, Entostat and talc (Table 2). The treatments with conidia and the higher amount of Entostat and talc (510.sup.9/m.sup.2 6:40 and 110.sup.10/m.sup.2 3:20) gave a significantly higher mortality than the treatment with the lower amount of Entostat and talc (510.sup.9/m.sup.2 3:20) (P<0.05). Similarly, a significant difference (P<0.05) in mortality of O. surinamensis was observed between the carrier low and carrier high treatments, with a lower level of mortality observed with the lower amount of carrier (Table 1). Treatments with the conidia alone were not significantly different to the control (P>0.05, Table 2).

(149) The highest mortalities for C. ferrugineus were seen in the treatments with IMI398548, Entostat and talc (Table 2). These treatments gave significantly greater mortality than the control and treatments with the conidia alone (P<0.05, Table 2). The carrier alone (Entostat and talc) also gave significantly greater mortality than the control and conidia only treatments. A significant difference (P<0.05) in mortality of C. ferrugineus was observed between the carrier low and carrier high treatments, with a lower level of mortality observed with the lower amount of carrier (Table 2).

(150) TABLE-US-00009 TABLE 2 Derived mean % mortality of insects 7 days after exposure to different treatments. % mortality is expressed in terms of the number of insects recovered for each species. Figures in parentheses are the derived 95% confidence intervals. O. surinamensis C. ferrugineus Control 3.7a 2.1a (0.9, 14.0) (0.4, 9.5) Carrier low 35.2b 70.9b (24.5, 47.7) (60.5, 79.4) Carrier high 53.8c 88.2c (41.4, 65.8) (79.3, 93.6) .sup.5 10.sup.9/m.sup.2 5.1a 10.5a (1.7, 13.9) (5.5, 19.2) 1 10.sup.10/m.sup.2 10.0a 5.7a (6.1, 22.4) (2.4, 13.0) .sup.5 10.sup.9/m.sup.2 6:40 93.6d, e 100 (82.1, 97.9) .sup.5 10.sup.9/m.sup.2 3:20 80.2d 97.6d (69.1, 88.1) (91.5, 99.3) 1 10.sup.10/m.sup.2 3:20 95.6e 99.5c, d (85.1, 98.8) (88.7, 99.9) In each column means followed by the same letter are not significantly different (GLM; P > 0.05)

6. Conclusions

(151) High levels of mortality (80-100%) were observed for O. surinamensis and C. ferrugineus in treatments with B. bassiana IMI398548, Entostat and talc after 7 days exposure in treated arenas. For O. surinamensis the mortality observed in the B. bassiana IMI398548, Entostat and talc treatments was greater than the additive mortality of the conidia only and carrier only treatments.

(152) The Entostat and talc carrier alone resulted in significant levels of mortality for O. surinamensis and C. ferrugineus compared with the control and conidia only treatments. The greater amount of the carrier gave the higher level of mortality.

Experiment 5

Field Scale Efficacy Testing of Beauveria bassiana Formulations Against Stored Grain Pests

Summary

(153) A powder formulation of a biopesticide based on an isolate (IMI398548) of the entomopathogenic fungus Beauveria bassiana, which is active against stored grain pests, was tested in an on-farm grain store facility. The treatment was applied to the floor of two empty grain silos and then target beetle pests (Oryzaephilus surinamensis and Cryptolestes ferrugineus) were introduced to discrete arenas installed in the silos and monitored over time to measure the effects of the treatment on insect mortality. Mortality was compared to two grain silos that were split so that half received no treatment (untreated controls) and half received kaolin only (vehicle control). There was one outdoor and one indoor (inside a barn) silo for each treatment group. The trial was done in the autumn (September/October). Efficacy was determined by comparing the % total mortality of each species in the treated outdoor or indoor silo with the % total mortality of each species in the untreated control outdoor or indoor silo at each monitoring point using the Schneider-Orelli formula (Pntener, 1981 Manual for field trials in plant protection second edition. Agricultural Division, Ciba-Geigy Limited.).

(154) The % efficacy in indoor and outdoor treated silos at each time point and for each species after adjustment for control mortality is given in the table below. Efficacy was very high (>90% death due to treatment) for the two species within 14 d post treatment.

(155) TABLE-US-00010 O. surinamensis C. ferrugineus Adjusted by 7 days Indoor 51.58 76.83 untreated Outd'r 5.34 61.14 control 14 days Indoor 100 99.05 Outd'r 100 97.69 Adjusted by 7 days Indoor 42.86 71.16 vehicle Outd'r 0 36.16 control 14 days Indoor 100 97.82 Outd'r 100 90.93

(156) In conclusion, under the conditions of this trial the biopesticide showed a high level of efficacy for the two target species.

Objective

(157) To determine the efficacy of a dry powder formulation of Beauveria bassiana strain IMI398548 against stored product pest species.

(158) Introduction

(159) The purpose of this study was to examine the efficacy of a powder formulation of a biopesticide based on an isolate (IMI398548) of the entomopathogenic fungus Beauveria bassiana against stored grain pests under realistic field conditions.

(160) This trial used realistic pest control operator (PCO) application equipment for applying the product to the whole floor of grain silos. This trial included a vehicle control treatment for the kaolin, monitoring mortality at 7 and 14 days and by testing the spore concentration at a low target rate (510.sup.9 total conidia per m.sup.2). We aimed for a minimum deposition of 110.sup.9 CFU/m.sup.2.

(161) Twenty-four hours following treatment, sample groups of grain beetles Oryzaephilus surinamensis (saw-toothed grain beetle) and Cryptolestes ferrugineus (rust-red grain beetle) were added to discrete arenas within the treated areas and monitored over time for mortality.

1. Test Item Details

(162) Test item type: Dry powder formulation containing conidia of Beauveria bassiana strain IMI398548, Sylvan batch: 2112003 (CABI batch code: 142/12; FERA batch code TA 2672). Test item contents: Fungus, Entostat and Kaolin Test item rate: 510.sup.9 total conidia/m.sup.2 (aiming for minimum 110.sup.9 CFU/m.sup.2) Batch 2112003 contains 5.2510.sup.10 conidia/g (calculated at CABI) so we needed to apply 0.095 g of the dry spores per m.sup.2. Entostat, 0.768 g/m.sup.2 and kaolin=5.120 g/m.sup.2. Supplier: IMI398548 was supplied by CABI of Bakeham Lane, Egham, Surrey, TW20 9TY). The kaolin is opal omega grade sourced from Goonvean Ltd (St Stephen, St Austell, Cornwall, United Kingdom, PL26 7QF). The Entostat was manufactured at Exosect (batch W1307). The formulations were prepared at CABI using calibrated balance equipment, mixed thoroughly and sealed in a foil sachet.

2. Test Sites

(163) The trial included four cylindrical grain storage silos. All of the silos had metal corrugated walls and solid concrete floors with side access through hatches or doorways. Two of the silos were located inside a barn (5 and 6) and were 3.6 m diameter and two were located outside (7 and 8) and were 5.7 m in diameter.

(164) Insects were cultured and supplied by the invertebrate supply unit at FERA. Mortality counts were done at the FERA site.

(165) The formulation was mixed at the CABI site. Calibration and validation work was also carried out at CABI.

3. Methodology

(166) 8.1. Experimental Design

(167) There were four silos available for treatment, two inside a barn and two outside. One indoor silo (6) and one outdoor silo (7) were used for control treatments and the remaining two silos (5 and 8) were treated with the fungal formulation. Forty stainless steel arenas to contain the insects were present in each silo. In the control silos twenty of the arenas were covered over before treatment (so they could be kept as untreated controls and the carrier powder kaolin was applied to the whole silo. Insects were introduced to twenty of the arenas in the treated silos and all forty of the arenas in the control silos one day after the treatment applications. Insects were collected from ten of the untreated control arenas, vehicle control arenas and treated arenas after 7 d and the remaining arenas after 14 d. The insects were subjected to mortality checks. The trial period extended over a 2 week period.

(168) 8.2 Study Schedule

(169) TABLE-US-00011 25/09/2012 - D0 Vacuum silos, cover untreated control arenas, apply kaolin and test item treatments 26/09/2012 - D1 Uncover untreated control arenas, introduce insects and refuges to all arenas, cover all arenas with mesh, set-up data loggers 03/10/2012 - D8 Collect insects from ten arenas in each treatment group 04/10/2012 - D9 FERA count live and dead insects 10/10/2012 - D15 Collect insects from remaining ten arenas in each treatment group, collect data loggers 11/10/2012 - D16 FERA count live and dead insects 30/10/2012 Trial clean-up
8.3 Site Preparation

(170) Before trial commencement the treated grain silos were vacuumed using a Numatic HZQ200-2 Hazardous Dust Vacuum Cleaner. The arenas were checked and thoroughly vacuumed to remove any remaining insects or treatment powder from previous trial work.

(171) 8.4 Arena Installation

(172) Forty arenas in silos 6 and 7 (control silos) and twenty arenas in silos 5 and 8 (treatment silos) were used in this trial. The arenas were already in place on top of concrete paving flags (Marshalls FL1200600; 6060 cm), four arenas per flag (ten flags per silo) in a 22 arrangement. Two arenas on each flag in silos 5 and 8 were not used. The arenas were made from stainless steel rings (Instant Fabrications Ltd, Chandlers Ford, Hants, UK; 20 cm diameter, 5 cm deep, 0.9 mm thick). The inside surfaces of the arenas were coated with Fluon (Blades Biological, UK) to prevent escape of the insects. The arenas were sealed to the concrete flag using aquarium sealant (Geocel Aquaria Aquarium Sealant: Sealants and Tools Direct Ltd) so that insects could not get underneath. A covering made from fine mesh material and held down with an elastic band was placed over each arena after insect introduction.

(173) In silos 6 and 7 the arenas in blocks 3, 4, 5, 6 and 10 were covered over with clingfilm and taped down before treatment. These were the untreated controls and were covered to protect them from kaolin powder deposition. In silos 5 and 8 only two arenas, A and B, were used; A was sampled at 7 d and B at 14 d. In silos 6 and 7, A and B were sampled at 7 d and C and D at 14 d. Within blocks the codes were randomly assigned to the arenas using a table of random numbers.

(174) 8.5 Equipment Calibration

(175) Application of the formulations and carrier powders were made using B&D Mini DustR applicators (Killgerm), one per silo. A calibration for the applicator was made.

(176) The nature of the pump mechanism meant that there may have been variations in the amount of powder applied from each pump, thus a standard position was utilised when applying and calibrating. The applicator was held at a 45 angle with the application nozzle in the lowest position nearest to the floor. This ensured the feeder tube inside the powder chamber was covered with the maximum amount of powder at all times, thus minimizing variance in application rates. To ensure an even application, the powder applicator was moved in an arc motion by the applicator with each puff. This was shown to be the most effective way to ensure even coverage during powder room trials at CABI facilities. The applicator calibration results showed that the amount of powder per puff dropped as the powder applicator emptied, thus to ensure an even spread of powder the standardised pattern of application involved covering the entire floor of the silo in less than 10 puffs, then repeating this standard pattern. This ensured a more even application of powder than if the applicator had systematically covered the floor of the silo once until the powder applicator was empty.

(177) 8.6 Environmental Data Collection

(178) Mean, max and min daily temperature and humidity readings for each grain silo were recorded from the day of insect introduction. Calibrated Lascar temperature and humidity loggers (model EL-USB-2) were positioned next to block 9 in each silo. Readings were collected every 60 min. The data was downloaded and analysed using Lascar software.

(179) 8.7 Application of Treatments

(180) A separate applicator was used for each treatment (four in total: two test treatments and two vehicle control treatments).

(181) The applications were made from the far side of the silo (opposite the entry) from applicator height with the nozzle directed downwards at a 45 angle in a standardized pattern, repeated until the applicator was empty. Treatment using kaolin only were made in silos 6 and 7 to the whole floor also, starting from the far side again. Only arenas in blocks 1, 2, 7, 8 and 9 received the carrier treatmentthe other arenas were covered over with clingfilm so that they could be used for the untreated controls.

(182) Before treatment the hatches of the outdoor silos were sealed shut. The indoor silos have doorways and the doors have been removed so the silos were sealed closed with polythene sheeting, cardboard and duct tape. Any ventilation pipes or holes in silos were covered with polythene sheeting and duct tape.

(183) 8.8 Insect Application and Monitoring

(184) Two species of insect were tested: Oryzaephilus surinamensis strain Tram (saw-toothed grain beetle), and Cryptolestes ferrugineus strain Stow (rust-red grain beetle). Insects were of mixed age and sex. Insects were provided by the Invertebrate Supply Unit at FERA and reared according to FERA Standard Operating Procedures (ISU/018, ISU/023, ISU/025, ISU/026, ISU/034).

(185) Insects were added 24 h after application of the treatments. In silos 6 and 7 (controls) each species was added to all of the arenas. In silos 5 and 8 only arenas A and B received insects. Each arena contained twenty of each species. A refuge made from a piece of electrical conduit (2516100 mm), containing kibbled wheat to provide food for the insects, was placed in the centre of each arena approximately 30 min before introduction of the insects.

(186) Insects were collected at 7 or 14 d using battery operated pooters and transferred to glass vials. Refuges were removed from the rings and emptied into labeled zip-lock bags. The vials and bags were boxed and couriered overnight to FERA for counting.

(187) 8.9 Data Analysis

(188) An adjustment for control mortality to the mean mortalities for each insect species in each treatment silo at each time point was calculated and is presented.

4. Results

(189) 9.1. Environmental Conditions

(190) Conditions within the silos were quite different between the indoor and outdoor silos, with temperature and humidity fluctuating to a much greater degree in the outdoor silos; probably due to exposure to sun and lack of insulation outside (Table 1). Temperature minimums and maximums were lower and higher respectively in outdoor silos. The mean temperatures and humidity however were similar between indoor and outdoor silos. Conditions were also similar between silos 5 and 6 (indoor) and between 7 and 8 (outdoor).

(191) 9.2. Insect Survival

(192) When the collected insects were being checked for mortality some of them were missing. It seemed unlikely that they had been able to climb out but in some arenas, due to the slight uneven surface, there was sufficient gap under the steel rings to allow some of the smaller beetles to take refuge and avoid being collected into the pooter. Because fewer than 10 beetles from one species were occasionally collected, and the arenas are not true replicates but samples, the total mortality in the silo from all arenas was calculated, rather than the mean mortality per arena.

(193) Mortality in the untreated control arenas was notable at each time point for O. surinamensis (10-20%) and C. ferrugineus (10-30%), particularly in silo 7 where conditions were warmer and fluctuated to a greater degree. This could be attributed to the fact that the insects are laboratory reared and used to constant controlled conditions. There was no acclimation period because conditions at the site were variable so they may not have been able to adapt to such an abrupt change. An additional possibility is that the warm conditions caused volatilisation of toxic residues (confirmed to be present during pre-trial work) from the surfaces of the store so that even though insects were not in physical contact with the floor of the silo, they were still exposed to low levels of volatile compounds that could have affected their survival.

(194) Survival of O. surinamensis and C. ferrugineus did seem to be affected by the carrier powders as mortality was greater than the untreated controls (27-60% and 28-82% respectively), particularly in Silo 7 where temperatures were warmer during the day.

(195) Although some mortality may be attributed to the carrier powder kaolin, mortality in arenas which received the full formulation treatment experienced the highest levels of mortality. This would seem to indicate that the combination of B. bassiana isolate IMI398548 and kaolin with Entostat provided the best level of control. Apart from lower than expected mortality (24%) in the outdoor treated silo at day 7, the mortality of O. surinamensis exposed to treatment was high (58% at day 7 in silo 5 and 100% in both silos at day 14). Mortality of C. ferrugineus was also high, even after just 7 days (7 d: 72-79% and 14 d: >98%).

(196) The % efficacy in indoor and outdoor silos at each time point and for each species was calculated using the untreated control mortality and the vehicle control mortality with Schneider-Orelli's formula (Pintener, 1981 Manual for field trials in plant protection second edition. Agricultural Division, Ciba-Geigy Limited.), which adjusts for control mortality (see Table 2). Because the carrier kaolin caused some mortality the adjusted % efficacy using these figures are lower. To examine the overall effect of the formulation on the insects it is best to examine the % efficacy adjusted by the untreated controls. To understand the benefit of the fungus in the formulation it is best to examine the % efficacy adjusted by the vehicle control mortality.

5. Discussion

(197) This trial is a series of trials designed to assess the mortality of insects exposed to a novel biopesticide treatment in grain storage silos. Insects were collected after 7 and 14 d and the concentration of spores added to the formulation was as stated above. The decision to check mortality at 7 d was beneficial as we were able to show that mortality of O. surinamensis was 20-60% and of C. ferrugineus was 80-90% in treatment silos. Clearly some mortality may be attributable to factors other than the treatment because mortality in the untreated controls was between 10-30% for these two species at 7 d. This may be largely attributable to the shock of being introduced to the arenas since untreated control mortality did not rise noticeably between 7 and 14 d.

(198) The environmental conditions in the silos were suitable for both insect survival and fungal germination. Temperatures did not drop below 5 C. and did not rise above 22 C. The isolate survives well in a broad range of temperatures. High humidity (60-90% RH) will have aided the initial germination of the fungus, although this isolate has lower requirements for water activity than other B. bassiana isolates.

(199) For a biological treatment the % efficacy achieved was high for C. ferrugineus after only 7 d, particularly in the indoor silo, and very high (>90%) after 14 d. For O. surinamensis the % efficacy was low in outdoor silos after 7 d but notable in the indoor silo (>50%). By 14 d the % efficacy for O. surinamensis was exceptional at 100% in both indoor and outdoor silos. We conclude that the efficacy for the two species O. surinamensis and C. ferrugineus after only 14 d post treatment was very good.

(200) Tables

(201) TABLE-US-00012 TABLE 1 Max, min and mean temperature and humidity in each of the silos during the trial Temperature % RH Silo Max Min Mean Max Min Mean 5 treated indoor 15.00 9.00 13.02 89.00 71.50 82.13 6 untreated indoor 15.00 9.00 12.92 89.00 71.00 82.34 7 untreated outdoor 21.00 4.50 12.99 92.00 54.50 82.56 8 treated outdoor 20.50 4.50 12.96 93.50 61.50 84.96

(202) TABLE-US-00013 TABLE 2 % efficacy in indoor and outdoor treated silos at each time point and for each species after adjustment for untreated control and vehicle control mortality using Schneider-Orelli's formula (Pntener, 1981) O. surinamensis C. ferrugineus Adjusted 7 days Indoor 51.58 76.83 by Outd'r 5.34 61.14 untreated 14 days Indoor 100 99.05 control Outd'r 100 97.69 Adjusted 7 days Indoor 42.86 71.16 by Outd'r 0 36.16 vehicle 14 days Indoor 100 97.82 control Outd'r 100 90.93

(203) In the context of this specification the term electret particles is to be understood as embracing electrostatically charged particles, including (but not restricted to) wax particles produced by the methods hereinbefore described.